[CANCER RESEARCH 63, 965–971, March 1, 2003] Experimental Treatment of Epstein-Barr Virus-associated Primary Central Nervous System Lymphoma 1 Sameek Roychowdhury, 2 Ruoqi Peng, 2 Robert A. Baiocchi, Darshna Bhatt, Srinivas Vourganti, John Grecula, Nilendu Gupta, Charles F. Eisenbeis, Gerard J. Nuovo, Weilian Yang, Petra Schmalbrock, Amy Ferketich, Melvin Moeschberger, Pierluigi Porcu, Rolf F. Barth, and Michael A. Caligiuri 3 Department of Molecular Virology, Immunology, Medical Genetics [S. R., M. A. C.], Division of Hematology/Oncology, Department of Internal Medicine [R. P., R. A. B., D. B., S. V., C. F. E., P. P., M. A. C.], Department of Pathology [G. J. N., W. Y., R. F. B.], Department of Radiology [J. G., N. G., P. S.], Comprehensive Cancer Center [C. F. E., M. M., R. F. B., M. A. C.], and Center for Biostatistics [A. F., M. M.], The Ohio State University, Columbus, Ohio, 43210 ABSTRACT Primary central nervous system lymphoma (PCNSL) that arises in immune-deficient patients is an aggressive B-cell neoplasm that is univer- sally associated with the EBV. Patients with EBV  PCNSL face a partic- ularly poor prognosis with median survival times of 2–12 months despite aggressive management with radiation therapy. We have developed a preclinical model of EBV  PCNSL to explore strategies that specifically target EBV-infected B lymphoblasts in vivo. Stereotactic implantation of EBV-transformed human lymphoblastoid B-cell lines into the caudate nucleus of the nude rat resulted in lethal CNS tumor burden manifested by the onset of focal neurological symptoms within 21 days. Histological evaluation at autopsy revealed a multifocal, perivascular human EBV  lymphoblastic B-cell infiltrate that displayed a latency type III EBV gene expression profile similar to PCNSL that develops in some immune- deficient patients. Radiation (1600 cGy) of lymphoblastoid B-cell lines resulted in up-regulation of the EBV thymidine kinase (EBV-TK) tran- script and sensitization of these cells to drug-induced apoptosis using nucleoside analogs. Enhanced expression of EBV-TK mRNA in EBV  PCNSL tumors by radiation therapy occurred in a dose-dependent fash- ion. In vivo trials using the nude rat PCNSL model demonstrated signif- icantly improved mean survival time (MST) with single fraction whole- brain radiotherapy (WBRT) and antiviral therapy consisting of zidovudine (AZT) and ganciclovir (GCV; MST 41.3  3.3 days; P  0.05), compared with either antiviral therapy (MST 32.1  1.1 days) or WBRT alone (MST 22  0.8 days). We found constitutive and abundant EBV-TK mRNA expression in a stereotactic core biopsy specimen from a solid organ transplant patient with EBV  PCNSL. Withdrawal of immunosup- pression did not result in disease regression. This patient achieved a complete response after therapy with high-dose AZT and GCV in the absence of WBRT, and remains in remission on oral maintenance AZT/ GCV therapy 3 years after diagnosis. These results suggest that antiviral therapies can be effectively explored in vivo using a preclinical animal model of human EBV  PCNSL with subsequent translation to patients with EBV  PCNSL. INTRODUCTION Individuals with acquired, iatrogenic, or congenital immunodefi- ciency are at increased risk for PCNSL, 4 which commonly presents as a monoclonal, B-cell neoplasm classified as either diffuse large-cell or immunoblastic non-Hodgkin’s lymphoma associated with the EBV (1, 2). The reported incidence of PCNSL in 1999 was 2–11% of patients with AIDS, 1–7% of post-transplant patients with iatrogenic immune suppression, and 4% of those with congenital immune deficiencies (3). Patients with PCNSL face high mortality and a median survival time of 2–12 months after diagnosis (4 – 6). For PCNSL tumors associated with AIDS or iatrogenic immune suppression, standard treatment is chemotherapy and WBRT, which at best provides patients with a minimal improvement in survival (7, 8). WBRT-induced neu- rotoxicity, including symptoms of cognitive impairment and ataxia, occurs in a substantial fraction of PCNSL patients and is progressively debilitating (9). PCNSL in immune-deficient patients is unique among intracranial neoplasms because of the strong association with EBV. These tumors demonstrate specific patterns of latent viral gene expression that likely influence tumor development and pathogenesis (10). The presence of viral gene expression in tumors may represent a unique opportunity to develop targeted therapeutic strategies. In the past 10 years, antiviral therapy with nucleoside analogs, mainly GCV, have targeted EBV- associated diseases with a pattern of EBV lytic gene expression (11). Nucleoside analogs require phosphorylation by the lytic viral gene product TK before being incorporated into viral and cellular DNA. During lytic infection, the EBV open reading frame BXLF1 encodes a full-length TK that has a higher affinity for nucleoside analogs than its cellular counterpart, thus creating the possibility of selective tox- icity for the virus-infected cells of the host (12, 13). However, because most EBV-associated neoplasms typically express latent genes and lack lytic gene expression, antiviral therapy is largely ineffective. Induction of endogenous EBV-TK has been combined recently with antiviral therapy to treat several EBV-associated cancers (14). Phe- nylbutyrate has been shown to activate EBV-TK expression in EBV- associated nasopharyngeal carcinoma and to enhance antiviral activity of GCV (15). Several agents, including phenylbutyrate and ionizing radiation, have successfully induced de novo EBV lytic gene expres- sion in EBV + LCLs in vitro and in vivo (16). More recently, we have shown that EBV-TK is constitutively expressed in some systemic EBV-associated post-transplant lymphomas (17). To our knowledge, an in vivo, preclinical model to evaluate these strategies for immunodeficiency-associated PCNSL has not been de- scribed. Clinical studies of AIDS-PCNSL patients have been difficult to undertake and interpret because of the late presentation of this disease and the recent decline in its incidence since the introduction of highly active antiretroviral therapy (18). Likewise, the relatively low incidence of PCNSL in other immune-deficient patients has made assessment of novel therapeutic approaches difficult. In the present study, we report on the development of a preclinical animal model of human EBV + PCNSL in the nude rat. Here we show, both in vitro and in vivo, that radiation can up-regulate EBV-TK expression that, in turn, can sensitize EBV + tumor cells to the cytotoxic effects of antiviral therapy. Our results suggest that radiation-induced EBV-TK expression followed by antiviral therapy in EBV + PCNSL may be an effective therapeutic strategy to reduce morbidity and mortality in EBV + PCNSL. Finally, we document the regression of EBV + PC- Received 10/29/02; accepted 1/3/03. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported in part by USPHS R01 CA65670 and T32 CA09338 2 These authors contributed equally to this work. 3 To whom requests for reprints should be addressed, at Department of Internal Medicine, Ohio State University, A458 Starling Loving Hall, 320 West 10 th Avenue, Columbus, OH 43210. Phone: (614) 293-7521; Fax: (614) 293-7522; E-mail: caligiuri- 1@medctr.osu.edu. 4 The abbreviations used are: PCNSL, primary central nervous system lymphoma; WBRT, whole brain radiotherapy; GCV, ganciclovir; TK, thymidine kinase; LCL, lym- phoblastoid B-cell line; AZT, zidovudine; RT-PCR, reverse transcription-PCR; CNS, central nervous system; CSF, cerebrospinal fluid; IHC, immunohistochemical; LMP, latent membrane protein; MRI, magnetic resonance imaging; DFS, disease-free survival. 965 Research. on December 10, 2021. © 2003 American Association for Cancer cancerres.aacrjournals.org Downloaded from