Advances in Brief Diagnostic and Prognostic Implications of Circulating Cell-free Epstein-Barr Virus DNA in Natural Killer/T-Cell Lymphoma

Purpose: Natural killer/T-cell (NK/T-cell) lymphoma is a highly aggressive tumor for which no serological tumor marker has yet been established to be useful clinically. We investigated the potential of circulating EBV DNA as a tumor marker for this malignancy. Experimental Design: A real-time quantitative PCR assay was used to measure circulating EBV DNA. Results: Plasma EBV DNA levels were measured in 18 patients with NK/T-cell lymphoma at presentation and during therapy. Plasma EBV DNA was detected in 17 of the 18 patients (median, 659 copies/ml; interquartile range, 181– 17,042 copies/ml) but in none of 35 control subjects (p < 0.0001). Serial measurements of plasma EBV DNA levels during therapy showed a close correlation between clinical response and changes in plasma EBV DNA levels. Clinically responding patients showed a fall of plasma EBV DNA levels to low or undetectable levels, whereas those who failed therapy showed a rapid increase in plasma EBV DNA levels. Most importantly, patients with high baseline plasma EBV DNA levels (>600 copies/ml) demonstrated a significantly inferior survival than those with low baseline plasma EBV DNA (<600 copies/ml; 21% versus 78%; P 0.024). Conclusions: Plasma EBV DNA, as measured by realtime quantitative PCR, is a useful tumor marker for diagnosis, disease monitoring, and prediction of outcome in patients with NK/T-cell lymphoma. Introduction NK/T-cell lymphomas represent a distinct clinicopathological entity characterized by the presence of progressive necrotic lesions, the expression of NK cell-associated marker, CD56, in tumor cells, and its strong association with the EBV (1, 2). The disease is rare in the United States and Europe but much more common in Asians and South Americans. Most patients present with a midfacial destructive disease in the nasal cavity, nasopharynx, or palate (referred to as nasal NK/T-cell lymphoma), but a minority have tumors that arise from other extranodal sites, including the skin, upper airway, gastrointestinal tract, and testis. The disease generally pursues an aggressive clinical course that is associated with a poor outcome (2–5). Currently, no serological tumor marker has yet been identified for disease monitoring and prediction of outcome in this group of patients. Circulating cell-free EBV DNA has been detected in the plasma/serum from patients with EBV-associated tumors, including Hodgkin’s disease, posttransplant lymphoproliferative disease, AIDS-related lymphoma, and nasopharyngeal carcinoma (6–11). In our recent study, plasma EBV DNA was also found in four patients with “nasal-type” NK/T-cell lymphoma. In this small series, plasma EBV DNA levels appeared to correlate with therapeutic response. However, its impact on prognosis has not yet been determined (11). In the present study, we aimed to investigate the dynamics of plasma EBV DNA levels in patients with nasal and nonnasal types of NK/T-cell lymphoma prior to and in the course of therapy using real-time quantitative PCR to correlate the changes of plasma EBV DNA levels with clinical response during therapy and to evaluate the prognostic value of plasma EBV DNA levels. Materials and Methods Patients. Between December 1998 and March 2001, plasma samples of 18 patients of newly diagnosed or relapsed NK/T-cell lymphoma managed in the Department of Clinical Oncology, Prince of Wales Hospital, were enrolled in this study, including the 4 patients described in our previous study (11). The diagnosis of NK/T-cell lymphoma was established histologically using the criteria described by Jaffe et al. (1) and the Revised European American Lymphoma Classification (12). The presence of EBV in tumor cells was assessed by in situ hybridization on paraffin-embedded tissue sections using a fluorescein-conjugated oligonucleotide probe for EBERs (Novocastra, Newcastle upon Tyne, United Kingdom) as described Received 9/4/01; accepted 10/17/01. 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 P. J. J. and Y. M. D. L. are supported by the Hong Kong Research Grants Council, the Direct Grants Scheme of the Chinese University of Hong Kong and the Industrial Support Fund (AF/90/99). They are also members of the Hong Kong Cancer Genetics Research Group supported by the Kadoorie Charitable Foundations. 2 To whom requests for reprints should be addressed, at Department of Chemical Pathology, Room 38023, 1/F Clinical Sciences Building, Prince of Wales Hospital, 30-32 Ngan Shing Street, Shatin, Hong Kong Special Administrative Region, China. Phone: 852-2632-2563; Fax: 852-2194-6171; E-mail: [email protected]. 3 The abbreviations used are: NK, natural killer; EBER, EBV-encoded small RNA; CR, complete response; PR, partial response; m-BACOD, methotrexate, bleomycin, doxorubicin, cyclophosphamide, vincristine, and dexamethasone; DHAP, dexamethasone, cytarabine, and cisplatin; LDH, lactate dehydrogenase; IPI, International Prognostic Index. 29 Vol. 8, 29–34, January 2002 Clinical Cancer Research Research. on April 12, 2017. © 2002 American Association for Cancer clincancerres.aacrjournals.org Downloaded from previously (13). Patients were staged according to the Ann Arbor Staging System (14). Investigations included a complete history, physical examination, full blood counts, blood chemistry, serum lactate dehydrogenase levels, bone marrow aspiration and biopsy, chest radiography, and computed tomographic scan of the thorax, abdomen, and pelvis. Endoscopic examination of the primary tumor was performed in patients with tumor involvement of the nasal/nasopharyngeal regions and gastrointestinal tract. All patients were treated with uniform departmental protocols according to the primary disease site. Blood samples were taken for EBV DNA analysis at presentation and serially during the course of therapy and subsequent follow-up visits. In addition to this cohort, we also searched for additional plasma samples of NK/T-cell lymphomas stored in our serum bank between 1995 and 1998. Eight cases of NK/T-cell lymphoma collected at presentation were identified and included for survival analysis. Circulating EBV DNA levels were determined. As controls, blood samples from 35 healthy subjects were also analyzed. The study was approved by the Ethics Committee of the Chinese University of Hong Kong, and informed consent was obtained from all subjects. DNA Extraction from Plasma Samples. Plasma samples were collected from patients and healthy subjects according to the protocol described previously (10, 15). The samples were stored at 20°C until further processing. DNA from the plasma samples was extracted using a QIAamp Blood kit (Qiagen, Hilden, Germany), according to the “blood and body fluid protocol” as recommended by the manufacturer (16, 17). Real-Time Quantitative EBV DNA PCR. Circulating EBV DNA concentrations were measured using a real-time quantitative PCR system toward the BamHI-W fragment region of the EBV genome as described previously (10). All plasma DNA samples were also subjected to real-time quantitative PCR analysis for the -globin gene (17), which served as a control for amplifiability of plasma DNA. Multiple negative water blanks were included in every analysis. A calibration curve was run in parallel and in duplicate with each analysis, using DNA extracted from an EBV-positive cell line Namalwa (American Type Culture Collection no. CRL1432; Ref. 18) as standard. Results were expressed as copies of EBV genomes/ml of plasma. Amplification data were collected using an ABI Prism 7700 sequence Detector (PE Applied Biosystems, Foster City, CA) and were analyzed using the Sequence Detection System software developed by PE Applied Biosystems. The mean quantity of each duplicate was used for further concentration calculation. The concentration expressed in copies/ml was calculated using the following equation (19):