Epstein-Barr virus (EBV), a linear double-stranded DNA

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1 Epstein-Barr Virus as a Paradigm in Nasopharyngeal Cancer: From Lab to Clinic Radha Raghupathy, MD, Edwin Pun Hui, MD, and Anthony Tak Cheung Chan, MD, FRCP OVERVIEW Nasopharyngeal carcinoma (NPC) of the undifferentiated subtype remains endemic in southern China, with a peak incidence in this region approaching 30 cases per 100,000 population per year. Despite advances in chemotherapy and radiation delivery techniques in localized disease, distant metastasis is still common and NPC remains the seventh leading cause of cancer death in the region. There is great need for early diagnosis, developing novel therapies, and identifying patients with localized disease at higher risk of future recurrence or metastasis to appropriately tailor their treatment and improve outcomes. Knowledge of the integral involvement of Epstein-Barr virus (EBV) in the pathogenesis of undifferentiated NPC has been of seminal importance in developing strategies to optimize disease management. The close association with EBV is being evaluated in multiple settings including screening of at-risk populations, disease prognostication, development of targeted therapies, optimizing adjuvant treatment, and early recurrence detection. These translational studies are likely to have an enormous effect on management of undifferentiated NPC and significantly improve the landscape of the disease in years to come. Epstein-Barr virus (EBV), a linear double-stranded DNA virus, infects over 90% of the world s population before adolescence. During acute infection B lymphocytes are targeted and lytic phase replication occurs, resulting in the release of more infectious virions. A small number of B cells retain the infection in latent phase with minimal antigen expression. This latent episomal virus has oncogenic potential and many cancers have been associated with EBV latency. 1 Undifferentiated nasopharyngeal cancer (NPC) is the most consistently associated cancer, with latent EBV occurring in more than 95% of cases irrespective of geographic or ethnic origin of the disease. The earliest association between EBV infection and NPC was demonstrated in 1966 by Old et al. Increased titers of immunoglobulin (Ig) G against viral capsid antigen (VCA) and diffuse-early antigen (D-EA) as well as aberrant immune response with IgA antibodies were later identifıed in patients with NPC. 2 Latent EBV is present in high-grade dysplasia and NPC cells, but not in normal epithelium or low-grade dysplasia. 3,4 Monoclonality of the viral genome in NPC has been shown by Southern blot hybridization, suggesting that EBV latency in these cells occurred before clonal expansion. 5 EBV DNA is also consistently shed from tumor tissue into the plasma and is a valuable biomarker. 6,7 Knowledge of this integral role of EBV in NPC carcinogenesis is being translated into clinical practice with the goal of improving disease outcomes. EPSTEIN-BARR VIRUS IN DIAGNOSIS AND MANAGEMENT OF NASOPHARYNGEAL CANCER Outcome in NPC is highly correlated with disease stage. In early localized disease, radiotherapy alone results in 80% to 90% 5-year survival. However, more than 60% of patients present with locally advanced disease; in this group 5-year survival rates drop to approximately 60% to 70%. 8,9 Early diagnosis may improve outcomes, and serologic and EBV DNA-based screening modalities have been studied for screening at-risk populations. High titers of IgA VCA, IgA D-EA, and EBV DNAse with increasing titers during follow-up correlate with subsequent diagnosis of NPC However, a false-positive rate of 2% to 18% has been noted with serologic testing alone. 13,14 Plasma EBV DNA is a very sensitive marker for NPC. EBV DNA is shed consistently from NPC tumor cells and can be routinely detected in the plasma of patients with NPC with more than 95% sensitivity. A recent large population-based study of 1,318 volunteers in Hong Kong evaluated plasma EBV DNA as a screening modality for NPC. Sixty-nine people (5.2%) had a baseline positive test among whom three early-stage NPC cases were identifıed by nasal endoscopy and magnetic resonance imaging (MRI). Only one of these patients had positive IgA VCA serology. Of the 69 people with an initial positive test, only 17 had persistent positive plasma EBV DNA at 18 months, and no new patients with NPC were identifıed among these peo- From the Partner State Key Laboratory of Oncology in South China, Sir Y K Pao Centre for Cancer, Department of Clinical Oncology, Hong Kong Cancer Institute and Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong Special Administrative Region, China. Disclosures of potential conflicts of interest are found at the end of this article. Corresponding author: Anthony Tak Cheung Chan, MD, FRCP, Department of Clinical Oncology, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong SAR; anthonytcchan@cuhk.edu.hk by American Society of Clinical Oncology. asco.org/edbook 2014 ASCO EDUCATIONAL BOOK 149

2 RAGHUPATHY, HUI, AND CHAN ple in 2 years follow-up. 15 In an expanded phase II project, the same group is studying about 20,000 healthy men between ages 40 and 60 screened by plasma EBV DNA (NCT ). Another serum-based polymerase chain reaction (PCR) test evaluating changes in the epigenome in seven candidate genes in NPC has also demonstrated some promise in identifying NPC cases. 16 Cases identifıed through such screening of asymptomatic patients may uncover earlier-stage disease amenable to curative therapy. Optimal cost-effective methods, a suitable target population for screening, and the survival benefıt of such screening must be evaluated. NPC therapy is tailored to disease stage: stage I disease is typically managed with radiotherapy alone; stage II may be treated with radiation alone or concurrent chemotherapy; and stage III, IVA, and IVB is typically managed with concurrent chemoradiation. 17 Phase III trials have shown an overall survival benefıt for concurrent chemoradiation over radiotherapy alone in locally advanced disease However, the role of adjuvant therapy for these patients remains debated. The U.S. Intergroup 0099 study evaluated concurrent high-dose cisplatin and radiation followed by adjuvant cisplatin and 5-fluorouracil (FU) for three cycles, which showed a survival benefıt compared to radiotherapy alone. However, the relative contribution of concurrent compared with adjuvant chemotherapy remains unclear, and only approximately 50% of patients were able to complete the planned adjuvant treatment. 21 In a recent randomized study from China, no signifıcant benefıt in failure-free or overall survival was seen for chemoradiation followed by adjuvant cisplatin/5-fu compared with chemoradiation alone. Distant metastasis remained the most common cause of treatment failure. 22 Identifying a subgroup of patients at high risk for distant metastasis and an adjuvant chemotherapy regimen with better tolerability is a high research priority. Plasma EBV DNA is an KEY POINTS Type II Epstein-Barr virus (EBV) latency is universally associated with the undifferentiated subtype (World Health Organization [WHO] type III) of nasopharyngeal carcinoma. Aberrant serologic responses to EBV are seen in patients with NPC as well as those at high risk for future disease development. EBV DNA detected in cell-free plasma in patients with NPC is an excellent marker of residual disease post-therapy and an independent predictor of disease-free and overall survival. Detectable plasma EBV DNA is being studied as a biomarker for screening and diagnosis in at-risk populations and determining adjuvant therapy options in locally advanced disease. EBV-based targeted therapies, including cytotoxic T lymphocytes, EBV-directed vaccines, and epigenetic treatments, are being investigated as stand-alone or combined modalities. excellent biomarker to identify patients at high risk of distant metastasis. During radiotherapy, plasma EBV DNA declines with a half-life of 3.8 days between the third and seventh weeks of treatment. Persistent positive plasma EBV DNA after completion of radiotherapy or concurrent chemoradiation is a strong predictor of residual disease and independently correlates with poorer disease-free and overall survival The Hong Kong NPC group is conducting a randomized phase III study in patients with locally advanced disease and detectable plasma EBV DNA after 6 to 8 weeks of completing radiation or concurrent chemoradiation, comparing adjuvant chemotherapy with cisplatin/gemcitabine to observation alone (NCT ). Harmonization of the plasma EBV DNA PCR assay across four centers in the United States, China, Hong Kong, and Taiwan has facilitated an international collaborative study. 26 In the forthcoming NRG oncology trial led by the RTOG (NRG-HN001), patients with persistent positive plasma EBV DNA postchemoradiation will be randomly assigned to receive cisplatin/5fu or gemcitabine/paclitaxel in a randomized phase II fashion to evaluate 2-year progression-free survival as a primary endpoint. Patients with post-chemoradiation negative plasma EBV DNA will be randomly assigned in a phase III fashion to adjuvant cisplatin/5fu or observation alone to evaluate 2-year overall survival in a noninferiority design (Fig. 1). Results of these trials will be crucial in identifying an appropriate population for adjuvant treatment of locally advanced disease and the optimal chemotherapy regimen for these patients. EPSTEIN-BARR VIRUS DIRECTED TARGETED THERAPIES FOR NASOPHARYNGEAL CANCER The use of targeted therapies against EBV as stand-alone or adjunctive treatments is being explored. Undifferentiated NPC is characterized by type II EBV latency where latent genes EBER, EBNA1, LMP1, LMP2A, LMP2B, and BARTs are expressed. EBNA1 protein is essential for stabilization of the viral episome and proliferation of the virus using the host DNA machinery. 27 Latent membrane protein 1 (LMP1)isthe major oncogene of EBV. It bears homology to the TNF receptor family (TNFR) and activates multiple downstream signaling pathways including MAPK, JNK, Pi3K/AKT, and NF-kB, resulting in inhibition of apoptosis, promotion of cellular proliferation, and possibly facilitation of metastasis. 28 LMP2A and LMP2B are more consistently expressed in NPC cells, however, their role in NPC oncogenesis is less clear. LMP2A is able to activate Akt and Pi3K pathways, resulting in cell division. Both LMP2A and LMP2B are involved in modulating cell adhesion and motility, potentially contributing to metastatic spread. 29 BART mirnas may facilitate immune evasion and inhibit apoptosis of tumor cells. 30 The host immune response against these EBV latency antigens is suboptimal. A higher number of circulating regulatory T cells (Tregs) has been demonstrated in patients with NPC. These Tregs have been shown to suppress the CD8 T-cell response to these already weakly immunogenic anti ASCO EDUCATIONAL BOOK asco.org/edbook

3 FIG 1. NRG-HN001 study: Biomarker-driven adjuvant therapy for locally advanced NPC. gens. 31 Although tumor-infıltrating lymphocytes appear to lack EBV-specifıc cytotoxicity, anti-ebv responses can still be generated from circulating CD8 lymphocytes. 32 The ability to stimulate anti-ebv responses in circulating CD8 T cells has been used as a basis for adoptive immunotherapy and vaccine development. Adoptive T-Cell Therapy Autologous cytotoxic T lymphocytes (CTL) expanded and developed against LMP2 antigens have shown some durable clinical responses with minimal toxicity. 33 A phase II study of gemcitabine/carboplatin-based chemotherapy followed by LMP2-specifıc CTL infusions revealed promising results. 34 The NATELLA study is a phase I trial looking at LMP1- and LMP2-specifıc CTLs in relapsed or refractory disease. An adenovirus vector based polysaccharide vaccine combining the antigenic epitopes of EBNA1, LMP1, and LMP2 has shown high potency in rapid expansion of autologous T cells and further clinical studies are awaited. 35 Autologous cytokine-induced natural killer (NK) cells have also shown effıcacy in treatment of metastatic disease in combination with chemotherapy. 36 Epstein-Barr Virus Directed Vaccines Vaccines have been developed against the latent EBV antigens. EBNA1, LMP1, and LMP2 antigens are not dominant targets of T-cell response, however, certain epitopes can be presented to the CD4 or CD8 T lymphocytes. EBNA1 predominantly induces a CD4T cell response; LMP2, a weak CD8 response. Both antigens have been combined in the modifıed vaccinia virus vaccine MVA-EL which expresses the CD4 epitope-rich C-terminal domain of EBNA1 fused to full-length LMP2 with CD8 epitopes. The combination facilitates CD4 mediated CD8 immune responses, which are more robust. The vaccine was well tolerated in a phase I study in EBV-positive patients with NPC in remission after primary therapy. T-cell responses to one or both vaccine antigens were increased in 15 of 18 patients. The range of these responses suggested a direct relationship with vaccine dose, with all six patients at the highest dose level having strong EBNA1/LMP2 responses. 37,38 This vaccine may be better targeted for patients with persistent positive EBV DNA in plasma following fırst-line therapy. An ongoing phase II trial is addressing this question (NCT ). In a phase II study of an autologous dendritic cell vaccine developed by adenoviral transduction of LMP2 and truncated LMP1 antigenic domains, in vitro expansion of immunoreactive T cells was seen, but similar fındings were not seen in vivo and effıcacy was limited. 39 In another trial of LMP2Adirected dendritic cell vaccination in localized NPC postradiotherapy, patients who developed a delayed-type hypersensitivity response to the vaccine were able to mount a T-cell immune response and demonstrate a signifıcant decline in post-radiotherapy plasma EBV DNA levels (p ). 40 Genetic and Epigenetic Therapies Gene therapy targeted to EBV-positive NPC cells has been explored using transgenes whose transcription will be regulated by the origin of replication of EBV (ori-p). Both adenoviral and nonviral vectors have been developed in this fashion and different genes, including pro-apoptotic p53 or asco.org/edbook 2014 ASCO EDUCATIONAL BOOK 151

4 RAGHUPATHY, HUI, AND CHAN Fas-L and IFN-y, have been introduced selectively in EBVpositive cells, showing antitumor effıcacy in NPC mouse models. 41,42 Conversion of EBV from latent phase to lytic phase replication promoting cancer cell lysis and death remains another cornerstone for genetic and epigenetic therapies in NPC. Mouse models of NPC have shown effıcacy of adenoviral vectors expressing the EBV immediate early proteins (BZLF1 or BRLF1) in inducing lytic phase replication and inhibiting tumor growth. 43 Epigenetic therapies such as sodium butyrate and azacitidine have shown in vitro effıcacy in modulating methylation signatures, activating lytic phase genes including BZLF1 and promoting cell lysis. 44,45 A phase I study combining azacitidine with suberoylanilide hydroxamic acid (SAHA) in relapsed or refractory NPC has shown some clinical benefıt, with fıve of 11 patients showing prolonged stable disease (NCT ). CONCLUSION We are in an exciting era in the history of NPC. Basic scientifıc knowledge of EBV and NPC carcinogenesis is being rapidly translated into clinical practice. The development of EBV-based screening tools, EBV-directed therapies, and biomarker-driven adjuvant treatment are on the horizon. Results of later-phase clinical trials in these areas are eagerly awaited. This knowledge will no doubt be practice-changing and will guide further research in other oncogenic virus related malignancies, including human papillomavirus related head and neck cancers. ACKNOWLEDGEMENT We sincerely thank the Hong Kong Cancer Fund and the Li Ka Shing Foundation for their ongoing generous support. Disclosures of Potential Conflicts of Interest Relationships are considered self-held and compensated unless otherwise noted. Relationships marked L indicate leadership positions. Relationships marked I are those held by an immediate family member; those marked B are held by the author and an immediate family member. Relationships marked U are uncompensated. Employment or Leadership Position: None. Consultant or Advisory Role: None. Stock Ownership: None. Honoraria: Anthony T. Chan, Merck. Research Funding: Anthony T. Chan, Lilly. Expert Testimony: None. Other Remuneration: None. References 1. Young LS, Rickinson AB. Epstein-Barr virus: 40 years on. Nat Rev Cancer. 2004;4: Henle G, Henle W. Epstein-Barr virus-specifıc IgA serum antibodies as an outstanding feature of nasopharyngeal carcinoma. Int J Cancer. 1976; 17: Lo KW, To KF, Huang DP. Focus on nasopharyngeal carcinoma. Cancer Cell. 2004;5: Klein G, Giovanella BC, Lindahl T, et al. Direct evidence for the presence of Epstein-Barr virus DNA and nuclear antigen in malignant epithelial cells from patients with poorly differentiated carcinoma of the nasopharynx. Proc Natl Acad Sci U S A.1974;71: Raab-Traub N, Flynn K. The structure of the termini of the Epstein-Barr virus as a marker of clonal cellular proliferation. Cell. 1986;47: Lo YM, Chan LY, Lo KW, et al. Quantitative analysis of cell-free Epstein-Barr virus DNA in plasma of patients with nasopharyngeal carcinoma. Cancer Res. 1999;59: Lo YM, Chan LY, Chan AT, et al. Quantitative and temporal correlation between circulating cell-free Epstein-Barr virus DNA and tumor recurrence in nasopharyngeal carcinoma. Cancer Res. 1999;59: Yi JL, Gao L, Huang XD, et al. Nasopharyngeal carcinoma treated by radical radiotherapy alone: ten-year experience of a single institution. Int J Radiat Oncol Biol Phys. 2006;65: Lee AWM, Lau WH, Tung SY, et al. Preliminary results of a randomized study on therapeutic gain by concurrent chemotherapy for regionallyadvanced nasopharyngeal carcinoma: NPC-9901 trial by the Hong Kong Nasopharyngeal Cancer Study Group. J Clin Oncol. 2005;23: Zong YS, Sham JS, Ng MH, et al. Immunoglobulin A against viral capsid antigen of Epstein-Barr virus and indirect mirror examination of the nasopharynx in the detection of asymptomatic nasopharyngeal carcinoma. Cancer. 1992;69: Cao SM, Liu Z, Jia WH, et al. Fluctuations of Epstein-Barr virus serological antibodies and risk for nasopharyngeal carcinoma: a prospective screening study with a 20-year follow-up. PLoS One. 2011;6:e Chien YC, Chen JY, Liu MY, et al. Serologic markers of Epstein-Barr virus infection and nasopharyngeal carcinoma in Taiwanese men. N Engl J Med. 2011;345: Zeng Y, Zhang LG, Li HY, et al. Serological mass survey for early detection of nasopharyngeal carcinoma in Wuzhou City, China. Int J Cancer. 1982;29: Neel HB 3rd, Pearson GR, Taylor WF. Antibodies to Epstein-Barr virus in patients with nasopharyngeal carcinoma and in comparison groups. Ann Otol Rhinol Laryngol. 1984;93: Chan KC, Hung EC, Woo JK, et al. Early detection of nasopharyngeal carcinoma by plasma Epstein-Barr virus DNA analysis in a surveillance program. Cancer. 2013;119: Tan Y, Alfanta EM, Lopa RAB, et al. A blood-based epigenetic test for early detection of nasopharyngeal carcinoma (NPC). J Clin Oncol. 2013;31 (suppl; abstr 6063). 17. Chan AT, Grégoire V, Lefebvre JL, et al. Nasopharyngeal cancer: EHNS- ESMO-ESTRO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol. 2012;23 Suppl 7:vii83-vii Chan AT, Leung SF, Ngan RK, et al. Overall survival after concurrent cisplatin-radiotherapy compared with radiotherapy alone in locoregionally advanced nasopharyngeal carcinoma. J Natl Cancer Inst. 2005; 97: Zhang L, Zhao C, Peng PJ, et al. Phase III study comparing standard radiotherapy with or without weekly oxaliplatin in treatment of locore ASCO EDUCATIONAL BOOK asco.org/edbook

5 gionally advanced nasopharyngeal carcinoma: preliminary results. J Clin Oncol. 2005;23: Lin JC, Jan JS, Hsu CY, et al. Phase III study of concurrent chemoradiotherapy versus radiotherapy alone for advanced nasopharyngeal carcinoma: positive effect on overall and progression-free survival. J Clin Oncol. 2003;21: Al-Sarraf M, LeBlanc M, Giri PG, et al. Chemoradiotherapy versus radiotherapy in patients with advanced nasopharyngeal cancer: phase III randomized Intergroup study J Clin Oncol. 1998;16: Chen L, Hu CS, Chen XZ, et al. Concurrent chemoradiotherapy plus adjuvant chemotherapy versus concurrent chemoradiotherapy alone in patients with locoregionally advanced nasopharyngeal carcinoma: a phase 3 multicentre randomised controlled trial. Lancet Oncol. 2012;13: Lo YM, Chan AT, Chan LY, et al. Molecular prognostication of nasopharyngeal carcinoma by quantitative analysis of circulating Epstein- Barr virus DNA. Cancer Res. 2000;60: Chan AT, Lo YM, Zee B, et al. Plasma Epstein-Barr virus DNA and residual disease after radiotherapy for undifferentiated nasopharyngeal carcinoma. J Natl Cancer Inst. 2002;94: Lin JC, Wang WY, Chen KY, et al. Quantifıcation of plasma Epstein- Barr virus DNA in patients with advanced nasopharyngeal carcinoma. N Engl J Med. 2004;350: Le QT, Zhang Q, Cao H, et al. An international collaboration to harmonize the quantitative plasma Epstein-Barr virus DNA assay for future biomarker-guided trials in nasopharyngeal carcinoma. Clin Cancer Res. 2013;19: Yates J, Warren N, Reisman D, et al. A cis-acting element from the Epstein-Barr viral genome that permits stable replication of recombinant plasmids in latently infected cells. Proc Natl Acad Sci U S A.1984; 81: Raab-Traub N. Novel mechanisms of EBV-induced oncogenesis. Curr Opin Virol. 2012;2: Allen MD, Young LS, Dawson CW. The Epstein-Barr virus-encoded LMP2A and LMP2B proteins promote epithelial cell spreading and motility. J Virol. 2005;79: Lo AK, Dawson CW, Jin DY, et al. The pathological roles of BART mir- NAs in nasopharyngeal carcinoma. J Pathol. 2012;227: Fogg M, Murphy JR, Lorch J, et al. Therapeutic targeting of regulatory T cells enhances tumor-specifıc CD8 T cell responses in Epstein-Barr virus associated nasopharyngeal carcinoma. Virology. 2013;441: Li J, Zeng XH, Mo HY, et al. Functional inactivation of EBV-specifıc T-lymphocytes in nasopharyngeal carcinoma: implications for tumor immunotherapy. PLoS One. 2007;2:e Straathof KC, Bollard CM, Popat U, et al. Treatment of nasopharyngeal carcinoma with Epstein-Barr virus-specifıc T lymphocytes. Blood. 2005; 105: Chia WK, Teo M, Wang WW, et al. Adoptive T-cell transfer and chemotherapy in the fırst-line treatment of metastatic and/or locally recurrent nasopharyngeal carcinoma. Mol Ther. 2014;22: Smith C, Tsang J, Beagley L, et al. Effective treatment of metastatic forms of Epstein-Barr virus-associated nasopharyngeal carcinoma with a novel adenovirus-based adoptive immunotherapy. Cancer Res. 2012;72: Li JJ, Gu MF, Pan K, et al. Autologous cytokine-induced killer cell transfusion in combination with gemcitabine plus cisplatin regimen chemotherapy for metastatic nasopharyngeal carcinoma. J Immunother. 2012; 35: Taylor GS, Haigh TA, Gudgeon NH, et al. Dual stimulation of Epstein- Barr virus (EBV)-specifıc CD4- and CD8-T-cell responses by a chimeric antigen construct: potential therapeutic vaccine for EBV-positive nasopharyngeal carcinoma. J Virol. 2004;78: Hui EP, Taylor GS, Jia H, et al. Phase I trial of recombinant modifıed vaccinia ankara encoding Epstein-Barr viral tumor antigens in nasopharyngeal carcinoma patients. Cancer Res. 2013;73: Chia WK, Wang WW, Teo M, et al. A phase II study evaluating the safety and effıcacy of an adenovirus-lmp1-lmp2 transduced dendritic cell vaccine in patients with advanced metastatic nasopharyngeal carcinoma. Ann Oncol. 2012;23: Li F, Song D, Lu Y, et al. Delayed-type hypersensitivity (DTH) immune response related with EBV-DNA in nasopharyngeal carcinoma treated with autologous dendritic cell vaccination after radiotherapy. J Immunother. 2013;36: Li JH, Chia M, Shi W, et al. Tumor-targeted gene therapy for nasopharyngeal carcinoma. Cancer Res. 2002;62: Zuo Y, Wu J, Xu Z, et al. Minicircle-oriP-IFN: A novel targeted gene therapeutic system for EBV positive human nasopharyngeal carcinoma. PLoS One. 2011;6:e Feng WH, Westphal E, Mauser A, et al. Use of adenovirus vectors expressing Epstein-Barr virus (EBV) immediate-early protein BZLF1 or BRLF1 to treat EBV-positive tumors. J Virol. 2002;76: Li L, Su X, Choi GC, et al. Methylation profıling of Epstein-Barr virus immediate-early gene promoters, BZLF1 and BRLF1 in tumors of epithelial, NK- and B-cell origins. BMC Cancer. 2012;12: Chan AT, Tao Q, Robertson KD, et al. Azacitidine induces demethylation of the Epstein-Barr virus genome in tumors. J Clin Oncol. 2004;22: asco.org/edbook 2014 ASCO EDUCATIONAL BOOK 153