Study of Cytolytic Viral Activation Therapy (CVAT) for Recurrent/Metastatic Nasopharyngeal Carcinoma
Study of Cytolytic Viral Activation Therapy (CVAT) for Recurrent/Metastatic Nasopharyngeal Carcinoma
Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus(EBV) related malignancy and is an
 endemic disease in Southeast Asian countries. EBV had been identified as a therapeutic target
 in some EBV related cancer such as lymphoma and NPC. In cancer cell, EBV was in latent phase
 and expressed 8-11 genes for maintaining EBV proliferation. After switching to lytic phase,
 almost all the EBV encoding genes were expressed including thymidine kinase (TK) and some
 highly immunogenetic genes. These latent-lytic phase swifter included DNA methyltransferase
 inhibitors, various histone deacetylase (HDAC) inhibitors, radiotherapy and chemotherapy.
 Recently, combined chemotherapy and viral lytic therapy, cytolytic viral activation therapy
 (CVAT) had been shown some promising result in pilot study of NPC. In our patient derived
 xenograft (PDX) animal model drug sensitivity screening, gemcitabine (GEM) was shown to be
 the most effective drug. Furthermore, CVAT with GEM + Valproic acid (VPA) + ganciclovir (GCV)
 maintaining chemotherapy may benefit but reduce chemotherapy related side effect and
 prolonging treatment response duration. The following phase I clinical trial will be proposed
 to test the optimal combination of these drugs.

 1. Number of patients: total 18 patients are needed

 2. Inclusion criteria:(1) used as 2nd line regimen in recurrence/metastasis NPC patients
 with tissue proved of World Health Organization (WHO) type II or type III.(2)
 Performance status: eastern cooperative oncology group performance status (ECOG PS) ≤2.

 3. Chemotherapy regimen: Gemcitabine (GEM, TTY) + Valproic acid (VPA, generic medicine) for
 viral activation + Valganciclovir (VGC, Roche) for antiviral medication

 4. This treatment cycle of 28 days was repeated maximum 6 times. (Q4wks/cycle, max: 6
 cycles)

 5. Dosage:

 (1) GEM: 600, 800, 1000, 1250 mg/m^2, D1 & D8, intravenously. (2) VPA 12.5 mg/kg/day D1~14,
 per os. (3) VGC (2-3) x 450 mg/day D9~15, per os. 6. Objectives:

 1. primary: to find the best combination of these 3 drugs in recurrent/metastatic NPC
 patients.

 2. second: to evaluate the response and disease control rate in this pilot study.

 Key words: NPC, cytolytic viral activation therapy, gemcitabine, valproic acid, ganciclovir.
1. Introduction to investigational treatment(s) and other study treatment(s)

 1.1 Overview of gemcitabine Gemcitabine (29,29-difluoro 29-deoxycytidine, dFdC) which
 developed from cytosine arabinoside (Ara-C) is an nucleoside analog used as
 chemotherapy. It was intended as an antiviral drug in initial, but preclinical testing
 showed that it killed leukemia cells and a spectrum of solid tumor in vitro. In cell,
 gemcitabine undergoes complex intracellular conversion to the active forms of
 nucleotides gemcitabine diphosphate (dFdCDP) and triphosphate (dFdCTP) that will
 influence DNA synthesis. dFdCTP competes with deoxycytidine triphosphate (dCTP) and
 dFdCDP is a potent inhibitor of ribonucleoside reductase (1). Gemcitabine is
 administered by the intravenous route with dose ranges from 0.6-1.2 g/m^2 of body
 surface area (2), and has been used in a broad spectrum cancer management including
 lung, breast, pancrease, and bladder (3).

 The toxic profile of gemcitabine was major in hematopoietic system especially
 neutropenia and thrombocytopenia (4). In NPC treatment, gemcitabine had been the focus
 of several reports, with interesting OR rates in the range of 23-48% and median
 progression-free survival (PFS) of between 3.6 and 5.1 months (5). In combination
 chemotherapy with cisplatin, the OR rate 42.7 to 73% had been reported and median PFS
 were 5.6 to 10.6 months (6). In combined with oxaliplatin, the OR rate was 56.1 % and
 median PFS was 9 months (6). In combined with vinorelbine, OR rate 36 to 37.7% had been
 reported and median PFS were 5.2 to 5.6 months. In multiple drugs combination with
 carboplatin, paclitaxel, 5-Fluoro-Uracil/LeucoVorin, high OR rate with 86% had been
 reported but with median PFS of 8 months (6). Still these trials were done in small
 series and lacking randomized large scale phase III trial.

 1.2 Valproic acid (VPA) Valproic acid (VPA), a branched short-chain fatty acid, is
 widely used in clinical as an antiepileptic drug and a mood stabilizer, primarily in the
 treatment of epilepsy, bipolar disorder, and prevention of migraine headaches (7). The
 antiepileptic properties of VPA have been attributed to inhibition of Gamma Amino
 Butyrate (GABA) trans aminobutyrate and of ion channels.

 VPA was recently classified among the Histone Deacetylase (HDAC) inhibitors, acting
 directly at the level of gene transcription by inhibiting histone deacetylation and
 making transcription sites more accessible. Chromatin is formed of DNA packaged in
 nucleosome structures. The condensed form of chromatin (heterochromatin) is inactive in
 terms of transcription whereas the decondensed form (euchromatin) corresponds to an
 active form. The histone acetylation leads to relaxation of the nucleosome structure,
 releasing the DNA and allowing transcription. Inhibition of histone deacetylases (HDACs)
 promotes decondensed chromatin formation, thereby promoting the expression of genes (7).

 Valproic acid (VPA), as a HDAC inhibitor, can specifically target at class I a, I b, and
 II a HDACs (8). VPA also down regulates expression of proteins essential for chromatin
 maintenance: Structural Maintenance of chromatin (SMC), DNA methyl transferase-1
 (DNMT1), and heterochromatin protein-1 (HP1) (9). VPA had been shown to induce histone 3
 methylation which would increase transcriptional activity (8). VPA had been shown some
 anticancer effect, major through its HDAC inhibitor, in single agent or combined with
 other anticancer medication including myeloid and lymphoid malignancies, breast cancer,
 prostate cancer, and NPC (10-13) Long-term VPA treatment may cause central nervous
 system (CNS) dysfunction, liver toxicity, and coagulopathy including thrombocytopenia
 and platelet dysfunction (7).

 1.3 Ganciclovir (GCV) and Valganciclovir (VGC) GCV was an antiviral agent had been used
 in treatment or prophylaxis of cytomegalovirus infection in solid organ transplantation
 recipients or bone marrow transplantation (14). GCV is a synthetic analogue of
 2'-deoxy-guanosine and can be phosphorylated to ganciclovir triphosphate, a competitive
 inhibitor of deoxyguanosine triphosphate (dGTP) incorporation into DNA and
 preferentially inhibits viral DNA polymerases more than cellular DNA polymerases, by
 viral and cell kinase. In addition, ganciclovir triphosphate serves as a poor substrate
 for chain elongation, thereby disrupting viral DNA synthesis by a second route (15).

 VGC, a valyl ester prodrug of GCV, had a high oral bioavailability of about 60% with
 similar efficacy of GCV in management of cytomegalovirus infection (16). The most common
 side effect of IV GCV is fever and leukopenia (16).

 1.4 Combination of GEM, VPA and GCV in NPC treatment In EBV-related malignancy,
 antiviral drugs exhibit no direct effect on cancer cell except when used combined with
 epigenetically active agents (17, 18). Recently, Wildeman et al, had shown some efficacy
 by combining chemotherapy of GEM with VPA and GCV in control of locally
 advanced/metastatic NPC patients (13). In this article, both GEM and VPA could shift EBV
 from latent phase into lytic phase and had synergetic effect when combined used. Further
 adding GCV in this regimen could suppress virion formation. The combination therapy had
 been tested in three locally advanced/metastatic NPC patients showing promising results
 with tumor regressing/stable in image and plasma EBV DNA load monitoring with few side
 effects Similar manageable side effects of these three drugs combined treatment were
 also proved by Stoker et al (19). These results encouraged us to develop a more
 practical regimen in this trial.

 2. Test products, dosage, and mode of administration:

 2.1. Chemotherapy regimen: Gemcitabine (Gemmis injection，200 mg) (GEM, TTY) + Valproic acid
 (Depakine gastro-resistant tablet，200 mg) (VPA, Sanofi) for viral activation + Valganciclovir
 (Valcyte film-coated tablets，450 mg) (VGC, Roche) for antiviral medication

 2.2. Dosage GEM: 600~1250 mg/m^2, D1 & D8, intravenously. VPA: 12.5 mg/kg/day D1~14, per os.
 VGC: (2~3) x 450mg/day D9~15, per os.

 1. This treatment cycle of 28 days will be repeated maximum 6 times. (Q4wks/cycle, max: 6
 cycles)

 2. Four dosage of GEM combined with fixed dosage of VPA and VGC will be tested

 3. The rationale of seven days treatment duration of VGC come from (A) 7 days treatment
 duration of valacyclovir in herpes zoster in immunocompetent patients (20) (B) overlap
 side effect of myelosuppression between GEM and VGC(13).

 4. Efficient dose intensity chemotherapy (gemcitabine) is essential in this three combined
 drugs regimen (21) and this trial will be started with dose level 0 (Gemcitabine 800
 mg/m^2)

 2.3. Concomitant treatment

 2.3.1 Permitted: The related treatment for relieve symptoms caused from tumor.

 2.3.2 Prohibited:

 1. Radiation therapy, operation, and other chemotherapy for eradicating tumor

 2. Valganciclovir concomitant with Imipenem-cilastatin could result in convulsion; with
 zidovudine could result in neutropenia; with Probenecid would increase toxicity of
 ganciclovir

 3. Co-administration of valproate with amitryptyline/nortryptyline, and warfarin need to be
 adjusted if necessary

 4. Co-administration of valganciclovir with didanosine, Mycophenolate mofetil, and bone
 marrow suppression drugs (e.g., dapsone, pentamidine, flucytosine, vincristine,
 vinblastine, adriamycin, amphotericin B, nucleoside analogues, hydroxyurea) would need
 to be monitored more seriously because of their toxicity might increase

 5. All other drugs prohibited co-administration with valproic acid absolutely

 3. Duration of treatment: This treatment cycle of 28 days will be repeated maximum 6 times.
 (Q4wks/cycle, max: 6 cycles) A standard 3 + 3 phase I dose escalation study design was used
 (22). A minimum of three evaluable patients were to be treated at each dose level. According
 to Worst Toxicity CTCAE v4.03 Grade and FDA indication of gemcitabine, the dose limiting
 toxicity (DLT) of this trial was determined in the first treatment cycle.

 In the absence of DLT, patients were enrolled in the next dose level. If 1 of three patients
 had a DLT, the cohort was expanded to include six patients. If ≥2 patients experienced DLT,
 maximum tolerated dose (MTD) was exceeded and further enrollment at that dose level was
 stopped. MTD was defined as the highest dose level at which ≤1 of 6 patients experienced a
 DLT. Only DLT that occurred during the first treatment cycle were used to determine the MTD.

 4. Patient examination and re-evaluation: Laboratory data of complete blood count
 (CBC)/differential count (DC), creatinine, alanine aminotransferase (ALT) will be routine
 checked every weeks during the first 3 treatment cycles. During all treatment courses, if
 grade 4 neutropenia or grade 4 thrombocytopenia attacked, the laboratory will be checked
 every 3 days until recovered to pre-treatment baseline. Around 70% responsive and stable
 cases could be enrolled in the 4th treatment cycle will be routine checked the laboratory
 data before gemcitabine administration. Plasma EBV DNA copies number will be monitored before
 each cycle treatment. Systemic re-evaluation will be performed after every three cycles
 treatment. Re-evaluation including physical examination, image studies including CT/MRI for
 head and neck area, chest x-ray, abdominal echo, and Gallium whole body tumor scan, and blood
 exam including complete CBC count, biochemical profile, and plasma EBV DNA copies number.
Inclusion Criteria:

 1. Have the ability to understand and willingness to sign a written informed consent
 document

 2. Identified as recurrent nasopharyngeal carcinoma or distant metastases of male or
 female subjects who had failed in 1st line therapy including radiotherapy, not
 suitable for radiotherapy or unwilling to receive radiotherapy, 1st line chemotherapy
 excluding gemcitabine, and no curative treatment options

 3. Biopsy confirmed belong to World Health Organization classification of nasopharyngeal
 carcinoma type II or type III

 4. Men and women between aged 20 to 80 years of age; female patients with childbearing
 potential will routinely consult obstetric doctor for contraception and need to have
 contraception at least 6 months after finished this trial

 5. Adequate internal organs including liver, kidney and bone marrow function

 -  white blood cell count of >3,000/µL; platelet count of ≥100,000/µL; absolute
 neutrophil count >1,500/µL

 -  total bilirubin <2.0 mg/dL, aspartate aminotransferase (AST), alanine
 transaminase(ALT) <2.5x upper limit of normal range (ULN)

 -  serum creatinine <2.0 mg/dL

 6. The daily performance status ECOG ≤ 2 points

 Exclusion Criteria:

 1. Pregnancy or breast-feeding women, and plans within six months of pregnancy

 2. Contraindication to Gemmis injection, Depakine gastro-resistant tablet, and Valcyte
 film-coated tablets, including:

 -  Allergy to Gemmis injection, Depakine gastro-resistant tablet, Valcyte
 film-coated tablets, and other similar drugs

 -  Patients with hepatic B or C, patients with human immunodeficiency virus, or
 viral related disease receiving anti-viral treatment

 -  Acute or chronic hepatitis not related to NPC with liver metastasis

 -  Using drugs which ineligible combination with valproic acid, including
 mefloquine, St.-John's-Wort, lamotrigine, Topiramate, quetiapine, cyclosporin,
 hepatic enzyme-inducing drugs (e.g., phenytoin, carbamazepine phenobarbital,
 primidone, rifampin), enzyme inhibitors (e.g., felbamate), aspirin, cimetidine,
 erythromycin, carbapenem (e.g., Panipenem, Aztreonam, Imipenem, Meropenem),
 diazepam, ethosuximide, lamotrigine, phenytoin, nimodipine

 3. With insomnia, anxiety or spiritual concerns, or are receiving mental illness
 treatment

 4. Has been diagnosed with a second cancer, except to basal cell carcinoma

 5. Patients unsuitable to this trial, including:

 -  Patients with significant disease

 -  PI evaluated with high risk group patients

 -  Patients not recovered from previous anti-cancer treatment

 -  Recent major surgery

 6. Patients with creatinine clearance rate <40 ml/min