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. 2015 Apr 29;141(10):1845–1857. doi: 10.1007/s00432-015-1969-3

Epstein–Barr virus-targeted therapy in nasopharyngeal carcinoma

Sharon D Stoker 1,✉,#, Zlata Novalić 2,#, Maarten A Wildeman 1,3, Alwin D R Huitema 4, Sandra A W M Verkuijlen 2, Hedy Juwana 2, Astrid E Greijer 2, I Bing Tan 1,5,6, Jaap M Middeldorp 2, Jan Paul de Boer 7
PMCID: PMC11823716  PMID: 25920375

Abstract

Purpose

Despite successful primary treatment of nasopharyngeal carcinoma (NPC), the incidence of distant metastasis remains 25–34 %. Treatment options are limited, and survival is poor. Intratumoural Epstein–Barr virus (EBV) was used as treatment target. In NPC, EBV is present in a latent state, expressing only few non-immunogenic viral products. Gemcitabine and valproic acid can trigger EBV to the lytic state, wherein viral kinases are expressed, making EBV-positive tumour cells susceptible for antiviral therapy with, i.e. valganciclovir, and inducing an EBV-specific immune response.

Methods

This drug combination was applied in eight patients with EBV-positive NPC, refractory to conventional treatment. The primary endpoints were safety, tolerability and clinical response. Secondary endpoint was to get proof of concept based on biomarkers, i.e. pharmacokinetics, EBV-DNA load in whole blood and nasopharyngeal brushes, EBV-RNA profiling for proof of lytic induction, EBV-IgG and EBV-IgA levels and diversity and EBV-specific T cell response.

Results

The best observed clinical response was partial in two patients (25 %) and stable disease in three patients (37.5 %). The median survival was 9 months (95 % confidence interval 7–17 months). Effective dose levels were reached. Peaking of EBV-DNA loads in blood and brush proved the biological effect on EBV during most treatment cycles. In one patient, RNA profiling confirmed lytic EBV induction. EBV-IgG and EBV-IgA antibody levels were already high before treatment and did not change during treatment. No changes in EBV-specific T cell response were detected.

Conclusion

The treatment was safe with manageable side effects, clinical response was observed, and viral activation corroborated.

Keywords: Nasopharyngeal carcinoma, Epstein–Barr virus, Targeted therapy, Metastatic disease, Advanced disease

Introduction

During the last two decennia, treatment of nasopharyngeal carcinoma (NPC) has improved considerably by the introduction of concurrent chemo-radiation. However, the overall incidence of distant metastasis remains 25–34 %, and survival of these patients is poor (Chen et al. 2013; Lee et al. 2012). Geographical regions with high incidence of NPC include developing countries with low health expenditures. The incidence of distant metastases in these regions is high, due to late diagnosis and limited treatment availability. In Indonesia, for example, around 14 % of patients have distant metastasis at diagnosis, and of the patients without distant metastases only 29 % shows a complete response after treatment, compared to up to 70–95 % in the literature (Adham et al. 2014; Wildeman et al. 2013). In case of distant metastasis at diagnosis, the median survival is 10–26 months, depending on dissemination pattern and choice of palliative treatment (Chen et al. 2013; Bensouda et al. 2011). When distant metastases occur after platinum-based regimens, palliative treatment with gemcitabine, capecitabine or docetaxel results in a median survival of 9.5–15 months (Bensouda et al. 2011). However, this type of treatment is expensive and only available in advanced care facilities, mostly out of reach of NPC patients living in rural areas in developing countries. The short survival, in combination with the relatively young age at primary diagnosis of NPC, and the lack of cheap and readily available drugs emphasizes the need for research for better or new targets for therapy.

NPC can be distinguished histologically in keratinizing tumours and non-keratinizing tumours. The latter can further be divided into differentiated and non-differentiated tumours. Non-differentiated NPC is for almost 100 % related to EBV and is the most frequently observed type of NPC both in endemic (>95 %) and in non-endemic (44–63 %) regions (Wei and Sham 2005; Arnold et al. 2013). Since EBV is clonally present in all tumour cells, it is considered as a potential target for treatment. This is exploited by recent clinical trials utilizing infusion of ex vivo activated T cells directed to EBV antigens to combat the virus-infected NPC tumour cells (Louis et al. 2010; Secondino et al. 2012; Chia et al. 2014; Lutzky et al. 2014).

In NPC tumour cells, EBV persists in a latent phase, wherein only a few viral proteins and non-coding small RNAs are expressed, essential for EBV maintenance and tumour growth. These viral products are non-immunogenic and contribute to NPC tumour immune escape (Middeldorp et al. 2003; Middeldorp and Pegtel 2008; Keryer-Bibens et al. 2006; Li et al. 2007a; Yip et al. 2009). When EBV is triggered to enter the reproductive lytic phase, additional and more immunogenic proteins are expressed, which can provoke a stronger and more effective immune response (Hislop et al. 2007). Importantly, viral kinases are induced rendering tumour cells sensitivity for antiviral treatment, e.g. (val)ganciclovir (Moore et al. 2001).

Virus-targeted lytic induction treatment in EBV-associated malignancies aims to evoke more potent immune responses and induce susceptibility for antiviral treatment. This strategy was initially pioneered in EBV-associated lymphoma by Faller et al. (2001) and has now been studied for several years (Perrine et al. 2007; Ghosh et al. 2012a). Different agents, such as histone deacetylase (HDAC) inhibitors, chemotherapeutics, radiation, phorbol esters and butyrates, have been found active for inducing the lytic phase of EBV in various latent virus carrying tumour cell lines (Ghosh et al. 2012a, b; Lima et al. 2011; Li et al. 2000). Until now, only a few clinical proof-of-principle studies on viral lytic induction have been performed (Perrine et al. 2007; Stevens et al. 2006a). EBV-targeted therapy approaches for NPC were recently reviewed by Hutajulu et al. (2014).

Studies in NPC cell lines and related mouse tumour models showed that a combination of gemcitabine (GCb) and valproic acid (VPA) acts synergistically in inducing the EBV lytic phase, and the addition of ganciclovir (GCV) as antiviral drug proved to further inhibit tumour growth (Feng et al. 2004; Wildeman et al. 2012; Feng and Kenney 2006). VPA has an HDAC inhibitory function and may reactivate transcription of multiple genes, which are silenced in cancer, possibly enhancing the GCb uptake. One such reactivated group of genes may be coding for hENTs, which are crucial for GCb uptake into the (tumour) cell. hENT1-deficient cells are highly resistant to GCb (Nordh et al. 2014).

GCb is an approved second-line chemotherapeutic agent for treating NPC and is in general associated with manageable side effects. GCb has been proven as potent lytic inducer of EBV (Wildeman et al. 2012). A combination of GCb, VPA and GCV (cytolytic virus activation therapy, CLVA) was previously applied by us in three patients with EBV-positive NPC for whom no curative treatment options were available (Wildeman et al. 2012). In these patients, the CLVA regime proved to be safe and tolerable. Treatment resulted in increased levels of viral DNA originating from apoptotic tumour cells with disease stabilization and improved quality of life. These observations formed the rationale to conduct a formal phase I–II study to further explore safety, tolerability and preliminary efficacy. Secondary aim of the study was to get proof of concept of the therapy by measuring several biomarkers; i.e. drug pharmacokinetics, EBV-DNA load in the nasopharyngeal area and whole blood to show peaking fluctuations as response to treatment, RNA profiling to proof lytic induction, plasma IgG and IgA levels and diversity for an increased antibody response and EBV-specific T cell response analysis for an enhanced cell-mediated immunity.

Patients and methods

Study design and subjects

This phase I–II study was performed at the department of head and neck oncology and surgery of the Netherlands Cancer Institute, Amsterdam, in collaboration with the pathology department of the VU University Medical Centre, Amsterdam. Institutional ethical approval and written informed consent from all patients were obtained. Candidate patients were identified in a multi-centre diagnostic survey programme (MS Submitted). Patients were eligible, if they had distant metastatic disease or refractory or relapsed locoregional EBV-positive NPC. EBV positivity was determined by in situ hybridization for EBV-encoded RNAs (EBER-RISH). Patients had to have radiological measurable disease, according to RECIST 1.1 criteria and WHO performance status of ≤2. Adequate organ and haematological function was required (neutrophil count of >1.5 × 109/L, platelet count of >100 × 109/L, haemoglobin level of >10 g/dL (≥6.2 mmol/L), serum bilirubin of <1.25 times the upper limit of normal (ULN), liver transaminases of <2.5 times ULN, serum creatinine of <1.25 times ULN). Patient’s age had to be between 18 and 70 year, and life expectancy should exceed 3 months.

Patients were excluded if they had an active infection, were pregnant, were treated (or within 4 weeks before inclusion) with any other anti-cancer therapy, had severe and/or uncontrolled concurrent medical disease, had a second malignancy or were legally incapable. All patients (n = 8) were treated between October 2011 and March 2013.

Treatment

The CLVA therapy comprised a 6-week cycle, including a 3-week drug treatment followed by a 3-week recovery period, with a total of 6 cycles (Wildeman et al. 2012). In each cycle, at day 1 and 8, GCb 1250 mg/kg2 was administered intravenously, from day 1 till day 14, daily VPA 12,5 mg/kg was given orally and from day 8 till 21, daily GVC 3 times 450 mg was given orally. Patients were treated in the outpatient clinic. The full treatment duration was approximately 9 months.

Safety and tolerability and dose modifications

Starting from baseline, patients underwent a weekly evaluation that included anamnesis, physical examination and a complete blood cell count with differential and serum chemistry analyses. Adverse events (AE’s) and serious adverse events (SAE’s) were evaluated according to the National Cancer Institute Common Toxicity Criteria, version 4.0 (CTCAE). In case patients experienced grade 3 or 4 toxicity, a dose reduction was performed. Patients who had a treatment delay of more than 2 weeks were withdrawn from the treatment and went off study.

Clinical response

The extent of disease was assessed by imaging with magnetic resonance imaging (MRI) of the head and neck region and full-body fluorodeoxyglucose positron emission tomography/CT scan (PET/CT scan). Clinical tumour response was evaluated after every two cycles. MRI was performed if the target lesion was located in the nasopharynx and CT scan in case of pulmonary, mediastinal, abdominal or bone metastases. Post-treatment and 3 months after treatment patients received also a full-body PET/CT scan. Tumour response was assessed by RECIST criteria 1.1. In case of progressive disease (PD) during treatment (according to RECIST criteria or by the investigator opinion), the patient was withdrawn from the study. Overall survival was calculated from the date of start therapy till the date of death.

Proof-of-concept analysis

Pharmacokinetic assessments

Pharmacokinetic (PK) sampling was performed in cycle 1. PK parameters were calculated using non-compartmental methods. At day one, plasma samples for determination of the concentration of GCb (dFdC) and its metabolite (dFdU) were taken pre-infusion and at 1, 2, 4, 8 and 24 h following the administration. Intracellular concentrations of gemcitabine triphosphate (dFdCTP) were determined in white blood cells (WBC) collected before, 2 and 24 h after infusion, as described previously (Veltkamp et al. 2006). The steady-state level of VPA was determined on day 8. GCV PK was performed at day 21; plasma samples were collected pre-intake and 1, 4 and 8 h following the first intake and stored at –80 °C until analysis. GCb and its metabolite dFdU and intracellular concentrations of dFdCTP were measured according to previously published LC–MS/MS methods (Veltkamp et al. 2006; Vainchtein et al. 2007). VPA level was measured using a homogenous enzyme immunoassay employed for routine therapeutic drug monitoring (EMIT(R), VIVA-E, Siemens). GCV level was determined using a validated LC–MS/MS assay. This assay fulfilled all validation requirements as stated in Food and Drug Administration (FDA) guidelines.

EBV monitoring

The effect of treatment on EBV was determined by weekly EBV-DNA load monitoring in whole blood (WB) and every 2–3 weeks in the nasopharyngeal (NP) brush (when feasible). Blood samples were collected in heparin-/EDTA-coated tubes. Three aliquots of 100 µl were mixed with 900 µl NucliSenslysisbuffer (LB) (Biomerieux) and stored at –80 °C until nucleic acid isolation. The remaining blood was centrifuged, and the plasma fraction was collected and stored at −20 °C. Nasopharyngeal brushes were collected as described previously, added directly in 4 ml LB and stored at −80 °C (Stevens et al. 2006b).

Quantitative EBV-DNA load in whole blood and nasopharyngeal brush

Nucleic acids were isolated from WB and the NP brush samples with silica-/guanidium thiocyanate-based method as described earlier (Boom et al. 1999). Viral load was determined by quantitative real-time PCR using the LightCycler480 (Roche) as described before (Wildeman et al. 2012; Stevens et al. 2005a). For amplification, a 99-bp fragment of EBNA1-encoding BKRF1 gene was used and a standard curve of BKRF1-containing plasmid was used for quantification. All samples were spiked with 1000 copies of EBV plasmid to analyse potential PCR inhibition. Data were analysed with absolute quantification method using a second Derivative Max LC480 software (Roche). A cut-off value (CoV) of 2000 copies per 1 ml WB and 2300 copies per brush was used (Stevens et al. 2005b, 2006b). The number of cells per sample was defined by PCR quantification of the beta-globin gene as before (Stevens et al. 2005a; Hesselink et al. 2005).

RNA profiling in the nasopharyngeal brush

RNA was treated with RQ RNase-free DNase (Promega) according to manufacturer’s protocol, followed by RNA precipitation with 3M NaAc pH 5.3, linear acrylamide and 100 % EtOH. Reverse transcription by target-specific cDNA synthesis was performed as described before (Wildeman et al. 2012). cDNA was diluted 10 times for use in SYBR green-based real-time PCR quantification (LightCycler480, Roche). To determine the exact number of molecules per sample, serial dilutions of plasmid pool containing all target genes were used to obtain a standard curve. Melting temperatures were analysed for specificity control of the PCR products. Quantification was done using absolute quantification/second Derivative Max LC480 software (Roche).

EBV-specific antibody responses in serum

Immunoglobulin A (IgA) reactivity was determined by synthetic peptide-based ELISA using immunodominant epitopes derived from EBNA1 and VCA-p18 as described previously (Fachiroh et al. 2006). Optical density was determined at 450 nm (OD450), and cut-off value (CoV) was calculated as mean OD450 + 2× standard deviation (SD). Immunoblot analyses of IgG diversity profiles were performed on strips containing HH514.c16 nuclear antigens after lytic induction with TPA and sodium butyrate and analysed as described previously (Middeldorp and Herbrink 1988).

EBV-specific T cell response

Frequency of EBV-specific CD8+ T cell populations was assessed before and after treatment for three patients positive for HLA-A*02.01 pMHC multimers. Viable PBMCs were collected at inclusion, after cycle 1 and after cycle 6 by Ficoll centrifugation and CD8+ T cell reactivity against a panel of 24 EBV-derived latent, and lytic epitopes was assessed making use of UV-induced ligand exchange and pMHC combinatorial coding (Andersen et al. 2012; Kvistborg et al. 2014).

Results

Patients

In total, eight patients could be enroled. Baseline patient characteristics are shown in Table 1. Three patients (patient 1, 4 and 8) were initially diagnosed with distant metastases and had received radiotherapy to the nasopharynx, neck and distant metastases. Patient 1 and 8 had also received prior treatment with GCb; patient 1 with a good response, and progression after ending GCb, and patient 8 had initially good response on GCb, but showed progression of disease during subsequent treatment.

Table 1.

Baseline patient characteristics

Sex Origin Age ECOG Initial stage Previous treatment Disease at inclusion CLVA treatment
Curative intent Palliative intent Nr. of cycles Reason for ending Best observed response OS
1 m North Africa 53 1 T2aN3M1 RT NP, neck and DM; GCb DM 6 SD 22†
2 m Surinam 58 1 T3N3M0 CRT ND, PDT Local 6 SD* 11†
3 m North Africa 63 2 T2N2M0 CRT ND, PDT, Cisp Locoregional and DM 2 PD 7†
4 m Surinam 64 2 T4N2M1 RT NP, neck and DM; Locoregional and DM 2 CVA PR 5†
5 m Dutch 47 0 T4N2M0 CRT DM 6 PR 19a
6 m North Africa 35 0 T3N2M0 CRT RT NP Local 2 PD SD** 9†
7 m Dutch 44 0 T4N0M0 CRT RT DM; GCb, Cisp DM 2 PD 13†
8 f Dutch 61 0 T3N1M1 RT NP, neck and DM; GCb DM 1 PD 5†
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ECOG performance stage, m male, f female, CRT chemo-radiotherapy, RT radiotherapy, NP nasopharynx, DM distant metastasis, GCb gemcitabine, ND neck dissection, PDT photodynamic therapy, Cisp cisplatin, CVA cerebrovascular incident, PD progressive disease

* According to RECIST, this was stable disease, clinically he had a partial response after cycle 2

** According to RECIST this was stable disease, but clinically progressive, wherefore treatment was ended

†Died

aAlive

Five patients (patient 2, 3, 5–7) were initially diagnosed with locoregional disease and had received chemo-radiation with curative intent. Two of them (patient 2 and 3) had locoregional tumour relapse, patient 2 received a successful neck dissection and local photodynamic therapy (PDT) with only partial local response, and patient 3 received a neck dissection, PDT and cisplatin, but only with partial locoregional response. Patient 5 developed distant metastasis 3 months after primary treatment. Patient 6 had local persistent disease after primary chemo-radiation and received successful re-irradiation, but relapsed locally 9 months later. Patient 7 developed distant metastases 3 years after primary treatment and received several courses of palliative radiation and chemotherapy (i.e. gemcitabine and cisplatin). On GCb, he had a good response, wherefore GCb was ended. He relapsed 11 months later. In summary, at time of inclusion, two patients had local relapse and six patients had distant metastatic disease with or without locoregional disease.

Treatment and dose modifications

Patients 1, 2 and 5 received the full schedule of six cycles, patient 8 received one cycle and the others received two cycles. Reasons for early treatment cessation were disease progression in four cases and a cerebrovascular accident in one patient (number 4). Dose modifications were performed in four patients. Patient 1 incorrectly received a dose reduction in GCb in cycle 5. Patient 5 did not receive the second dose of GCb in cycle 1, due to a grade 3 neutropenia. In the second cycle, GCb was given with a dose reduction, but again he developed a grade 3 neutropenia. Therefore, GCV dose was also reduced. Patient number 6 had a grade 3 neutropenia in cycle 1, which resolved within 7 days. GCb was postponed by 1 week. Patient 7 suffered from pleural oedema and nausea probably related to pneumonia (grade 3 toxicity) in cycle 1. GCb was reduced, and in the second week of cycle 2, GCb was postponed due to nausea and vomiting (grade 3 toxicity).

Pharmacokinetics

All patients (n = 8) were evaluable for pharmacokinetics (PK) of GCb and dFdU, from six patients steady-state VPA levels were available, and from four patients evaluable PK data for GCV were available. Table 2 shows the estimated PK values of GCb, GCV and VPA. In all evaluable cases, the intended effective drug concentrations were achieved.

Table 2.

Pharmacokinetics

Gemcitabine (dFdCTP) Gemcitabine (dFdC) Gemcitabine (dFdU) Valganciclovir Valproic acid
C2hours (pmol/106cells in PBMC) Cmax (mg/L) AUC0-last (mg*h/L) Cmax (mg/L) AUC0-last (mg*h/L) Cmax (mg/L) AUC0-last (mg*h/L) Css mg/L
Median 127 0.780 1.05 29.3 187 3.98 17.1 25.0
Range 111–157 0.439–1.49 0.561–1.81 21.7–39.0 162–469 3.33–6.69 13.3–23.1 7.0–35.0
n 5 8 8 8 8 4 4 6
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C 2hours concentration after 2 h, Cmax maximal concentration, AUC0-last area under the curve from t = 0 until last observation, Css steady-state concentration

Safety and tolerability

The most common grade 3 toxicity was neutropenia. Patient 7 was admitted to the hospital because of suspected pneumonia with some pleural oedema and nausea; therefore, all scored as grade 3. Treatment with antibiotics improved the clinical condition. Patient 2 complained for a period of impaired hearing and nasal congestion. Patient 4 had headache and fatigue, limiting his daily activities, these complaints resolved after blood transfusion. Patient 8 had increased liver enzymes, most likely related to the progression of the liver metastases. Grade 4 toxicities were not seen. Patient 4 had a cerebral accident and refused any further treatment.

Clinical response

The median survival was 9 months (95 % confidence interval 7–17 months). The best observed response was partial response in two patients (25 %) and stable disease in three patients (37.5 %). Patient 1 had measurable disease in the lungs; during the study, these were stable and no new lesions were observed. Three months after treatment he had disease progression. Patient 2 only had disease in the nasopharynx and showed a clear reduction in tumour volume after two cycles. However, according to RECIST, this was still regarded as stable disease (Fig. 1). Patient 3 and 7 both had clinically and according to RECIST progressive disease after the first two cycles. Patient 4 had a partial response on the liver metastases, but had a cerebrovascular accident and was withdrawn from the study. Patient 5 had a lesion in the hilum of the lung, which partially responded after two cycles. Three months after treatment, this lesion progressed again, a second lesion in the hilum was visible and a suspect lesion in the femur was shown. Patient 6 had tumour in the nasopharynx only; after two cycles, this tumour was increased in volume and extended towards surrounding structures (orbit, base of the scull and both maxilla sinus). Since the maximal tumour diameter was not increased, he had stable disease according to RECIST. Patient 8 developed increased liver enzymes, and CT scan showed progressive disease in the liver.

Fig. 1.

Fig. 1

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Tumour response of patient 2 (a) before treatment, (b) after the second cycle. The diameter in this direction is decreased and in the centre of the tumour is less enhancement, suggesting a necrotic process

EBV-DNA load in NP brush

Viral DNA load determined in NP brushes was used to monitor the presence and quantity of EBV-containing tumour cells in the NP region before and during treatment. The mean value of cells per brush (based on beta-globin PCR) was 2 × 106 (±3 × 106), indicating proper sampling in each brush. The range of viral load in NP brushes is shown for all patients in Table 3. Patient 1 (distant metastasis) had a high EBV-DNA load in the brush at inclusion, which strongly decreased after fifth cycle of treatment (Fig. 2a). After administration of the last cycle, a rise in viral load was observed. At that time, no disease progression appeared clinically, but 3 months later, a suspect lesion was visible in the nasopharynx and the lung lesions had progressed. Patient 2 (local disease) had very high viral load levels in all NP brushes, reaching the maximum after second cycle of treatment, slowly decreasing thereafter (Fig. 2b). EBV-DNA load in brushes collected from patient 5 (distant metastasis) below the CoV at all times (Fig. 2c). EBV-DNA load in all brushes from patient 3, 4 and 6 (all with local disease) was above the CoV reflecting local disease activity. Arise in viral load was observed during the first cycle of treatment in patient 6, but no samples were available for follow-up (Table 3). All patients with local disease showed disease progression despite treatment.

Table 3.

EBV-DNA load in NP brush and WB

Patient (a) EBV-DNA load NP brush (copies/brush) (b) EBV-DNA load WB (copies/ml) (c) EBV-DNA load NP brush (copies/brush) (d) EBV-DNA load WB (copies/ml)
Min Max Min Max Before After Before After
1 2 × 103 2 × 106 <1 × 102 3.1 × 104 5 × 105 2 × 106 1 × 102 1 × 102
2 1.3 × 105 9 × 106 <1 × 102 1.9 × 104 2.9 × 106 1.4 × 106 1 × 102 1.4 × 103
3 1.7 × 103 2.6 × 106 3.9 × 102 2.2 × 104 2.6 × 106 5.2 × 103 2.6 × 103 5.7 × 102
4 5.3 × 103 1.1 × 105 <1 × 102 7.4 × 103 2 × 104 5.3 × 103 7.4 × 103 8 × 102
5 0 1 × 103 5.2 × 102 1.3 × 103 1 × 102 1 × 102 1 × 102 1 × 102
6 2.3 × 104 3.7 × 106 <1 × 102 1 × 103 6.5 × 104 3.7 × 106 9.3 × 102 1 × 103
7 0 0 <1 × 102 1 × 103 0 0 0 <1 × 102
8 0 1.7 × 105 3 × 103 3.5 × 105 1.7 × 105 0 3 × 103 1.4 × 105
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Lowest and highest levels measured (a, b); levels before the start of first cycle and after the latest cycle administered (c, d) are indicated per patient

Fig. 2.

Fig. 2

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EBV-DNA load dynamics during treatment. Viral load in WB (black line) and in NP brush (grey line) of patients who completed the full treatment is presented. Administration of VPA and GCV is depicted with blocks, and GCb is represented as black arrows. Dose reductions are indicated by grey arrows and dose delays by stars (*). In patient 1 (a) and patient 2 (b), the EBV-DNA levels are fluctuating, while in patient 5 (c) all samples were negative from the start of second cycle of CLVA

EBV-DNA load in whole blood

During treatment, all patients showed wide variation in EBV-DNA load levels in whole blood (Table 3; Fig. 2). In patient 1, there was a general decline visible (Fig. 2a). Clinically, this patient had SD during treatment. Patient 2 also showed a small overall decrease (Fig. 2b). In samples, from patient 5 EBV-DNA load levels did not exceed the CoV (Fig. 2c).

Lytic transcripts detected in tumour cells during CLVA treatment

EBV-RNA profiling could be performed in two patients (1 and 2) who received complete therapy and had enough NP brush samples with high viral load. In brushes from patient 1, no lytic transcripts were detected. For patient 2, RNA profiling revealed viable cells in 77 % of samples and indicated viral presence by detectable RNA transcripts, reflecting the NPC-specific EBV latency type-II (data not shown). In two samples, lytic transcripts of early viral kinases TK and PK were detected, paralleled by a peak in EBV-DNA load in the brush, which appeared precisely at the end of the second cycle of CLVA (Fig. 3). Immediate early transcripts Zebra and Rta remained below detection level. Transcripts of late lytic phase marker VCA-p18 were undetectable at all-time points, as expected due to antiviral GCV treatment. Simultaneous to the burst of viral lytic RNA expression, a significant decrease in the tumour mass in the nasopharynx was observed on MRI (Fig. 1).

Fig. 3.

Fig. 3

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Lytic transcripts in tumour cells during treatment in NP brush samples of patient 2. Presented are the numbers of molecules per brush. Expression of viral kinases PK (black-dotted line) and TK (grey-dotted line) is elevated after the second cycle of CLVA and is accompanied by an increase in EBV-DNA copies per brush (black solid line)

EBV-specific IgA and IgG serology

EBV-specific immune reactivity is reflected indirectly by plasma IgA responses towards viral proteins EBNA1 and VCA-p18, which parallels the NPC development. In this patient series, the levels of IgA against EBNA1 and VCA-p18 were high at intake and in general did not significantly change in time (Fig. 4a–c). The diversity of IgG response against immunodominant EBV epitopes of latent and lytic proteins was assessed by immunoblot analyses in sera of 6 patients, before and after treatment (Fig. 4d). No changes in IgG reactivity or diversity were observed in patient 1, 2, 6, 7 and 8. Patient 5 showed a slight increase in IgG to TK, VCA-p40 and VCA-p18 bands, together with a new response to a yet unspecified viral protein at 30 kDa.

Fig. 4.

Fig. 4

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Antibody responses against EBV latent and lytic proteins. IgA responses against EBNA1 (grey line) and VCA-p18 (black line) by single antigens are presented before and during treatment as detected by ELISA in patients 1 (a), 2 (b) and 5 (c). IgA levels in patient sera were normalized to the CoV as defined by healthy controls. d Immunoblot analysis of EBV-specific IgG response in 6 patients prior and after CLVA therapy. Epstein–Barr nuclear antigen 1 (EBNA1); immediate early protein Zebra (ZEBRA); early antigens (EA): p138, TK, DNAse, p47–54; late proteins: VCA-p40 and VCA-p18. Legend: NPC patient (N); healthy control (C); p = patient number; (B/A) = anti-EBV-IgG reactivity before (b) and after (a) the latest cycle of therapy

EBV-specific T cell response

Three patients (number 3, 5 and 6) were positive for HLA-A*02.01 and were included in the analysis of specific T cell responses against a set of EBV latent and lytic epitopes, before treatment and after two cycles (patient 3), after 1 and 6 cycles (patient 5) and after three cycles (patient 6). No increase was detected in number of the EBV-specific T cell responses post-therapy, and the magnitude of pre-existing responses was unaltered.

Discussion

This phase I–II study aimed to use EBV in the tumour cells as target for the treatment of patients with recurrent and/or metastatic NPC, for whom no other curative treatment options were available. In NPC, EBV is present in a latent state and only non-immunogenic viral antigens are produced, while active immune evasion strategies are operational in the tumour microenvironment (Middeldorp and Pegtel 2008; Li et al. 2007b). In this study, gemcitabine (GCb) and valproic acid (VPA) were used to trigger endogenous EBV into the lytic stage, wherein viral kinases are produced, making the responsive tumour cells susceptible for antiviral therapy, i.e. valganciclovir (GCV). In addition, newly induced lytic and immunogenic viral antigens were expected to trigger additional B cell- and T cell-mediated immune responses against EBV-positive NPC, leading to more and longer lasting tumour destruction (Hislop et al. 2007; Merlo et al. 2011).

The combined CLVA treatment proved safe with manageable and reversible side effects. One patient died subsequent to a cerebrovascular accident, which was considered unlikely to be treatment related. One patient had persistent increased liver enzymes (grade 3), most likely related to the progression of liver metastases. The other grade 3 toxicities resolved, and no grade 4 toxicities were observed. Dose modifications due to grade 3 toxicities were performed in 3 (37.5 %) patients. Pharmacokinetics, measured in the first cycle of treatment, showed that the doses of the different drugs resulted in drug exposures in the published effective (VPA, GCV, dFdC and intracellular dFdCTP) and safe (dFdU) ranges for all drugs and relevant metabolites. The best observed clinical response was partial in 2 patients (25 %) and stable disease in 3 patients (37.5 %). Three patients (37.5 %) had progressive disease despite the treatment. After treatment cessation, all patients developed disease progression.

To assess the clinical efficacy of the treatment, the number of patients in this study was too low. In the Netherlands, approximately 50–60 new NPC patients are diagnosed yearly. A fraction of these will have distant metastasis at diagnosis, and some will develop recurrent disease. Despite multiple presentations at different head and neck clinics in the Netherlands to refer these patients to our hospital, it was not possible to include more patients. One of the referred patients refused to participate for personal reasons; furthermore, no patients were excluded for study participation.

From the included patients, most were heavily pre-treated. They had rapid recurrent disease or even progressed during the previous treatments, which might reflect the aggressiveness of the tumours and resistance to chemotherapeutics. Three of the eight patients were previously treated with GCb. All three had initially partial response on GCb. One had disease progression 6 months after ending the GCb, one after 11 months, and one patient progressed during the GCb courses, suggesting drug resistance. The latter two patients showed no response on the CLVA treatment. If there is disease progression during treatment with GCb, it is assumable that the CLVA treatment will be unsuccessful as well. The intended reactivation of EBV will be ineffective due to the insensitivity of the tumour cells for GCb. In a future study, it would be advisable to exclude patients who had progression on GCb treatment.

To estimate the biological response of CLVA treatment, we analysed the EBV-DNA load levels in repeated NP brushings and whole blood during treatment. The EBV-DNA load in blood showed a repetitive peaking pattern during each cycle indicating release of apoptotic DNA. Patients with clinical response showed a gradual decrease in brush EBV-DNA load over the time. Such a pattern may reflect induction of the lytic phase by GCb and VPA, and the subsequent clearance of EBV by GCV with concomitant local release of EBV DNA was measurable in the brushed mucosa. A prior study confirmed that repeated NP brushing is well tolerated by patients and that pre- and post-treatment level of EBV DNA in NP brush is a good indicator for local treatment effect (Adham et al. 2013). None of the patients with local disease in this study had a complete local response; therefore, brush dynamics could not be related to clinical response. Interestingly, patient 1 had an increase in the brush viral load at the end of therapy. At that time, no clinical suspicion for local disease was seen, but 3 months later a suspect lesion in the nasopharynx appeared. This suggests that a raise in brush viral load precedes recurrence and warrants close investigation for early detection.

The peaking of EBV-DNA levels in blood is assumed to reflect the release and subsequent rapid clearance of apoptotic fragments in circulation as a response to therapy (Stevens et al. 2005b, 2006a; Chan and Lo 2002). A dynamic alteration was observed in EBV-DNA levels in blood during every cycle, which might reflect response to treatment, or changes in EBV clearance. Advanced NPC stages or larger tumour volumes have been shown to correlate with increased EBV-DNA load in plasma of the majority of NPC cases, although this has not been confirmed consistently when using whole blood as specimen (Adham et al. 2013; Sun et al. 2014; Leung et al. 2006). Technical variation in PCR methodology and data interpretation may underlie this difference (Le et al. 2013). In stem cell transplant recipients receiving rituximab treatment for EBV-driven post-transplant lymphoproliferative disease (PTLD), the treatment efficacy is reflected in rapid and complete EBV-DNA clearance from circulation within days (van Esser et al. 2002; Greijer et al. 2012). In NPC, where spontaneous EBV-DNA release by the tumour may be highly variable and chemo-radiation treatment is more gradually affecting tumour viability and is variable between patients, a slower release of apoptotic DNA is seen, reflecting the balance between production and clearance. The peaks of EBV-DNA levels noticed in our patients are therefore considered to directly derive from tumour cells driven into apoptosis by CLVA therapy. (Wang et al. 2010) proposed the use of plasma EBV-DNA clearance rate (t(1/2)) as an indicator of tumour response and survival (Wang et al. 2010). In 34 patients with metastatic/recurrent NPC, the EBV plasma levels were measured during treatment. A slow clearance rate was associated with poor outcome, suggesting that for these patients a change in chemotherapy regimen should be considered. This is confirmed in recent independent studies (Adham et al. 2013; Leung et al. 2014) supporting the initial finding of van Esser et al. (2002) in EBV-associated PTLD, where the fast clearance of plasma EBV-DNA load in the first 72 h of therapy indicated beneficial clinical outcome (van Esser et al. 2002).

Apart from monitoring of EBV-DNA load dynamics, NP brush samples were used to prove EBV reactivation by a novel quantitative RT-PCR assay. This RNA profiling was earlier performed in CLVA-treated NPC cell lines (Wildeman et al. 2012). Upon CLVA induction, RNA transcripts specific for lytic phase were detectable after second cycle of treatment in the patient with the highest viral DNA load measured. Lack of local tumour mass in the nasopharynx and poor general RNA quality of NP brush samples may have limited validation of this process in other patients. Interestingly, in our feasibility study we have also detected presence of early lytic transcripts as well as higher EBV-DNA copies in NP brush of a CLVA-treated patient indicating occurrence of virus reactivation upon second/third cycle of CLVA (Wildeman et al. 2012). As expected, due to the antiviral therapy after initial lytic induction regime, no late lytic viral transcripts were detected (no virus spreading).

NPC patients have aberrant IgG and IgA antibodies to a range of latent and lytic EBV antigens, indicative of a broadly triggered anti-EBV immune response (Fachiroh et al. 2006; Fachiroh et al. 2008). By using ELISA and immunoblot analyses, we observed that all patients in this study had already strongly aberrant IgG responses with high diversity to multiple EBV antigens, reflecting a long-term exposure and memory to EBV lytic antigens and EBNA1. During and post-CLVA treatment, no significant increase in specific B cell response to viral neo-antigens was observed, indicating that no new drug-induced immune response against viral neo-antigens was formed.

Although only measurable in 3 patients, the focussed analysis of T cell responses using pMHC multimer technology did also not show a new or stronger immune response against EBV. CLVA-mediated activation of EBV-specific T cells could be a part of reactivation of existing, but locally suppressed immune responses (Li et al. 2007b), which may be modulated by T cell response, triggered through apoptotic tumour release and selective regulatory T cells elimination mediated by GCb treatment (Zheng et al. 2015). The study population might have been too small to proof the expected phenomenon, since only one of the patients indeed had a clinical response. On the other hand, the immunomodulatory effects may predominantly operate at the tumour level and not be measurable in circulating T cells by tetramer technology (Li et al. 2007b). Nevertheless, it seemed that there was an existing immune response against EBV, which might have been suppressed by the microenvironment of the tumour cells and therefore ineffective.

To date, the suppression of the tumour infiltrating lymphocytes in the tumour’s microenvironment has the attention of many researchers. Effective modulation of these immune suppressors might enhance the anti-tumour effect by the immune system itself. This can be a potential addition to the treatment used in the current study. Another alternative therapeutic approach is the administration of autologous EBV-specific cytotoxic T cells (Hsu et al. 2010). The strategy of transferring (autologous) EBV-specific cytotoxic T lymphocytes (EBV-CTL) into NPC patients was beneficial in several phase I–II trials (Louis et al. 2010; Chia et al. 2014; Lutzky et al. 2014). However, this strategy is difficult to realize in developing countries, where most NPC burden is present. An overview of this and alternative EBV-targeted therapies in NPC was recently published (Hutajulu et al. 2014).

In conclusion, treatment for recurrent and/or metastatic NPC remains a challenge, in particular when previous (multiple) lines of chemotherapy have failed. Many studies are performed to identify novel targets for therapy for NPC, including EBV-targeting therapies with so far no clinical breakthrough yet (Hutajulu et al. 2014). The survival benefits have to outweigh the side effects, but these depend on many factors, like the number of previous treatments, the tumour extension and the patient’s performance status. This also explains the wide range in response and survival rates in all studies, making it difficult to interpret the results and compare them with our results, and proof the benefit of CLVA therapy above the mechanism of solely GCb. The EBV-targeting therapy by GCb, VPA and GCV combination proved to be safe in these 8 patients with recurrent and/or metastatic NPC. Further clinical studies are required to demonstrate whether CLVA therapy is a safe treatment regimen. Preliminary signs of clinical efficacy have been obtained. In some patients, evidence for the proof of concept was demonstrated, although the biomarker response was difficult to interpret. Alternative HDAC inhibitors and EBV reactivating drug combinations together with GCV, as well as dose optimization of the CLVA approach, should be evaluated in further studies. An interesting additional aspect could be the enhancement of the immune response against EBV by suppressing the immunosuppressive microenvironment of the tumour.

Acknowledgments

This study was supported by Grant ZonMW 95110069 from the Netherlands Government and Grants KWF VU2011-4555, KWF-VU2010-4809 from the Netherlands Cancer Society.

Conflict of interest

The authors declare that they have no conflict of interest.

Footnotes

Sharon D. Stoker and Zlata Novalić have contributed equally.

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