Cytolytic Virus Activation Therapy for Epstein-Barr Virus Driven … · primer QP3 and 4, and...

Cancer Therapy: Clinical

Cytolytic Virus Activation Therapy for Epstein-BarrVirus–Driven Tumors

Maarten A. Wildeman1, Zlata Novali�c3, Sandra A.W.M. Verkuijlen3, Hedy Juwana3, Alwin D.R. Huitema4,I. Bing Tan1, Jaap M. Middeldorp3, Jan Paul de Boer2, and Astrid E. Greijer3

AbstractPurpose: Nasopharyngeal carcinoma (NPC) is causally linked to Epstein–Barr virus (EBV) infection.

Because all tumor cells carry EBV, the virus itself is a potential target for therapy. In these tumor cells, EBV

hides in a latent state and expresses only a few non-immunogenic proteins for EBV maintenance and

contributes to tumor growth. We developed a cytolytic virus activation (CLVA) therapy for NPC treatment,

reactivating latent EBV, triggering immune recognition, and inducing susceptibility to antiviral therapy.

Experimental Design: CLVA therapy combines gemcitabine (GCb) and valproic acid (VPA) for virus

activation and tumor clearance with (val)ganciclovir (GCV) as the antiviral drug to block virus replication

and kill proliferating virus-infected cells. CLVA treatment was optimized and validated inNPC cell lines and

subsequently tested in 3 Dutch patients with NPC that was refractory to conventional treatment.

Results: In NPC cell lines, both GCb and VPA can induce the lytic cycle of EBV. Their combination

resulted in a strong synergistic effect. The addition of GCV resulted in higher cytotoxicity compared with

chemotherapy alone,whichwasnot observed inEBV-negative cells. CLVA therapywas analyzed in3patients

with end-stage NPC. Patients developed increased levels of viral DNA in the circulation originating from

apoptotic tumor cells, had disease stabilization, and experienced improved quality of life.

Conclusions: Our results in the initial CLVA-treated patients indicate that the therapy had a biological

effect and was well tolerated with only moderate transient toxicity. This new virus-specific therapy could

open a generic approach for treatment of multiple EBV-associated malignancies. Clin Cancer Res; 18(18);

5061–70. �2012 AACR.

IntroductionEpstein–Barr virus (EBV) is causally linked to multiple

cancers, including several lymphomas, undifferentiatednasopharyngeal carcinoma (NPC), and approximately10% of gastric cancers worldwide. Although NPC is anuncommon disease in most countries, NPC is the 4th mostcommon tumor among males in Indonesia and has a highincidence in southern China, northern Africa, and Alaska(1). Early diagnosis is essential because disease-free survivaldeclines for patientswith late-stageNPC(2).Unfortunately,early symptoms for NPC, such as epistaxis and tinnitus,are nonspecific and the majority of patients come to the

hospital with advanced stage disease. In Indonesia, 87% ofpatients have NPC stage III or higher and 17% have distantmetastasis at first presentation (3).

Because all NPC tumor cells harbor EBV, this virus itself isa possible target for therapy. In these tumor cells, EBV"hides" in a latent state due to methylation of the viralpromoters, expressing only a few non-immunogenic viralproteins that are essential for EBV maintenance and con-tribute to tumor growth (4). This state of latency enablesNPC tumors cells to evade the immune system, even in thepresence of an existing strong immune response tomultipleviral antigens (5, 6). Recent studies have shown that the lyticcycle of EBV can be efficiently induced by using chemo-therapeutic agents effectingDNA synthesis and drugs affect-ing host DNA methylation and histone deacetylation(HDAC; refs. 7–11). The reactivation of EBV leads to theexpression of proteins involved in viral genome replicationand formation of new virions. These newly expressed pro-teins are highly immunogenic and could induce a powerfulimmune response toward the tumor cells containing thereactivated virus (5). In addition, lytic induction enablesexpression of viral kinases sensitizing the cells to antiviraltreatment (9).

Inducing the lytic phase with chemotherapy in combi-nation with a HDAC inhibitor caused reduced tumor vol-ume in EBV-positive tumor cells in a mouse lymphoma

Authors' Affiliations: Departments of 1Head and Neck Oncology andSurgery and 2Medical Oncology, The Netherlands Cancer Institute-Antonivan Leeuwenhoek Hospital; 3Department of Pathology, VU UniversityMedical Center; and 4Department of Pharmacy and Pharmacology, TheNetherlands Cancer Institute/Slotervaart Hospital, Amsterdam, TheNetherlands

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Astrid Greijer, VU University Medical Center, DeBoelelaan 1117, 1081 HV Amsterdam, The Netherlands. Phone: þ31-20-4444052; Fax: þ32-20-4442964; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-12-0574

�2012 American Association for Cancer Research.

ClinicalCancer

Research

www.aacrjournals.org 5061

on March 11, 2021. © 2012 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst July 3, 2012; DOI: 10.1158/1078-0432.CCR-12-0574

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model (8). The addition of an antiviral component to thiscombination therapy did further decrease tumor develop-ment. This concept was first used in 1998 for the treatmentof a patient with EBV-positive lymphoma in which a com-bination of an HDAC inhibitor with antiviral therapy wasadministered (12). This was followed by a study usingvalproic acid (VPA) instead of arginine butyrate (13). Thecombination of chemotherapy (5-FU) with an HDACinhibitor (HDACi) for a stronger induction of the lytic cyclewas evaluated in a Dutch patient with end-stage NPC. After5 days, the patient received antiviral treatment with valgan-ciclovir (GCV). This treatment resulted in an increase ofviral DNA in the circulation, reflecting shedding of apopto-tic fragments from the tumor, which was not observedbefore treatment (14).

In this study, a novel combination therapywas developedtargeting EBV within the tumor cell by combining EBVreactivation with subsequent antiviral therapy. After vali-dation in a single naturally EBV-infected NPC cell line andEBV-positive gastric cancer cell lines, this cytolytic virusactivation (CLVA) therapy, comprising gemcitabine (GCb),VPA, and GCV, was applied as a new treatment modality inpatients with EBV-positive NPC for which no curable treat-ment options were available. In addition to clinical para-meters, biological response was analyzed by EBV viral loadand serology.

Materials and MethodsCell lines

The EBV-positive NPC cell line C666.1 was cultured inDulbecco’s Modified Eagle’s Medium (DMEM; BioWittha-ker) containing 10% fetal calf serum (FCS) and 1% peni-

cillin/streptomycin/glutamine (P/S/G). Cell culture plateswere first coated with fibronectin (Calbiochem) inPBS(100mg/mL) for1hourat roomtemperature (rT).Gastriccarcinoma cell lines of AGS and the EBV-positive cell lineAGS-BX1were cultured in F12HAM(BioWitthaker) contain-ing 10% FCS and 1% P/S/G. AGS-BX1 cells were culturedunder G418 (geneticin; Life Science Technologies) selection.The AGS-BX1 harbors the EBV genome with an insertion ofthe neomycin-resistance gene and theGFP gene that disruptsthe TK gene (kindly provided by L. Hutt-Fletcher).

Western blot analysisProtein samples were obtained by lysing cells in a RIPA

buffer in the presence of a protease inhibitor cocktail(Roche) for 30 minutes at 4�C. Total protein concentrationwas determined with the BCA protein assay kit (ThermoScientific). Proteins were mixed with 4� sample buffer andboiled for 5 minutes at 95�C. Protein samples were run onSDS-PAGE and transferred to nitrocellulose sheets by blot-ting. Immunoblots were blocked with 5% dried milk pow-der in PBST (0.05% Tween20 in PBS) and incubated withthe primary antibodies BZ-1 for detecting ZEBRA (a giftfrom P. Farrell) and actin-HRP (C4; Santa Cruz). Afterincubation with horseradish peroxidase (HRP)-conjugatedsecondary antibody (DAKO) the proteins were visualizedby the ECL detection kit (GE Healthcare) according toinstructions provided in the manual.

ImmunofluorescenceC666.1 cells were cultured on 10-mm coverslips in 24-

well plates. Cells were induced with 3 mmol/L GCb and 0.3mmol/L VPA for 2 days. Cells were fixed by incubation withmethanol-acetone for 10 minutes. Subsequently, nonspe-cific binding was blocked by incubation in PBS containing10% FCS. Glass slides were incubated with BZ1 antibodyand, after washing with PBS containing 0.05% Tween-20,incubated with the secondary antibody conjugated withfluoroscein isothicyanate (FITC). Slides were mounted inVectashield (Vector Laboratories Inc.) containing 0.3 mmol/L 40,6-diamidino-2-phenylindole (DAPI). Immunofluores-cence was analyzed by using a Leica microscope.

Cytotoxicity analysisFor CLVA treatment cytotoxicity screens, 25,000 C666-1

cells were seeded in a 96-well plate in 100 mL medium andwere allowed to adhere for 24 hours at 37�C in 5%CO2. Thenext day, serial dilutions of GCb in the presence of 0.3mmol/LVPAwere added to cells in triplicate for 6days. Afterdrug incubation, 5 mL of MTT (5mg/mL; Roche) was addedto each well and the mixture was incubated for 3 hours.Thereafter, 100 mL of isopropanol–HCl solubilization buff-er was added and the plates were incubated at 37�C over-night. TheOD595 was determined using amicroplate reader(Molecular Devices).

PatientsThree patients with histologically confirmed residual,

recurrent, and metastatic EBV-positive NPC were included

Translational RelevanceUndifferentiated nasopharyngeal carcinoma (NPC) is

causally associatedwithEpstein–Barr virus (EBV). As thisvirus is present in all tumor cells, EBV could serve as atarget for therapy. In NPC cells, EBV hides in latency,expressing only a few essential proteins contributing totumor formation although escaping immune elimina-tion. Inducing the viral lytic phase makes tumor cellssusceptible for immune recognition and antiviral ther-apy. The concept was confirmed in NPC cell lines usinggemcitabine combined with a host DNA methylationand histone deacetylation (HDAC) inhibitor as lyticinducer. Subsequent addition of antiviral therapy (gan-ciclovir) increased specific cytolysis. The cytolytic virusactivation (CLVA) therapy was evaluated in 3 patientswith progressive end-stage NPC. All patients had stabledisease during and for more than 6 months aftertherapy with improved quality of life. CLVA therapyresulted in increased shedding of viral DNA in thecirculation. These data indicate that CLVA therapy mayopen up a new therapeutic approach for EBV-drivenmalignancies.

Wildeman et al.

Clin Cancer Res; 18(18) September 15, 2012 Clinical Cancer Research5062




in this study after they had failed conventional curativetreatment options and were deemed incurable. All patientssigned an informed consent for this experimental pilotstudy. Before treatment, patients had a strong and broadlyreactive humoral immune response against EBV antigens,measured by ELISA and immunoblot as described below.

CLVA therapyOne treatment cycle extends over 42 days. Patients

received GCb (1250 mg/m2; Fresenius Kabi Oncology Plc)on days 1 and 8 intravenously. Valproic acid (genericmedicine) was administered orally (12.5 mg/kg per day)during the first 14 days of the treatment cycle. From days 9to 22, patients were treated with GCV (Roche) daily (3 �450 mg/day orally). This treatment cycle of 42 days wasrepeated 6 times.

Clinical monitoring of patientsAll patients had a baseline magnetic resonance imaging

(MRI) scan of the head-and-neck region, an ultrasound ofthe neck region, and complete body PET-CT scan. Afterevery 2 treatment cycles, the tumor response was measuredby imaging. In case of response or stable disease, patientsreceived a total of 6 cycles. For the nasopharyngeal and neckregion, MRI was carried out. Distant metastases in thethoracic region were monitored with CT-imaging. Afterfinishing the treatment, a PET-CT scan was conducted.Adverse events were graded on the basis of the NationalCancer Institute Common Toxicity Criteria version 3.0.Blood samples for measuring EBV viral load and humoralantibody response were obtained every week. Brush sam-pling for measuring EBV DNA load was carried out every3 weeks.

Nucleic acid isolationDNA was isolated from nasopharyngeal brushing sam-

ples by silica-based nucleic acid extraction as describedpreviously (15). One milliliter of lysate was used as inputfor the isolation procedure, and the nucleic acids wereeluted in 100 mLwater. Reagents for the isolation procedurewere obtained from BioMerieux.RNA was isolated from cell lines by addition of 250 mL

TRIzol to 300,000 cells according to the manufacturer’sinstructions (Invitrogen).

EBV viral load by real-time quantitative PCRThe EBV DNA load in whole blood was determined by a

quantitative LightCycler480 amplifying a 99-bp part ofEBNA1. The probemastermix used was supplemented withprimer QP3 and 4, and hybridization probes were used forquantification as described previously (16). Real-time PCR(RT-PCR) reagents were obtained from Roche Diagnostics.Then, 10-fold serial dilutions of spectrophotometricallyquantified plasmid DNA containing the EBNA1 targetsequence were used to create a standard curve. To checkfor putative inhibition of PCR, EBV DNA-negative sampleswere spiked with 1,000 copies of EBV plasmid DNA. b-Glo-bin PCR was carried out with the primers PCO3 (50-ACA-

CAACTGTGTTCACTAGC-30) and PCO5 (50-GAAACCCAA-GAGTCTTCTCT-30), which generate a 209-bp PCR product.Reaction conditions were as described previously (17).Quantification of the amplification products was carriedout with the second derivative software of the LC480(Roche).

cDNA synthesis and quantitative RT-PCRRNA was treated with 1 mL RQ RNase-free DNase (Qia-

gen), 1 mL RQ1RNase-free DNase 10� Reaction Buffer, andprecipitated with 1 mL 3mol/L sodium acetate, 0.5 mL linearacrylamide (Ambion; 5 mg/mL), and 25 mL of 100% eth-anol at �80�C. Precipitated RNA was reverse transcribedby gene-specific cDNA synthesis using a multiprimedapproach for 10 minutes at 65�C. Subsequently, 2 mL RTbuffer, 10mLdNTPs (2mMmol/L), 2mLDTT (100mmol/L),0.5 mL H2O, 0.25 mL RNAsin, and 0.25 mL AMV reversetranscriptase was added to each sample and incubated for 1hour at 42�C. EBV gene expression was quantified by quan-titative LightCycler PCR using an LC480 system (Roche).Quantitative PCR was carried out using a LightCycler 480SYBR Green I Master kit (Roche) in a total reaction mixtureof 10 mL containing 2.5 mL of 10-times-diluted cDNA, 5 mL2� LightCycler 480 SYBR Green Master Mix, 0.5 mL(10 pmol/mL) of the gene specific primers, and 1.5 mLH2O. Primers used in the quantitative LightCycler PCR arelisted in supplementary Table S1. Absolute quantificationwas determined using a standard curve of the plasmid poolcontaining all targets for quantifying the exact amount ofRNA molecules for each target.

SerologyHumoral antibody responsesweremeasured in the serum

of the patients with NPC. Immunoglobulin A (IgA) reac-tivity was assessed by a synthetic peptide-based ELISA usingimmunodominant epitopes derived from EBNA1 andVCA-p18 as described previously (18). Immunoblot anal-ysis was carried out on blot strips containing HH514.c16nuclear antigen induced by TPA and sodium butyrate toproduce the late lytic phase of EBV. Stripswere prepared andanalyzed as described previously (19, 20).

ResultsLytic inductionbyGCb incombinationwithVPA in vitro

To validate the concept of CLVA therapy, the combina-tion of a chemotherapeutic agent and a histone deactylationinhibitor was tested on human EBV-positive NPC (C666.1)and gastric carcinoma (AGS-BX1) cell lines. C666.1 cellscarry a tightly latent EBV, whereas AGS-BX1 cells are lesslatent, showing spontaneous lytic EBV reactivation inapproximately 5% of cultured cells. For inducing the EBV,replicative phase cells were treated with 3 mmol/L GCb and0.3 mmol/L VPA, either separately or simultaneously.Expression of lytic RNA and proteins was analyzed byquantitative RT-PCR and Western blot (Fig. 1). The ZEBRAprotein, acting as the EBV lytic switch, is upregulated mar-ginally by the HDAC inhibitor VPA alone in C666.1 cells.The addition of GCb to VPA resulted in a stronger lytic

New Therapy for Epstein-Barr Virus–Driven Tumors

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induction, as indicated by the increased level of ZEBRAprotein (30-fold) inC666.1 cells and a 3-fold increase in theAGS-BX1 lines, which already showed detectable ZEBRAexpression at baseline.

The combination GCb and VPA resulted in a steepincrease in ZEBRARNA levels in both theC666.1 (150-fold)and AGS-BX1 cell line (10-fold; Fig. 1C) and was dose-dependent for each drug (Fig. 2).Other EBV lytic cycle genesbesides the ZEBRA switch protein, such as protein kinase(PK), thymidine kinase (TK), and the structurally smallcapsid protein VCA-p18, were also induced by GCb andVPA in a dose-dependentmanner inC666.1 cells (Fig. 2). Toanalyze the percentage of cells entering the lytic phase, theZEBRA protein was stained in C666.1 cells induced for 2days by GCb and VPA. More than 80% of the cells showedZEBRA expression in a typical staining of nuclear dots(Fig. 3). The intensity of the staining varied among the cells;however, in more than 10% of cells, a strong immunoflu-orescence was observed.

Cytotoxicity of lytic induction increased by antiviraltreatment

Inductionof the EBV lytic cycle can create susceptibility toantiviral treatment and consequently increases antiviraldrug-induced cytotoxicity. In the early phase of EBV reac-tivation, the enzymes TK and PK are expressed, whichconvert the antiviral drug, ganciclovir, into its cytotoxicform (9, 21). The C666.1 cell line was used to induce thelytic phase of EBV by adding 0.3 mmol/L VPA and a serial

dilution of GCb (Fig. 4A). After 3 days, the cells werereseeded and analyzed for cytotoxicity in the absence andpresence of 20mmol/LGCV.Cytotoxicity (MTT) assayswereconducted 6 days after initial culture and the increase incytotoxicity caused by the addition of GCV to GCb wasdetermined. The IC50 values for GCb shifted under theaddition of GCV from 6.1 to 3.1 mmol/L. In AGS-BX1, theaddition of GCV lowered the IC50 values of GCb from4.3 to2.0 mmol/L (data not shown). The increased cytotoxicityprovided byGCVwasnot very high, butGCV showed a cleareffect on inhibition of EBV replication. We observed a totalblock in production of RNA encoding the viral capsidprotein VCA-p18 above the 7.5 mmol/L GCV level (Fig.4B). The absence of VCA-p18 reflects the inability ofinduced EBV to generate new virions, thus blocking virusspread.

CLVA therapy in patients with end-stage NPCPatient 1. The first patient treatedwith CLVA therapywas

a 48-year-old female (WHOperformance status 1). She hada local recurrence with extension into the retropharyngealrecess surrounding the internal carotid artery. Twenty-twomonths before commencement of the CLVA treatment, shereceived chemoradiation for T4N1M0NPC. Fifteenmonthsafter initial treatment, she received photodynamic therapyfor local recurrence in the nasopharynx, but only had apartial response. This patient received a total of 6 cycles ofCLVA therapy. During the first 2 cycles patient received 1.25mg/kg VPA instead of 12.5 mg/kg due to logistic failure.

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Figure 1. Lytic induction in cellslines. EBV-carrying NPC (C666.1)and gastric carcinoma (AGS-BX1)cell lines were cultured in thepresence of gemcitabine(GCb), valproic acid (VPA), and acombination of the 2 drugs.Lytic induction was indicatedby: A, increase of ZEBRAprotein on Western blot; B,quantification ZEBRA protein;and C, quantification of ZEBRAmRNA levels by RT-PCR.

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Tumor response. The patient showed a significantdecrease of tumor in the nasopharynx after 2 courses (Fig.5A and 5B). During the remaining treatment and for up to11 months of the follow-up period, the patient had stabledisease. An MRI scan after treatment showed disease pro-gression of the tumor in the nasopharynx (Fig 5B).Adverse events. This patient developed a grade 4 toxicity

with platelet count decrease after course 3, but recoveredcompletely before the start of course 4. After the first GCbcourse, the patient again developed a grade 3 toxicityplatelet count; therefore, she received 50% of the GCb dosefor the remaining courses. Due to grade 3 toxicity of hemo-globin levels, the patient received blood transfusions.Patient 2. A 52-year-old male (WHO performance

status 0) patient was referred to our hospital with diseaseprogression after palliative radiotherapy for a T2aN3M1NPC diagnosed 10 months previously. The disease pro-gression consisted of cytologically proven positive lymphnodes high in the mediastinal region and a costal metas-tasis. In addition, 2 regional lymph nodes in the leftneck were assessed and found to have persistent tumorafter radiotherapy. This patient received a total of 6cycles, with no dose reductions. Blood sampling for EBV

viral load and antibody response were collected every 2weeks.

Tumor response. After 2 courses of CLVA therapy, patient2was observed tohave respondedwith a reductionof oneofthe tumor positive lymph nodes in the neck to normal sizeon CT-imaging. After 6 cycles, imaging showed the absenceof PET activity. The remaining neck-node tumor remainedstable after 6 courses of treatment. As there was an increasein quality of life and improvement of well-being, patient 2received another course of adjuvant radiotherapy on theresidual positive lymph node (36 Gray) in the left neckregion and has been observed to have a persistent stabledisease for 6 months in the follow-up period.

Adverse events. Patient 2didnot encounter any grade3or4 adverse events.

Patient 3. The 3rd patient was a 48-year-old female(WHO performance status 1) with progressive disease inthe nasopharynx, which was histologically proven as recur-rent disease, and a cytologically proven lymph node in levelV of the neck region. IN addition, PET-CT showed supra-clavicular, intrathoracic, and left thyroid lobe metastases.Nine years previously, she underwent a modified radicalneck dissection for a tumor in the neck with an unknown

Figure 2. Dose-dependentinduction of EBV lytic cycle by GCband VPA in C666.1 cells. Doseescalation of GCb was analyzed forlytic induction. A, RNA levels of lyticgenes (ZEBRA, early genes TK, PK,and late gene VCA-p18 gene) weredetermined and normalized to acellular housekeeping gene. B,Western blot analysis of the level ofthe proteins expressed from theimmediate early gene ZEBRA wasassessed with the loading controlb-actin. Dose escalation of GCb wasanalyzed for lytic induction afteraddition of 0.3 mmol/L VPA. C, RNAlevels of lytic genes were determinedand normalized to a cellularhousekeeping gene. D, analysis andquantification of the ZEBRA proteinlevels wasmeasured onWestern blotassay. Dose escalation of VPA inpresence of 3 mmol/L GCb wasstudied. E, RNA levels of lytic geneswere determined and normalized to acellular housekeeping gene. F,analysis and quantification of theZEBRA protein levels was measuredon Western blot assay.

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primary site. This patient had already received palliativetreatment with capecitabine and radiotherapy before initi-ation of the present treatment with CLVA. Patient received atotal of 5 treatment cycles, with 25%dose reductions duringcourse 5. The patient refused to undergo cycle 6.

Tumor response. This patient had a significant decrease oftumormass in the nasopharyngeal region (Fig. 5C and 5D).The lymphnodes and distantmetastasis remained stable for6 months during the foloow-up.

Adverse events. At the start of therapy, patient 3 had agrade 3 anorexia for which she received tube feeding duringall courses of therapy and during follow-up. Patient 3developed aspiration pneumonia grade 3 after cycle 4.During the 5th cycle, this patient was admitted to thehospital twice with two grade 3 infections. During the 5thcourse, she also suffered a grade 3 hepatotoxicity (increasedSGPT).

EBV DNA load in the tumor cells and whole blood ofCLVA-treated patients with NPC

The presence of high levels of EBV DNA in minimallyinvasive brushings, taken from the nasopharyngeal muco-sa has been described to correlate with the presence ofviable NPC tumor cells (22). In contrast, EBV viral DNAdetected in the circulation of NPC patients may originatefrom apoptotic tumor cells rather than viable circulatingtumor cells (29). The 3 patients treated with CLVA ther-apy were monitored for EBV viral load in nasopharyngealbrushing samples and in whole blood, drawn before,during, and after CLVA treatment (Fig. 6). Patients 1 and2 did not have a visible tumor in the nasopharyngeal

mucosa and, therefore, brushing samples were taken atlonger intervals. The 3rd patient did have a locally visibletumor in the nasopharyngeal region and brushing sam-ples were obtained every 2 weeks. The viral load measuredin patient 3 before treatment was 4.3 � 106 EBV copies/brushing sample. After an initial 200-fold decrease in viralload during the second cycle of treatment down to 2.4 �104 EBV copies/brushing sample, the viral load showed arise again mainly during a prolonged interval in thetreatment schedule. Brushing samples of the otherCLVA-treated patients showed an occasionally positiveresult up to 104 copies/brushing sample.

The viral DNA levels in blood were monitored to analyzethe biological response to therapy. Before treatment, allpatients had EBV copies below the clinical cutoff level, thatis, 1.6 � 103 copies/mL of blood. At the start of therapy,patient 3 had slightly higher levels (1.1� 104 copies/mL ofblood). Viral load in blood during CLVA treatment showeda highly dynamic response, probably reflecting tumor

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Figure 3. Percentage of lytic-induced C666.1 cells by GCb VPA wasvisualized by ZEBRA protein staining. A, the percentage of ZEBRA-positive cells in untreated condition and after incubation for 48 hours with3 mmol/L GCb and 0.3mmol/L VPA. B, typical example of the punctuatedZEBRA staining in the lytically induced C666.1 cells (left), with the DAPInuclear control (right). Although ZEBRA expression is (weakly) induced inmost cells, brighter nuclear ZEBRA staining is observed in approximately11% of the cells in a typical experiment.

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Figure 4. Increased cytotoxicity and prevention of RNA expression of thestructural gene VCA-p18 by ganciclovir in lytically induced C666.1cellline. A, the additional toxic effect of GCV to GCb in the C666.1 cell line.IC50 for the C666.1 cells incubated with serial dilution of GCb in thepresence of 0.3 mmol/L VPA was 6.1 mmol/L GCb and, after addition of20mmol/LGCV, the IC50 valuedecreased to3.1mmol/LGCb; the solid lineshows group without GCV and the dotted line represents values inpresence of GCV. B, the expression of the structural gene VCA-p18 isdiminished by concentrations greater than 7.5 mmol/L GCV, indicatinginhibition of viral replication. The lytic induction is not hampered asreflected by the stable ZEBRA gene expression.

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apoptosis. The highest fluctuationswere observed in patient1, in whom it varied from below detection level up to 1.4�106 EBV copies/mL of blood. In patients 2 and 3, lower EBVDNA levels weremeasured in the blood. A common patternin the blood that was observed for all 3 patients was theappearance of viral DNA in the periods following treatment(Fig. 6).

Humoral immune responses of CLVA-treated patientswith NPCThe IgG response against immunodominant EBV epi-

topes was analyzed. The data confirmed that the patientshad a molecularly diverse anti-EBV immune responsebefore treatment, including reactivations against lytic viralantigens. A potent immune response is needed to allowidentification of therapy-induced new immunogenic viralproteins. Two patients showed a strong immune responseagainst a wide diversity of viral proteins (Fig. 5E). The first

patient had a weaker response, mainly directed againstZEBRA and VCA-p18. The level of the immune responseremained the same after treatment and the pattern ofproteins recognized did not change.

Further, the level of the humoral immune response wasdetermined by standardized peptide-based ELISA beforeand during therapy. High constant levels of VCA-p18 reac-tivity were observed, whereas the EBNA1 responsesremained at a constant low level (data not shown).

DiscussionA new therapeutic approach was explored on the basis of

reactivation of the latent viral genome in combination withantiviral therapy to target EBV within NPC tumor cells. ThisCLVA therapy proved to be effective in vitro and, subse-quently, CLVA therapy was administered to 3 patients withend-stage NPC disease. CLVA treatment in patients withend-stage NPC was well tolerated and showed a clinicalresponse and improvement in quality of life during andafter therapy in all patients. Side effects of CLVA therapywere transient and only moderate. These first in vivo resultsindicate that the concept of epigenetic induction of viralantigens with subsequent administration of antiviral ther-apymay be a potent approach in patients with EBV-positiveNPC, which can possibly be extended to other EBV-drivenmalignancies.

Gemcitabine is one of the most effective single-agenttherapies in the treatment of NPC (23), and was recentlystudied in 31 patients where initial platinum-based chemo-therapy failed and subsequent GCb therapy resulted inpartial response in 43.8% of the patients and stable diseasewith minimal side effects in 28.1% of patients (24). Besidestumor cell apoptosis induced by GCb, it could reactivateEBV within the tumor cells. The potential for EBV lyticinduction byGCb in EBV-positiveNPC cell lines was shownto be higher than by 5-FU, which was used in a previousjuvenile NPC case study (14, 25, 26). Activation of highlymethylated DNA of latent EBV can be enhanced by epige-netic chromatin remodeling of the EBV genome induced byHDAC inhibitors and DNA-demethylating agents (7–11).In the naturally EBV-carrying infected NPC cell C666.1, thecombination of histone deactylase inhibitor VPA with GCbshowed a clear synergistic action on the expression of EBVlytic genes, ZEBRA, TK, PK, and VCA-p18, which was con-firmed in the gastric carcinoma cell AGS-BX1 harboring arecombinant EBV (Figs. 1–3). Lytic induction sensitized theEBV-positive cell lines to the antiviral drug gancicolovir(GCV), which resulted in an increase of cytotoxicity com-pared to the toxicity induced by GCb and VPA alone (Fig4A). Further, EBV lytic gene expression is known to result inan increase of apoptosis in combination with GCV (27).This additional toxic effect was observed in an EBV-positivelymphoma animal model (25). In addition, enhancedtoxicity by GCV was observed in a recent study by Sidesand colleagues in a study wherein they provoked lyticinduction with low-dose arsenic (28). Importantly, GCVresulted in reduced production of RNA encoding the lateviral capsid antigen p18 (VCA -p18) in lytically induced

BA

DC

E 1 2

12 12 12

3CLVA-treated patient

EA-p138

EBNA1TK

EA(d)

ZEBRA

VCA-p18

Figure 5. MRI scan of patients before and after CLVA treatment. A, MRIscan, axial slide of nasopharyngeal region of patient 1 before treatmentand B, after treatment. C, MRI scan; axial slide of nasopharyngeal regionof patient 3 before treatment andD, after 4 treatment cycles; imagingwasnot repeated after complete treatment because of claustrophobia notedin the patient. Arrows indicate the relevant area of response. E, humoralimmune response against EBV antigens revealed by IgG immunoblotanalysis [20] of sera from the CLVA patients before (1) and after (2) thecompleted therapy.






C666.1 cells (Fig. 2). This clearly demonstrates the effect ofGCV treatment on inhibiting new viral particle formationafter lytic induction, which is necessary for the safety ofCLVA treatment in patients with NPC.

The choice of drugs already approved for human useresulted in the rapid translation of CLVA-based treatment

into the clinic. Therapy was administered to 3 patientswith end-stage NPC and was well tolerated. We observedsome toxicity, mainly consisting of neutropenia, in onlyone case. However, previous chemotherapy administeredto this patient may be more causal to the reduced bonemarrow capability to recover from GCb. The other 2

VPAGCV

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Figure 6. Effect of CLVA treatmenton the viral load in whole blood(black line) and nasopharyngealbrushings (gray line) of the CLVANPC-treated patients. In the boxesabove the individual graphs, thetherapy schedule is depicted. Thearrows indicate the GCb infusions,1250 mg/m2. However the last 3GCb infusions in patient 1comprised only half of the dose.The VPA was given at aconcentration of 12.5 mg/kg perday, except for the first 4 cycles forpatient 1, wherein 1.25 mg/kg perday was administered. The GCVconcentration was 3� 450 mg/dayand administered at the indicatedtimes. DNA load was determined inblood samples obtained weeklyand in the tumor cells obtained byNP brushing at the indicated timepoints.

Wildeman et al.





patients did not encounter any detectable side effects ofCLVA therapy.All 3 patients showed a biological response to CLVA

therapy indicated by an increase in viral DNA load in theblood, as observed in a previous case study (14). Thisincrease reflects fragmented EBV DNA derived from apo-ptotic tumor cells (29). The dynamics of theDNA load in allpatients showed a similar trend, that is, an immediatedecline of EBV DNA loads during therapy cycles and anincrease in EBV DNA load in the recovering periods. Adecline of EBV load in blood has been described to predicta good clinical response in patients with NPC (30). Theunexpected increase of viral load in the period withouttreatment could reflect the functional recovery of theimmune system and its effect in eliminating lytic inducedcells. We failed to detect an increase in anti-EBV immunereactivity as result of the CLVA therapy, although moredetailed analysis may be needed for this, including analysisof T-cell responses.A residual (recurrent) tumor in the nasopharynx was

only present in the 3rd patient. Viral load in noninvasiveNP brushings taken regularly during and after therapydecreased, but did not disappear completely. Repeatedbiopsies in the nasopharynx during and after treatmentwere considered too invasive; therefore, it is unclear wheth-er these viral loads represent shed tumor cells or viralreactivation in the nasopharynx.Despite the small number of patients in this pilot study,

which may prevent strong conclusions, we followed theclinical outcomes of these patients. All patients initiallyhaving progressive disease developed stable disease duringand after treatment, and, in addition, a clear improvementof their clinical condition was observed.In conclusion, virus-specific CLVA tumor therapy may

provide a new and generic approach for treatment of mul-tiple EBV-associated malignancies in both developed anddeveloping countries worldwide. The 3 patients with end-stage NPC all had a clinical response to CLVA therapy andan improved quality of life. A phase I/II trial was startedrecently (Eudract nr: 2010-022444-20). In this trial, addi-tional PBMCswill be collected to obtainmore insight in the

immunological aspects of the CLVA therapy response of thepatients.

The use of novel combination of existing drugs to activateEBV and eliminate virus-infected cells may open the way tomore simplified, possibly oral therapies that would greatlybenefit patients in developing countries where complexchemoradiation is not possible. Further studies on CLVAare needed for better insight in themolecular basis of tumoror EBV specific immune responses provoked by the lyticinduction and the long-term effect of the treatment. The useof EBV as target in therapy could open up new approachesfor other EBV-driven tumors.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: M. A. Wildeman, I. Bing Tan, J. M. Middeldorp,A. D. R. Huitema, J. P. de Boer, A. E. GreijerDevelopment of methodology: I. Bing Tan, J. M. Middeldorp, A. D. R.Huitema, J. P. de Boer, A. E. GreijerAcquisition of data: M. A. Wildeman, Z. Novali�c, S. A. W. M. Verkuijlen,H. Juwana, I. Bing Tan, J. P. de Boer, A. E. GreijerAnalysis and interpretation of data:M. A. Wildeman, Z. Novali�c, S. A. W.M. Verkuijlen, H. Juwana, A. D. R. Huitema, I. Bing Tan, J. M. Middeldorp,J. P. de Boer, A. E. GreijerWriting, review, and/or revision of the manuscript: M. A. Wildeman,A. D. R. Huitema, I. Bing Tan, J. M. Middeldorp, A. E. Greijer, J. P. de BoerAdministrative, technical, ormaterial support:M. A. Wildeman, S. A. W.M. Verkuijlen, A. D. R. Huitema, M. A. Wildeman, J. P. de Boer, J. M.Middeldorp, A. E. GreijerStudy supervision: M. A. Wildeman, J. M. Middeldorp, J. P. de Boer, A. E.Greijer

AcknowledgmentsThe authors thank the patients who participated in this trial. The authors

also thankMaxNobis and Chantal Kuijpers for excellent technical assistanceand Renske Fles for organizing the logistics of patient care. The donation ofthe AGS-BX1 cell line by Dr. L. Hutt-Fletcher is greatly appreciated.

Grant SupportThis study was financially supported by grants from The Netherlands

Organisation for Health Research and Development (95110069) and DutchCancer Foundation (NKI-2008-4233 and VU2010-4809).

The costs of publication of this article were defrayed in part by the pay-ment of page charges. This article must therefore be hereby marked adver-tisement in accordancewith 18U.S.C. Section 1734 solely to indicate this fact.

Received February 21, 2012; revised June19, 2012; accepted June 20, 2012;published OnlineFirst July 3, 2012.

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2012;18:5061-5070. Published OnlineFirst July 3, 2012.Clin Cancer Res Maarten A. Wildeman, Zlata Novalic, Sandra A.W.M. Verkuijlen, et al. Tumors

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