Characterization of Epstein Barr Virus Genotype in AIDS-Related Non-Hodgkin s Lymphoma

Transcription

1 AIDS RESEARCH AND HUMAN RETROVIRUSES Volume 18, Number 1, 2002, pp Mary Ann Liebert, Inc. Characterization of Epstein Barr Virus Genotype in AIDS-Related Non-Hodgkin s Lymphoma LUCIA FASSONE, 1 ANTONELLA CINGOLANI, 2 MAURIZIO MARTINI, 3 GIUSEPPE MIGLIARETTI, 4 PIER LUIGI ORESTE, 5 DANIELA CAPELLO, 1 ANNUNZIATA GLOGHINI, 6 DANIELA VIVENZA, 1 RICCARDO DOLCETTI, 7 ANTONINO CARBONE, 6 ANDREA ANTINORI, 8 GIANLUCA GAIDANO, 1 and LUIGI MARIA LAROCCA 3 ABSTRACT In the present study sequence variations at the C terminus of the Epstein Barr virus (EBV) nuclear antigen 1 (EBNA-1), EBV-encoded latent membrane protein 1 (LMP-1), and EBNA-2 and EBNA-3C genes were investigated in 64 cases of EBV-positive AIDS-related diffuse large cell lymphoma (AIDS-DLCL), both systemic (12) and localized primarily to the central nervous system (52), and in 12 cases of EBV-positive AIDS-related Burkitt s lymphoma (AIDS-BL). Sequence analysis of the EBNA-1 C-terminal region led to the distinction of two major unrelated EBV strains, termed strain P (prototype) and strain V (variant), and their related subtypes, namely P-ala, P-thr, V-leu, V-val, and V-pro. Analysis of the LMP-1 gene was performed to assess the frequency of the C-terminus deletion variant, whereas analysis of EBNA-2 and EBNA-3C genes led to the identification of the distribution of the EBV type 1 and type 2 strains. The frequency of EBNA-1 subtypes was assessed in 49 cases of AIDS-NHL, including 37 cases of AIDS-DLCL and 12 cases of AIDS-BL. The P strain was detected in 45 of 49 cases (91.8%) whereas the V strain was found in 4 of 49 samples (8.1%). A significant difference in the distribution of the P and V strains was found between AIDS-DLCL and AIDS- BL (p, 0.01), because of the exclusive infection by the P strain of the AIDS-DLCL samples analyzed. The frequency of LMP-1 deletion variants and of EBV type 1 and type 2 strains in AIDS-DLCL overlapped with that of the general population, and no correlation was found with the evaluated clinicoepidemiological data of patients, that is, disease site, tumor histology, CD4 1 cell counts, and HIV transmission route. In conclusion, we found that the distribution of the EBV genotype in all of the AIDS-NHL samples analyzed is similar to the viral representation found in control individuals of both immunocompetent and immunocompromised populations. INTRODUCTION THE EPSTEIN BARR VIRUS (EBV) is a human gammaherpesvirus widespread in the world population. Although healthy individuals carry EBV as an asymptomatic latent infection of the B lymphocyte pool, a role for the virus in the pathogenesis of malignant diseases has been suggested by in vitro and in vivo studies. 1 Natural EBV isolates display a certain degree of genetic variability and viral variants are frequently distinguished according to sequence polymorphisms in subgenomic regions. 2,3 Several EBV polymorphisms have been investigated in malignant diseases including those of the virus EBV nuclear antigen 2 (EBNA-2), EBNA-3C, EBV-encoded latent membrane protein 1 (LMP-1), and EBNA-1 genes. EBV 1 Division of Internal Medicine, Department of Medical Sciences, Amedeo Avogadro University of Eastern Piedmont, Novara, Italy. 2 Institute of Infectious Diseases, Catholic University of the Sacred Heart, Rome, Italy. 3 Institute of Pathology, Catholic University of the Sacred Heart, Rome, Italy. 4 Department of Public Health and Microbiology, Statistical Unit, University of Turin, Turin, Italy. 5 Division of Pathology, Niguarda Cà Grande Hospital, Milan, Italy. 6 Division of Pathology, Centro di Riferimento Oncologico, IRCCS, Istituto Nazionale Tumori, Aviano, Italy. 7 Division of Experimental Oncology 1, Centro di Riferimento Oncologico, IRCCS, Istituto Nazionale Tumori, Aviano, Italy. 8 National Institute for Infectious Diseases Lazzaro Spallanzani, IRCCS, Rome, Italy. 19

2 can be classified as a type 1 or type 2 strain, based on major sequence differences in the EBNA-2 and EBNA-3C genes. Type 1 EBV shows a more potent transforming efficiency in vitro than type 2 and appears to predominate in the general white and Asian populations, whereas both types are relatively common in equatorial Africa and New Guinea. 4 Independent of geographical and ethnic distribution, type 2 infection has been described to be more prevalent in immunocompromised patients from Western populations, particularly in immunoblastic lymphomas arising in HIV-positive homosexual patients. 5 LMP-1 is essential for B lymphocyte transformation and is believed to play an important role in the pathogenesis of many EBV-associated diseases. 6 Deletions from the 39 end of the LMP-1 gene have been suggested to be associated with nasopharyngeal carcinoma (NPC) and Hodgkin s disease (HD), displaying aggressive clinicopathologic features. 7,8 Also, these LMP-1 deletion variants appear to harbor increased tumorigenicity and reduced immunogenicity compared with wild-type genes. 7,9 These findings suggested that deletions within the LMP-1 gene may be responsible, at least in part, for the enhanced in vitro and in vivo growth potential induced by this viral strain in B lymphocytes. EBNA-1 is required for replication and maintenance of viral episomes and represents the sole viral protein consistently expressed in EBV-infected cells. Previous studies identified a high frequency of sequence variation at the DNA-binding and dimerization domain of EBNA-1. 10,11 Such EBNA-1 mutations cluster at amino acids 467 and 525 and identify two major unrelated EBV strains, designated strain P (prototype) and V (variant). Further, on the basis of amino acid signature at codon 487, five EBNA-1 subtypes may be recognized, namely P-ala, P-thr, V-leu, V-val, and V-pro. Work has investigated whether certain EBNA-1 subtypes preferentially associate with malignancies from distinct geographical regions or with different types of malignancies However, to our knowledge, a study of EBNA-1 variant distribution throughout the pathologic spectrum of AIDS-related non-hodgkin s lymphoma (AIDS-NHL) has not been performed. In this work, a well-characterized panel of AIDS-NHL, including AIDS-related diffuse large cell lymphoma (AIDS- DLCL), both systemic and primarily localized in the central nervous system (CNS), and AIDS-related Burkitt s lymphoma (AIDS-BL), was investigated in order to determine the EBV strain infecting the tumor clone. Further, the genotypic features of EBV were correlated to the epidemiological and clinical features of the tumor, as well as to the tumor phenotype and stage of differentiation. 20 MATERIALS AND METHODS Tumor panel and DNA extraction The present study is based on a panel of 76 EBV-positive AIDS-NHL, for which clinical and epidemiological data were available. In all instances, the specimens were collected at diagnosis before specific therapy. Diagnoses were based on analysis of histopathology and immunophenotypic analysis of cell surface markers. The fraction of malignant cells in the pathologic specimens was $60% and in most cases $75%, as determined by tissue section immunohistochemical analysis of cell surface markers. The panel of AIDS-NHL included 52 cases of AIDS-DLCL primarily localized to the CNS, 12 systemic AIDS-DLCL cases, and 12 samples of AIDS-BL. All but one patient were white, and their geographical origin was Italy. One patient with systemic AIDS-DLCL originated from Africa. The route for HIV transmission was available for all patients. The risk factor was represented by heterosexuality for 20 of 76 patients (26.3%), homosexuality for 21 of 76 patients (27.6%), and intravenous drug user (IVDU) for 35 of 76 patients (46.0%). Genomic DNA was prepared as previously reported. 15 Determination of EBNA-1 subtypes The distribution of EBNA-1 subtypes was determined by DNA direct sequencing of polymerase chain reaction (PCR)- amplified products, as previously reported. 10 DNA samples were amplified by a single-step PCR protocol using specific primers (EBNA-1/59 and EBNA-1/39) that amplify a 330-bbp fragment containing most of the polymorphisms observed in EBNA-1 variant subtypes. PCR was performed in a final volume of 50 ml containing 500 ng of DNA, 50 pmol of each primer, 100 mm dntps, 10 mm Tris-HCl (ph 8.8), 50 mm KCl, 1.5 mm MgCl 2, 0.01% gelatin, and 2.5 U of AmpliTaq polymerase (Perkin-Elmer, Norwalk, CT), using an Omn-E thermocycler (Hybaid, Middlesex, UK). The annealing temperature was 60 C. After PCR, the DNA amplimers were purified (QIAquick gel extraction kit; Qiagen, Chatsworth, CA) and subjected to DNA direct sequencing with a commercially available kit (Thermosequenase; Amersham Life Science, Rockville, MD). a- 33 P-labeled terminator dideoxynucleotides were included in the sequencing mixture. All sequences obtained were compared with the DNA sequence of B95.8, which represents the EBV prototype. 16 In selected cases of AIDS-NHL, the EBNA-1 amplimer was subcloned into a TA-cloning plasmid vector (TA cloning kit; Invitrogen, San Diego, CA) and multiple independent clones were subjected to DNA sequencing with an ABI 373 (Perkin- Elmer) automated sequencer with a dye-terminator cycle sequencing reaction kit (Perkin-Elmer) and the same primers utilized for PCR. Analysis of LMP-1 gene The LMP-1 genotype was investigated by PCR analysis, using primers specific for the C terminus of LMP-1, as previously reported. 17 The PCR was performed as described above. The EBV-positive cell lines B95.8 and AG876 were used as controls for wild-type LMP-1 and an LMP-1 variant harboring a 30-base pair deletion (D30LMP-1), respectively. As reported previously, 7,8 the D30LMP1 variant was defined as an LMP-1 gene harboring the deletion of codons , whereas the D69LMP-1 variant was defined as an LMP-1 gene harboring the deletion of codons PCR products were analyzed on a 3% agarose gel and the size of the amplimers was compared with that obtained from controls. Cases found to harbor LMP-1 variants differing from wild-type LMP-1 were further subjected to DNA sequence analysis for confirmation. Definition of EBV type 1/type 2 FASSONE ET AL. Definition of EBV type 1/type 2 was performed by using standard PCR assays across type-specific regions of the EBNA-

3 MOLECULAR PROFILE OF EBV IN AIDS-NHL 21 2 and EBNA-3C genes, as previously reported. 17,18 The EBVpositive cell lines B95.8 and AG876 were used as controls for EBV type 1 and EBV type 2, respectively. PCR products from tumor samples and from controls were analyzed on a 2% agarose gel. Immunohistochemistry All cases included in this study were EBV positive as assessed by in situ hybridization analysis of EBV-encoded small RNAs (EBER). 19 All antigens were tested on paraffin-embedded tissue sections. LMP-1 antigen was detected on paraffin sections using the alkaline anti-alkaline phosphatase (APAAP) method and a pool of four anti-lmp-1 monoclonal antibodies (MAbs) (CS 1 4; Dakopatts, Glostrup, Denmark) after enzymatic digestion with Pronase (Biomeda, Foster City, CA), as previously described. 19 The BCL-6 protein was detected by the PG-B6 MAb (Dakopatts). BCL-2 expression was assessed with the 124 MAb (Dakopatts). For BCL-6 and BCL-2 immunostaining, sections were treated in a microwave oven at 250 W for 30 min in EGTA solution (1 mm, ph 8) (for BCL-6) or citrate buffer (ph 6) (for BCL-2). Immunostaining was performed on an automated immunostainer (Techmate 500; Dakopatts) according to the manufacturer protocols. Statistical analysis Statistical analysis was performed to correlate EBV polymorphisms to clinical and epidemiological features of AIDS-NHL patients, including disease site, tumor histology, and risk factor for HIV infection. EBV polymorphisms were also compared with each other in order to investigate a preferential association between the different viral genotypic markers. Statistical analysis for the categorical variables was based on the x 2 test with Yates correction and, when the number of cases made it appropriate, the Fisher exact test. Differences were considered significant when p values were,0.05. SPSS (Chicago, IL) software (version 6.0 for Windows) was used for statistical elaboration. RESULTS Analysis of EBNA-1 variants in AIDS-NHL The C-terminal region of the EBNA-1 gene was investigated in a panel of 49 AIDS-NHL, inclusive of 37 AIDS-DLCL and 12 AIDS-BL. Results of EBNA-1 gene analysis are represented for each case in Tables 1 and 2 and are summarized in Table 3. Analysis by DNA direct sequencing showed the presence of a single EBNA-1 variant in 35 of 49 AIDS-NHL cases (71.4%). Conversely, the remaining 14 samples (28.5%) displayed multiple EBNA-1 sequences within the same tumor biopsy. In these cases, the heterogeneity of the EBNA-1 pattern appeared to result from the infection of different EBNA-1 sequences of the same prototype strain (n 5 7), or alternatively from simultaneous infection of both P-ala and P-thr strains (n 5 7) (Table 1). The amino acid sequences of the EBNA-1 C terminus (codons ) were classified on the basis of the residue at codon 487 and were compared with the B95.8 virus prototype (Fig. 1). Our results corroborate previous work suggesting that the classification of EBNA-1 subtypes should not be based solely on the amino acid at position ,20 In fact, different amino acid changes at codons 492, 499, 520, and 524 appear to be specifically associated with distinct EBNA-1 subtypes (Fig. 1). According to this observation, the sequence called P- ala variant V in Fig. 1 may result from a recombinant event between a P-ala subtype and a V-leu subtype. The P-ala and P-thr subtypes were equally represented in AIDS-NHL. Infection with P-ala alone was found in 18 of 49 samples (36.7%) and infection with P-thr alone was found in 19 of 49 samples (38.7%). Seven samples harbored coinfection by P-ala and P-thr (7 of 49; 14.2%). The V-leu subtype was present in 3 of 49 AIDS-NHL cases (6.1%), whereas a V-val sequence was detected in 1 of 49 cases (2.0%). One case (8877UC) harbored a P-ala variant that had recombined to a V- leu subtype. The frequency of EBNA-1 subtypes in AIDS-NHL samples was overall similar to the incidence found in EBV isolates from nonneoplastic B cells of HIV-infected individual, deriving from the same geographical region as donors of the tumor panel investigated in this study, and previously reported. 15 Notably, whereas AIDS-BL harbored both prototype and variant EBNA-1 major strains, AIDS-DLCL cases, either systemic or primarily localized to the CNS, were found to be consistently infected solely with prototype EBNA-1 strains, that is, P-ala and P-thr (p, 0.01) (Table 3; see also Tables 1 and 2). Most EBNA-1 patterns detected in AIDS-NHL were, in part or completely, known from previous studies of B cell tumors and normal or reactive B cells ,20 The presence of coresident EBV strains in the context of the same lymphoma biopsy was formally demonstrated by DNA sequencing of multiple (n 5 8) subclones obtained from two samples (cases 1683 and 2280) exhibiting heterogeneity at several EBNA-1 nucleotides. In both cases, the EBNA-1 pattern was represented by two different, independent EBNA-1 variants. In case 1683, and P-ala II and P-ala IV variants were found to infect the tumor clone, whereas sample 2280 showed the presence of the P-ala III and P-thr I variants. Moreover, in 4 of 16 subclones analyzed, additional mutations, not revealed by DNA direct sequencing of the PCR product, were detected along the EBNA-1 C-terminal region. Each mutation (n 5 6) was represented by single base substitutions and was detected in a unique subclone. Analysis of LMP-1 in AIDS-NHL The prevalence of deletions in the C-terminal fragment of the LMP-1 gene was investigated in 72 cases of AIDS-NHL. Results of LMP-1 gene analysis are represented for each case in Tables 1 and 2 and are summarized in Table 4. Sixty-eight of 72 cases harbored a single LMP-1 species. Of these, 37 of 72 cases (51.4%) harbored a full-length LMP-1 sequence, 24 of 72 cases (33.3%) harbored the D30LMP-1 variant, and 7 of 72 cases (9.7%) harbored the D69LMP-1 variant. Four AIDS- NHL samples carried the D30LMP-1 gene in association with the full-length LMP-1 gene or with the D69LMP-1 gene (Tables 1, 2, and 4). DNA sequencing analysis of D30LMP1 and D69LMP-1 variants confirmed that, in our tumor samples, deletions spanned codons and , respectively. The D30LMP-1 variants displayed point mutations identical to those frequently associated with D30LMP-1 identified in neoplastic as well as

4 TABLE 1. CLINICAL AND MOLECULAR CHARACTERISTICS OF AIDS-DLCL CASES CD4 1 Case Localization Histology Risk factor cells/ml EBV type LMP-1 EBNA-1 797UC Systemic DLCL Heterosexual 347 Type 1 wt P-ala/P-thr 1540UC Systemic DLCL IVDU 41 Type 1 wt P-thr 3259UC Systemic DLCL IVDU 36 Type 1 wt P-thr 5133UC Systemic DLCL IVDU 173 Type 1 D30 P-thr 5506UC Systemic DLCL Homosexual 227 Type 1 wt P-thr 6619UC Systemic DLCL IVDU 105 Type 1 wt P-ala a 8877UC Systemic DLCL IVDU 62 Type 1 D30 P-ala b 9205UC Systemic DLCL IVDU 18 Type 2 D30 P-thr 9479UC Systemic DLCL Heterosexual 270 Type 2 D69 P-thr 16903UC Systemic DLCL IVDU 71 Type 1 D69 P-ala/P-thr 17069UC Systemic DLCL Homosexual 3 Type 1 D30 P-ala/P-thr 18261UC Systemic DLCL Heterosexual 107 Type 1/type 2 wt/d30 P-ala/P-thr 1630 CNS DLCL Homosexual 4 Type 1 wt NA 1632 CNS DLCL IVDU 32 Type 1 wt NA 1634 CNS DLCL Homosexual 2 Type 1 wt P-thr 1635 CNS DLCL IVDU 3 Type 1 wt P-ala 1675 CNS DLCL Heterosexual 20 Type 1 wt NA 1676 CNS DLCL Homosexual 29 Type 2 wt NA 1677 CNS DLCL Homosexual 8 Type 1 D69 NA 1678 CNS DLCL IVDU 10 Type 1 wt NA 1679 CNS DLCL IVDU 4 Type 1 wt NA 1680 CNS DLCL Heterosexual 4 Type 1 wt NA 1681 CNS DLCL Homosexual 2 Type 2 D69 NA 1683 CNS DLCL IVDU 4 Type 2 D30 P-ala 2201 CNS DLCL Homosexual 24 Type 2 D69 P-thr 2202 CNS DLCL Heterosexual 4 Type 1 wt NA 2203 CNS DLCL Homosexual 11 Type 2 wt P-thr 2204 CNS DLCL Homosexual 12 NA wt NA 2205 CNS DLCL Heterosexual 5 NA wt NA 2206 CNS DLCL Homosexual 5 Type 1 wt P-thr 2207 CNS DLCL Heterosexual 5 Type 1 wt P-ala 2208 CNS DLCL IVDU 41 Type 1 wt P-ala 2209 CNS DLCL Homosexual 4 Type 2 wt P-ala 2210 CNS DLCL Heterosexual 19 Type 1 wt NA 2211 CNS DLCL Homosexual 3 Type 1 D30 P-ala 2212 CNS DLCL Heterosexual 152 Type 1 wt P-thr 2213 CNS DLCL Heterosexual 8 Type 1 wt P-thr 2214 CNS DLCL IVDU 2 Type 2 wt P-ala 2215 CNS DLCL Heterosexual 41 Type 2 wt P-thr a 2216 CNS DLCL IVDU 5 Type 1 wt P-ala 2217 CNS DLCL Heterosexual 280 Type 1 wt NA 2218 CNS DLCL Homosexual 30 Type 1 D30 P-ala 2244 CNS DLCL Homosexual 12 Type 1 D69 P-thr 2245 CNS DLCL Heterosexual 0 NA wt NA 2246 CNS DLCL IVDU 79 Type 1 wt P-thr a 2247 CNS DLCL IVDU 22 Type 1 D69 NA 2249 CNS DLCL IVDU 36 Type 1 wt P-ala a 2250 CNS DLCL Heterosexual 57 NA D30 NA 2251 CNS DLCL IVDU 15 Type 1 NA NA 2253 CNS DLCL Homosexual 8 Type 1 NA NA 2278 CNS DLCL IVDU 7 Type 1/type 2 D30/D69 P-ala a 2279 CNS DLCL IVDU 10 Type 2 D30 P-ala 2280 CNS DLCL IVDU 15 Type 1 D30 P-ala/P-thr 2281 CNS DLCL IVDU 33 Type 1 D30 P-ala/P-thr 2282 CNS DLCL Homosexual 20 Type 1/type 2 D30 NA 2283 CNS DLCL IVDU 18 Type 1 D30 NA 2284 CNS DLCL IVDU 30 Type 1 D30/D69 P-ala/P-thr 2285 CNS DLCL IVDU 2 Type 1 D30 P-ala 2359 CNS DLCL IVDU 43 Type 1 NA NA 2360 CNS DLCL IVDU UK Type 2 wt NA 2361 CNS DLCL IVDU 8 NA D30 NA 2363 CNS DLCL Heterosexual 34 Type 1 NA NA 2365 CNS DLCL IVDU 5 NA wt/d30 NA 2366 CNS DLCL IVDU 13 Type 1 wt NA Abbreviations: CNS, central nervous system; DLCL, diffuse large cell lymphoma; IVDU, intravenous drug user; UK, unknown; NA, not assessed; wt, wild type. a Case harboring infection by different EBNA-1 sequence of the same EBV prototype strain. b The EBNA-1 genotype in case 8877UC was represented by a recombination between P-ala and V-leu.

5 MOLECULAR PROFILE OF EBV IN AIDS-NHL 23 TABLE 2. CLINICAL AND MOLECULAR CHARACTERISTICS OF SYSTEMIC AIDS-BL CASES Case Localization Histology Risk factor CD4 1 cells/ml EBV type LMP-1 EBNA Lymph node BL IVDU UK Type 1 D30 V-leu 1246 Lymph node BL Heterosexual 472 Type 2 D30 P-ala 1249 Spleen BL Homosexual 399 Type 2 D30 V-val 1254UC Stomach BL IVDU 84 Type 1 D30 P-ala a 1900 Lymph node BL IVDU 245 Type 1 D30 V-leu 1903 Pleural effusion BL Homosexual 220 Type 1 D30 P-thr 1907 Lymph node BL Heterosexual 321 Type 1 wt P-ala 1908 Lymph node BL Heterosexual 421 Type 1 D30 P-thr 2139 Lymph node BL Homosexual 345 Type 1 wt P-ala 2582UC Lymph node BL IVDU 126 Type 1/type 2 D30 P-thr 3539UC Large intestine BL Homosexual 252 Type 1 wt P-thr 21678UC Lumbar spine BL Heterosexual 76 Type 2 D30 V-leu Abbreviations: BL, Burkitt s lymphoma; IVDU, intravenous drug user; UK, unknown; NA, not assessed; wt, wild type. a Case harboring infection by different EBNA-1 sequence of the same EBV prototype strain. in normal and reactive B cells. 21,22 The sequence analysis of the D69LMP-1 gene showed a number of point mutations, which identify at least three different viral strains (Fig. 2). In most D69LMP-1 variants detected in AIDS-NHL, the sequence is similar to the D30LMP-1 sequence of an EBV Mediterranean isolate obtained from the nasopharyngeal tumor cell line C Definition of EBV type 1 and type 2 infection in AIDS-NIH The frequency of EBV type 1 or type 2 infection was determined in 70 AIDS-NHL samples. Results are represented for each case in Tables 1 and 2 and are summarized in Table 5. Collectively, type 1 EBV was present in 51 of 70 AIDS-NHL samples (72.9%) and type 2 EBV was found to infect 15 of 70 samples (21.4%). Four cases (5.7%) displayed coresident type 1 and type 2 EBV strains in the context of the same tumor biopsy (Table 5). Tumor phenotype All AIDS-NHL samples tested expressed EBER and EBNA- 1. Expression of LMP-1 was detected in 29 of 38 AIDS-DLCL samples (76.3%), whereas all AIDS-BL samples were LMP-1 negative. In LMP-1 1 AIDS-DLCL, both the membrane surface and the cytoplasm of the neoplastic cells stained positive for LMP-1. Expression of the LMP-1 protein occurred independent of the LMP-1 genetic variant harbored by the lymphoma. As expected, 19,23 all LMP-1 1 AIDS-DLCL cases failed to express BCL-6 and expressed BCL-2. In all cases, the LMP-1 gene was found to modulate BCL-6 and BCL-2 independent of the LMP- 1 genetic variant present in the tumor clone. Correlation of EBV genotype with clinical and epidemiological features of AIDS-NHL The distribution of EBV polymorphic markers was analyzed according to the disease site (CNS vs. systemic localization of AIDS-NHL), the tumor histology (DLCL vs. BL), the peripheral blood CD4 1 cell counts, and the HIV transmission route (homosexual vs. heterosexual vs. intravenous drug user). While the frequency of EBNA-1 subtypes was found to be independent of disease site, HIV transmission route, and CD4 1 cell counts, a significant difference was found in the distribution of prototype and variant EBNA-1 strains within the histological groups of AIDS-NHL (p, 0.01) (Table 3). In particular, the variant EBNA-1 strains were restricted to AIDS-BL whereas they were consistently absent in AIDS-DLCL (p, 0.01) (Table 3). The distribution of LMP-1 variants and of the type 1 or type 2 EBV strain did not associate with any of the analyzed clinicoepidemiological data of AIDS-NHL patients. In particular, although type 2 EBV was found to be more prevalent in homosexual HIV-positive individuals (6 of 21; 28.5%) than in IVDUs (5 of 35; 14.2%), the difference was not significant. In heterosexual individuals type 2 EBV occurred in 4 of 20 cases (20%), a fraction similar to that detected in homosexual patients. Finally, statistical analysis showed that EBNA-1 subtypes detected in AIDS-NHL were casually associated with LMP-1 genotype variants and to type 1 or type 2 EBV strains. DISCUSSION In the present study, multiple polymorphic loci of the EBV genome, that is, EBNA-1, LMP-1, EBNA-2, and EBNA-3C genes, were screened in a well-characterized panel of AIDS- NHL cases associated with EBV infection of the tumor clone. TABLE 3. DISTRIBUTION OF EBNA-1 MAJOR STRAINS IN AIDS-NHL Prototype EBNA-1 Variant EBNA-1 AIDS-DLCL a 37/37 (100%) b 0/37 (n 5 37) AIDS-BL 8/12 (66.7%) 4/12 (33.3%) (n 5 12) a AIDS-DLCLs include 12 systemic cases and 25 cases primarily localized to the CNS. b p, 0.01, Fisher exact test.

6 24 FASSONE ET AL. FIG. 1. Alignments of EBNA-1 sequences in EBV-positive AIDS-NHL. Shown is the partial amino acid sequence of the C- terminal region of EBNA-1 amplified from EBV-positive AIDS-NHL. Amino acids are numbered on the basis of the B95.8 sequence. Codon changes relative to B95.8 are shown: dashes ( ) indicate wild-type sequence; asterisks (*) indicate silent mutations. EBNA-1 sequences were classified on the basis of the codon at residue 487. The different EBNA-1 subtypes and relative variants detected in AIDS-NHL are clustered and boxed. Overall, the results of the present study indicate that no specific EBV genotype is associated with the development of AIDS-DLCL or AIDS-BL. Nevertheless, the data provided herein do not rule out the possibility that different EBNA-1- or LMP-1-defined EBV variants share common pathogenic properties that may enhance the risk of lymphoma development in the HIV setting. The analysis of the polymorphic C-terminal region of the EBNA-1 gene showed a relatively high prevalence of the P-ala and P-thr subtypes, while the V-leu and V-val patterns were present in a minority of cases. One case displayed a P-ala variant that had recombined to a V-leu subtype. Events of interstrain homologous recombination during EBV coinfection and productive replication, leading to hybrid viral sequence variants, were previously reported to occur in the LMP-1 gene. 21 Overall, these results are consistent with previous work that defined the EBNA-1 pattern in B cell tumors of immunocompetent hosts as reflecting that of control individuals of the same ethnic and/or geographic derivation. 13,14,20 Nevertheless, AIDS- DLCL and AIDS-BL showed a significantly different distribution of EBNA-1 P and V variants, with AIDS-DLCL being exclusively infected by the P strain. In these cases, the presence of EBNA-1 P variants was not associated with particular EBNA-2, EBNA-3C, or LMP-1 polymorphisms, ruling out the existence of a disease-related EBV strain. The meaning of the strict association between AIDS-DLCL and EBNA-1 P variants is presently unknown. Further studies are needed to elucidate whether this is a differentiation-related phenomenon or whether the EBNA-1 V strains are associated with an extremely low risk to develop AIDS-DLCL. The presence of multiple EBNA-1 sequences in a fraction of the AIDS-NHL samples may be explained by the presence of TABLE 4. FREQUENCY OF LMP-1 VARIANTS OF EBV IN AIDS-NHL Double AIDS-NHL patients wtlmp-1 D30LMP-1 D69LMP-1 infection All patients (n 5 72) 37/72 (51.4%) 24/72 (33.3%) 7/72 (9.7%) 4/72 (5.5%) Systemic DLCL (n 5 12) 5/12 (41.6%) 4/12 (33.3%) 2/12 (16.6%) 1/12 (8.3%) PCNSL (n 5 48) 29/48 (60.4%) 11/48 (22.9%) 5/48 (10.4%) 3/48 (6.2%) BL (n 5 12) 3/12 (25.0%) 9/12 (75.0%) 0 0 Abbreviation: PCNSL, Primitive central nervous system lymphoma.

7 MOLECULAR PROFILE OF EBV IN AIDS-NHL 25 FIG. 2. Alignments of the amino acid sequence pattern identified in the C terminus of the LMP-1 gene in AIDS-NHL. For comparison, the LMP-1 patterns of EBV isolates B95.8, AG876, and C15 are also shown. Codons are numbered on the basis of the B95.8 sequence. Amino acid changes relative to B95.8 are underlined; dashes indicate sequence deletions. EBV-positive infiltrating lymphocytes and monocytes or, alternatively, by coinfection of the tumor clone with multiple EBV strains. 24 Notably, coinfection of the tumor clone with multiple EBV strains has also been reported in nasal T cell lymphomas and in some cases of B cell tumors. 20,25 Experiments involving the subcloning of the EBNA-1 C terminus revealed the occurrence of additional mutations that could not be detected by DNA direct sequencing. Because the sensitivity of DNA direct sequencing in our laboratory ranges from 10 to 20%, it is conceivable that these EBNA-1 subclones may represent a minor component of the tumor clone. Immunocompromised hosts provide an ideal environment for rapid EBV evolution and the presence of additional mutations may be ascribed to productive EBV replication, which increases the opportunity for new point mutations to arise in the context of prehexisting sequence patterns. 26 Our results indicate that the frequency of detection of EBV coresident strains varies depending on the polymorphic marker examined. In fact, combined analysis of the LMP-1, EBNA-2, and EBNA-3C genes revealed multiple EBV strains in only six AIDS-NHL samples. Conversely, the relatively higher fraction of cases with detectable intraclonal EBNA-1 heterogeneity suggests that the EBNA-1 C terminus is a highly polymorphic region capable of revealing more EBV variants than other markers usually employed for individual viral strain identification. The overall frequency of LMP-1 variants detected in our series of AIDS-NHL was similar to that found in the general population. When lymphoma histotypes were analyzed separately, however, AIDS-BL were shown to carry a high frequency of the D30LMP-1 variant (75%), substantially higher than that observed in PBMCs of HIV-seropositive Italian individuals (43.2%). 27 The presence of D30LMP-1 in AIDS-BL was not associated with a particular EBV subtype or EBNA-1 variant. Although ruling out the involvement of specific EBV strains in the development of AIDS-BL, these findings are in keeping with published data reporting a high prevalence of LMP-1 deletions in AIDS-associated lymphoid malignancies. 17,28 Further studies of larger series of cases are, however, required to verify whether particular AIDS-NHL histotypes are associated with LMP-1-deleted variants. Although the D69LMP-1 variant has been shown to have pathogenic relevance, more recent studies have described a relatively high frequency of this variant in HIV-negative lymphoproliferations, both malignant and benign, from Italian patients, suggesting that the D69LMP-1 frequency in AIDS-NHL reflects the incidence of the variant in the healthy population. 29 In AIDS-DLCL cases, with assessed morphophenotypic pattern, LMP-1 was found to modulate the BCL- 2/BCL-6 expression status of the tumor independent of the LMP-1 genotype. In conclusion, our analysis of several EBV polymorphic regions indicates that, when considering AIDS-NHL in general, the distribution of EBV genotypes is similar to the viral representation found in control individuals of both immunocompetent or immunocompromised populations. However, the segregation of EBNA-1 major strains among AIDS-DLCL and AIDS-BL samples showed a significant difference. The exclusive infection by EBNA-1 prototype strains of a relatively large cohort of AIDS-DLCL patients, coupled with the consistent absence of EBNA-1 variant strains in the same tumors, may suggest a correlation between the EBNA-1 major strain and the TABLE 5. FREQUENCY OF EBV TYPE 1/TYPE 2 VARIANTS IN AIDS-NHL AIDS-NHL patients Type 1 Type 2 Type 1/Type 2 All patients (n 5 70) 51/70 (72.9%) 15/70 (21.4%) 4/70 (5.7) Systemic DLCL (n 5 12) 9/12 (75.0%) 2/12 (16.7%) 1/12 (8.3) PCNSL (n 5 46) 34/46 (73.9%) 10/46 (21.8%) 2/46 (4.3%) BL (n 5 12) 8/12 (66.7%) 3/12 (25.0%) 1/12 (8.3%) Abbreviation: PCNSL, Primitive central nervous system lymphoma.

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