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Originally published as JCO Early Release 10.1200/JCO.2004.04.185 on March 8 2004 © 2004 American Society of Clinical Oncology. Azacitidine Induces Demethylation of the Epstein-Barr Virus Genome in TumorsFrom the Department of Clinical Oncology, Chinese University of Hong Kong, Prince of Wales Hospital, Shatin NT, Hong Kong SAR, China; Cancer Epigenetics/Tumor Virology Laboratory, Johns Hopkins Singapore, Singapore; Epigenetic Gene Regulation and Cancer Section, National Cancer Institute, National Institutes of Health, Bethesda; Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD; Department of Medicine, University of California San Francisco-Mt. Zion Hospital, San Francisco, CA Address reprint requests to Richard F. Ambinder, MD, PhD, Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Bunting-Blaustein Bldg Rm 389, 1650 Orleans St, Baltimore, MD 21231; e-mail: ambinri{at}jhmi.edu
PURPOSE: To determine whether therapy with a DNA methyltransferase inhibitor is effective in achieving demethylation and gene re-expression in tumor DNA in patients. METHODS: Biopsy specimens were obtained from patients with Epstein-Barr virus-associated tumors, enrolled on a clinical trial of 5-azacitidine, within 72 hours of the conclusion of the last infusion of the first cycle of therapy, and compared to pretreatment specimens. Methylation-specific polymerase chain reaction, bisulfite genomic sequencing, and immunohistochemistry were used to assess demethylation and gene re-expression. RESULTS: Substantial degrees of demethylation were detected in all latent and lytic Epstein-Barr virus promoters examined. Immunohistochemistry suggested activation of a previously silent viral antigen expression in one instance. CONCLUSION: Pharmacologic reversal of dense CpG methylation in tumor tissue can be achieved in patients.
CpG methylation plays several important roles in tumorigenesis, including the silencing of tumor suppressor genes, loss of imprinting, and failure to express DNA repair enzymes and other enzymes important for detoxification of carcinogens.1-5 CpG methylation also silences the expression of differentiation antigens important for immune evasion.6,7 Finally, epigenetic changes may contribute to resistance to particular therapeutic interventions. For example, silencing of estrogen receptor expression precludes certain hormonal therapies in breast cancer.8 Thus, pharmacologic reversal of CpG methylation may be therapeutically beneficial insofar as transcription of tumor suppressor genes reactivate sensitizing to immune surveillance or enhance the response to other therapeutic interventions. Consistent with this concept, various in vitro studies have shown the demethylation of specific cellular and viral genes in cell lines with demethylating agents. Moreover, clinical trials using the DNA methyltransferase suicide substrate inhibitors 5-azacitidine or 5-aza-2'-deoxycytidine (decitabine) have been carried out in patients with a variety of malignancies.9-12 However, the molecular impact of therapy with these agents in terms of demethylation of tumor DNA has not yet been investigated. Epstein-Barr virus (EBV) is a ubiquitous herpesvirus that is associated with a variety of malignancies. CpG methylation of the viral genome plays an important role in regulating viral latency and limiting viral gene expression in normal lymphocytes and in certain tumors including Burkitt’s, Hodgkin’s, AIDS, and nasal lymphomas, as well as nasopharyngeal carcinoma (NPC).13-22 CpG methylation is implicated in silencing expression of the immunodominant EBV nuclear antigens (EBNAs- 2, 3A, 3B, 3C), the latency membrane protein 1 (LMP1), lytic cycle immediate-early antigens Zta and Rta, and lytic cycle viral kinases that are implicated in the phosphorylation of ganciclovir and other antiviral nucleoside analogues.20-23 An intervention that led to activation of these silenced viral genes might facilitate immune-mediated destruction of tumor cells.24 Thus, as many as several percent of CD8+ T-cells in seropositive individuals target EBV antigens that are not expressed in Burkitt’s, Hodgkin’s, NPC, or most AIDS lymphomas.25,26 Whereas adoptive cellular immunotherapy has been shown to effectively treat or prevent EBV-associated tumors in immunocompromised patients,27,28 a change in the pattern of viral antigen expression might functionally accomplish the same thing in patients who were not profoundly immunocompromised.24 The latter include many HIV-infected patients who retain cellular immunity to immunodominant viral antigens not expressed in tumors until late in the disease process.29,30 In vitro, azacitidine, which has been used in clinical trials for more than two decades, leads to demethylation of a variety of genes. In patients with EBV malignancies, we undertook a clinical trial of azacitidine aimed at upregulating expression of silenced viral antigens. The sole and universal presence of hypermethylated EBV DNA in every tumor cell but rare normal cells in EBV-associated tumors has provided us with a suitable biologic model system to study the impact of demethylation drugs in tumor patients in vivo. Analyses of several EBV promoters at the molecular level before and after treatment in patients with NPC and AIDS lymphoma show demethylation to varying degrees in all latent and early lytic EBV promoters examined, though activation of viral gene expression was observed for only one antigen by immunohistochemistry. This is the first study demonstrating that it is feasible to achieve demethylation of tumor DNA in patients with azacitidine treatment.
Patients and Treatment Patients were treated at the Prince of Wales Hospital (Hong Kong, China), the Johns Hopkins Hospital (Baltimore, MD), and the Mt. Zion Hospital (San Francisco, CA). The trials were approved by human investigations committees and written informed consent obtained. The patient with HIV infection was treated under the auspices of the AIDS Malignancy Consortium (Trial 001). Patients had EBV-associated malignancies not expressing EBNA-2 and not curable with conventional therapy. Recurrent or residual disease had to be accessible to minimally invasive biopsy. Minimum parameters for bone marrow function (absolute neutrophil count, 1,000/mm3; and platelets, 75,000/mm3), renal function (serum creatinine <1.5 mg/dL), hepatic function (bilirubin <1.5 mg/dL; transaminases < 3 x control), and coagulation (prothrombin time and partial thromboplastin time <1.3 x control) were also required. Finally, patients had to agree to repeat biopsy of tumor within 72 hours after the seventh day of azacitidine administration in the first cycle. Azacitidine was given by intravenous infusion at a dose of 75 mg/m2/d for 7 days in patients without HIV and for 5 days in patients with HIV as a single daily 3-hour infusion. Biopsy was performed within 72 hours of the last infusion of the first cycle. Patients were to receive therapy every 28 days unless progression or toxicity occurred. Patients were maintained on therapy until progression.
Methylation Studies C promoter (Cp) methylation status was assessed using methylation-specific polymerase chain reaction (PCR).13 Primers mb3/mb4 selectively amplify the products of bisulfite-treated methylated EBV DNA, while primers ub3/ub4 selectively amplify the products of bisulfite-treated unmethylated DNA (Table 1).
W promoter (Wp) methylation status was assessed using strand-specific nested PCR amplification with bisulfite genomic sequencing. The outer primers used (WpOM1/WpOM22; Table 1) spanned the junction between the BamHI C and BamHI W fragments of the genome, while the nested primers (WpIM3/WpIM4; Table 1) targeted the regulatory region of Wp yielding a 595-bp fragment containing 22 CpG sites. The PCR conditions for WpOM1/WpOM22 were 94°C for 3 minutes, followed by 40 cycles of amplification (94°C for 30 seconds, 53°C for 30 seconds and 72°C for 1 minute and 15 seconds) and a final extension at 72°C for 10 minutes. Bisulfite-treated DNA (50 ng) was amplified by using 0.3U Taq polymerase (Promega, Madison, WI), 0.2 mmol/L dNTPs, 0.4 µmol/L primers, and 1.5 mmol/L MgCl2 in a 12.5 µL reaction. The nested PCR procedure involved an initial denaturation of 95°C for 10 minutes and 45 cycles of amplification (94°C for 30 seconds, 53°C for 30 seconds, and 72°C for 30 seconds) followed by a final 1-hour extension at 72°C. The nested PCR product was electrophoresed and cloned into the pCR2.1-TA cloning vector (Invitrogen, Carlsbad, CA). Plasmid DNA was extracted and sequenced. The methylation status of other EBV promoters (LMP1p, Zp [Z promoter], and Rp [R promoter]) was analyzed using bisulfite genomic sequencing.13,14,17 The corresponding primers are listed in Table 1.
Immunohistochemistry
Ten patients were treated; eight with NPC, one with Hodgkin’s lymphoma, and one with AIDS-associated diffuse small noncleaved lymphoma. All had failed prior standard therapies, including allogeneic bone marrow transplantation in the patient with Hodgkin’s lymphoma. Treatment was continued for one to six cycles. In most patients, myelotoxicity was modest with no myelotoxicity in five patients, grade 1 or 2 in three patients, grade 3 in one patient and grade 4 in one patient. In situ hybridization showed the presence of EBV in biopsy specimens. Tumors in nine clinically assessable patients did not respond to treatment. Technically adequate paired specimens from five patients (four with NPC, one with lymphoma) were studied. As planned in the protocol, biopsy specimens for the analysis of methylation were obtained within 72 hours after azacitidine administration in the first cycle. Thus, although treatment was continued in many instances, this laboratory data reflects only the immediate impact of the first course of therapy. EBV antigens are almost unique among antigens recognized by CD8+ cytotoxic T-cells in healthy seropositives for the intensity of the chronic response that they elicit. Thus, several different EBV antigens are targeted by up to several percent of CD8+ cytotoxic T-cells in healthy seropositive individuals. Other infections such as influenza may transiently elicit such response, but none other than cytomegalovirus are known to elicit such a sustained response. The particular viral antigens that we selected for study were selected because they are representative of families of immunodominant antigens, and well-characterized monoclonal antibodies were available for the analysis. The promoters that we selected for study each drive expression of more than one antigen. These were selected because they are the key regulatory elements for expression of the antigens studied and for regulation of gene expression in latency and lytic cycle. EBNA-2 is a key viral regulatory protein expressed in latency that activates expression of EBNA-3A, EBNA-3B, and EBNA-3C, each associated with intense cytotoxic T-cell responses in healthy EBV seropositive individuals. EBNA-2 and the other immunodominant EBNAs are not expressed in NPC, Hodgkin’s lymphoma, or the family of AIDS lymphomas studied in this protocol. Activation of EBNA-2 expression might have been expected to lead to immune killing of tumor cells. Laboratory analysis using immunohistochemistry confirmed that none of the tumors studied expressed EBNA-2 pretreatment. Treatment did not activate EBNA-2 expression. Zta is a key viral regulatory protein that initiates a cascade of viral gene expression resulting in replication of the viral genome and the production of new virions. As with the immunodominant EBNAs, expression of Zta would be expected to result in immune killing of tumor cells. By immunohistochemistry, it was not detected in any pretreatment biopsy specimen but was detected in scattered tumor cells in one patient (NPC3) following treatment (Fig 1).
In contrast to the apparent absence of effect of azacitidine treatment on viral antigen expression, it had profound effects on the methylation status of viral promoters (Table 2). The latency promoter in the BamHI-C region of the viral genome (Cp) drives expression of all of the EBNAs, including the immunodominant EBNAs. We analyzed the Cp by a methylation-specific PCR technique that targets a region of the promoter shown to be hypersensitive to the transcriptional inhibitory effects of CpG methylation.32 Methylation-specific PCR and bisulfite genomic sequencing yield results that correspond closely in the Cp region as we have previously demonstrated.13 We found dense methylation of pretreatment tumor DNA specimens as indicated by the presence of a band with mb, but not ub, primers (Table 1 and Fig 2). DNA from post-treatment specimens showed either a mix of methylated and unmethylated DNA, or just unmethylated DNA, as indicated by the presence of a band with ub primers. Thus, an unmethylated DNA band appeared or its relative proportion increased in each instance.
In some instances, expression of the immunodominant EBNAs may be driven by an alternative latency promoter, Wp. We also studied changes in the methylation status of Wp. After primary infection of lymphocytes in vitro and in vivo, Wp expresses transcripts. Thereafter, a promoter switch occurs. Wp transcription turns off and Cp transcription activates.22 For the analysis of Wp, we relied on standard bisulfite genomic sequencing to assess CpG methylation. Adequate material was available to compare patterns of methylation pre- and post- treatment in four patients. In each specimen we examined 22 CpG sites in four or more clones (Fig 3). We found that  80% of CpG sites were methylated in all of the pretreatment specimens (Table 2). In one case of NPC (NPC1) and in the patient with AIDS lymphoma, treatment led to demethylation of at least one-third of methylated CpG sites (83% to 27%; 92% to 55%), while in the other two cases, changes in methylation with treatment were negligible (87% to 88%; 80% to 85%).
Next we studied CpG sites in the vicinity of the LMP1 (ED-L1 and ED-L1-E promoters) and LMP2B promoters (Fig 4). Methylation analyses showed marked heterogeneity among pretreatment patient specimens. Therefore, two of the pretreatment NPC specimens (NPC1 and NPC3) were densely methylated ( 96%) while two specimens (NPC2 and NPC5) were minimally methylated (9% and 10%, respectively). The pretreatment AIDS lymphoma specimen showed almost no methylation in this region (2%). Following treatment, all NPC specimens demethylated by at least one-third of methylated CpG sites (to 17%, 23%, 5%, and 5%, respectively) and there was no evidence of any residual methylation in this region in the AIDS lymphoma specimen (Table 2).
Finally, we examined promoters for the early lytic cycle genes Zta and Rta (Fig 5). The availability of material limited this analysis so that Zp could only be studied in two NPC specimens but we observed demethylation of Zp in both. We studied Rp in four patients, and demethylation was seen in all of them. The NPC patient who activated Zta expression in scattered cells also demethylated Zp and Rp.
This study demonstrates that substantial demethylation can be achieved using a clinically well-characterized demethylating agent in tumor DNA and that in vivo, as in vitro, there is variability in the susceptibility to demethylation between loci. Two different methods were used to assess changes in methylation status. The methylation specific PCR technique used to characterize the methylation status of the Cp detects the methylation status of the primer binding regions only, and represents an average of the methylation status of the primer binding regions.13 It does not provide information about sites located between primers. However, as previously shown in tumors and tumor cell lines, the results of methylation specific PCR accurately reflect patterns of methylation in the vicinity of the primer binding sites in the Cp. Other regions were evaluated by bisulfite genomic sequencing. Whereas methylation specific PCR represents an average, genomic sequencing yields results that reflect the methylation pattern in a single strand of DNA. These two different methods yielded substantially similar results, both showing marked decreases in CpG methylation in tumor tissue with azacitidine treatment. The finding that demethylation can be achieved in vivo is consistent with expectations based on in vitro and animal experiments.33,34 However, the rapid breakdown of azacitidine in vivo, the limited pharmacokinetic data available, and the observation that tissue culture itself profoundly alters methylation patterns has made extrapolation from in vitro and transplantable tumors in murine models highly speculative.35,36 The occurrence of demethylation at several loci and the hint that lytic gene expression may have been activated in one instance points to possible future therapeutic maneuvers. In particular, evidence that modest reversal of dense CpG methylation can facilitate promoter activation with histone deacetylase inhibitors suggests the possibility that combinations of DNA methyltransferase inhibitors and histone deacetylase inhibitors might bring about expression of silenced genes.37,38
The authors indicated no potential conflicts of interest.
Research funded by National Institutes of Health grants PO1 CA15396 "EBV-related tumors" and UOI CA70062 "AIDS-associated malignancies clinical trials in a cooperative group." Anthony T.C. Chan and Qian Tao contributed equally to the work. Authors’ disclosures of potential conflicts of interest are found at the end of this article.
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Copyright © 2004 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
