Int. J. Cancer: zyxwvutsrqp 39,25-29 (1987) zyxwvuts 0 1987 Alan R. Liss, Inc. zyxwvutsrq Publication of the International Union Against Cancer Publication de I'Union Internationale Comre le Cancer zy THE DIFFERENTIATED FORM OF NASOPHARYNGEAL CARCINOMA CONTAINS EPSTEIN-BARR VIRUS DNA Nancy hAJ3-TRAUB1, Kathy FLY"', Gary &ARSON2, Andrew HUANG3, Paul LEVINE4, Anne LANIER5 and Joseph PAGANO' 'Lineberger Cancer Research Center, University zyxwvut of North Carolina, Chapel Hill, NC; 2Georgetown University, Washington, DC; 3Duke University, Durham, NC; 4National Cancer Institute, NIH, Bethesda, MD; 5Alaska Investigation Laboratory, Centersfor Disease Control, Anchorage, AK, USA. zyxwvuts Immunologic studies of Epstein-Barr virus (EBV) have impli- cated EBV in undifferentiated and partially differentiated, non-keratinizing nasopharyngeal carcinoma (NPC). Patients with the well-differentiated, keratinizing form of NPC have EBV serologic patterns similar to those of control populations. In addition, viral D N A has not been detected in the differen- tiated tumors using viral cRNA probes to DNA immobilized on filters. In this study we have tested for EBV DNA using recombinant D N A probes to Southern blots of D N A from 33 NPC specimens. The 24 undifferentiatedand 4 partially differ- entiated specimens generally contained a relatively high num- ber of EBV genome equivalents, while the 5 well-differentiated NPC all contained detectable EBV, but at low copy number. The viral DNA from one of the well-differentiated specimens was cloned into a cormid vector. Five recombinant clones representingthe fused viral termini were obtained, indicating the presence of episomal, intracellular DNA in the tumor. These findings indicate that all histologic subsets of NPC con- tain EBV DNA. Nasopharyngeal carcinoma (NPC), which arises in the sur- face epithelium of the posterior nasopharynx, has varying degrees of differentiation classified by the World Health Or- ganization (WHO) into 3 categories (Shanmugaratnam and Sobin, 1978). Squamous-cell carcinomas, WHO 1, are highly differentiated with characteristic epithelial growth patterns and intra- and extra-cellular keratin filaments. Non-keratinizing WHO 2 carcinomas retain epithelial cell shape and growth patterns. Undifferentiated carcinomas produce no keratin and have no distinctive growth pattern. The WHO 2 and 3 histo- logic subtypes have been associated with Epstein-Barr virus infection for many years; patients have elevated IgG and IgA responses to the viral capsid antigen (VCA) and diffuse com- ponent of the early antigen (EAd) (Henle and Henle, 1976). In addition, the antibody-dependent cellular cytotoxicity assay (ADCC) can predict the clinical course of patients with type-2 and type-3 NPC (Pearson et al., 1978, 1984). In contrast, patients with well-differentiated carcinomas, WHO 1, have EBV serologic profiles similar to those of control populations (Pearson et al., 1983) and are thought not to have a special association with EBV infection. Earlier studies with 32P-labelled EBV complementary RNA (cRNA) in filter hybridizations revealed a high number of genome equivalents in DNA from undifferentiated tumors (zur Hausen et al., 1970; Nonoyama and Pagano, 1973; Pagano et al., 1975; Desgranges et al., 1975) and no detectable EBV DNA in 2 of 4 tumors with some differentiation (Andersson- Anvret, 1977). One tumor that had differentiated foci within a largely undifferentiated carcinoma had 2 EBV genome equiv- alents. The availability of recombinant EBV DNA fragments for use as probes has made it possible to detect viral DNA with greatly increased sensitivity. This is particularly impor- tant when analyzing clinical specimens which may contain adjacent normal tissue. This study was undertaken to deter- mine if EBV DNA could be detected in tumors of differen- tiated and partially differentiated histology in order to ascertain the full range of NPC that might be correlated with EBV infection. MATERIAL AND METHODS Preparation of tissues and DNA extraction Tissue specimens were obtained through a cooperative study on the relation of EBV serology and histopathologic character- ization (Pearson et al., 1983). The histopathology of the na- sopharyngeal carcinoma tissue specimens was evaluated by z 4 pathologists and classified according to WHO standards. The 5 specimens of well-differentiated squamous-cell carcinomas, WHO 1, had characteristic epidermoid growth patterns with intra- and extra-cellular keratin filaments. All of the 5 WHO 1 patients were Caucasian. Specimens were divided and either prepared for histology or frozen and maintained at -70°C. Frozen specimens were pulverized in a microdismembrator (Braun, Fisher Scientific, Lexington, MA) and dissolved in 4M guanidine thiocyanate. RNA and DNA fractions were separated (Raab-Traub et al., 1983). The DNA fraction was dialyzed to remove CsCl and guanidine thiocyanate, treated with proteinase K, and ex- tracted twice with phenol and chloroform. Restriction enzyme digestion and hybridization DNA specimens were digested with BamHI, subjected to electrophoresis through a 0.6% agarose gel, and transferred to nitrocellulose membranes. The restriction fragment, BamHI V, representing the large internal repeat of EBV, was labelled by nick translation with 32P-labelled dCTP and hybridized to Southern blots (Dambaugh et al., 1980; Raab-Traub, 1980). Nitrocellulose filters containing BamHI-digested DNA pre- pared from Raji cells diluted 1:lO and 1 5 0 to represent ap- proximately 5 and 1 copies of EBV genome equivalents were included in hybridizations to obtain approximate EBV copy number. Cloning of EBV DNA from NPC Viral DNA from a well-differentiated NPC was enriched through two cycles of equilibrium buoyant density gradient centrifugation. EcoRI-restricted DNA was cloned into a cos- mid vector (Raab-Traub et al., 1980). Clones containing EBV fragments were identified and compared with EcoRI fragments of W91 viral DNA (Miller et al., 1974; Raab-Traub et al., 1980). RESULTS Detection of EBV DNA in NPC Thirty-three NPC specimens and 20 control tissues were tested for EBV DNA. All specimens were analyzed by South- ern blot hybridization with the recombinant DNA fragment, BamHI V. All NPC specimens were positive for EBV DNA, as summarized in Table I. In particular, all of the type-1 specimens contained detect- able EBV DNA. The control tissues, which included bone Received: May 28, 1986 and in revised form August 12, 1986.