Immune Activation and Microbial Translocation as Prognostic Biomarkers for AIDS-Related Non-Hodgkin Lymphoma in the AMC-034 Study.

PURPOSE: AIDS-related non-Hodgkin lymphoma (ARL) is the most common cancer in HIV-infected individuals in the United States and other countries in which HIV-positive persons have access to effective combination antiretroviral therapy (cART). Our prior work showed that pretreatment/postdiagnosis plasma levels of some cytokines, such as IL6, IL10, and CXCL13, have the potential to serve as indicators of clinical response to treatment and survival in ARL. The aims of this study were to identify novel prognostic biomarkers for response to treatment and/or survival in persons with ARL, including biomarkers of microbial translocation and inflammation. EXPERIMENTAL DESIGN: We quantified plasma levels of several biomarkers (sCD14, LBP, FABP2, EndoCab IgM, IL18, CCL2/MCP-1, sCD163, IP-10/CXCL10, TARC/CCL17, TNFα, BAFF/BLyS, sTNFRII, sCD44, and sIL2Rα/sCD25) by multiplexed immunometric assays (Luminex) or ELISA in plasma specimens obtained from ARL patients enrolled in the AMC-034 trial, which compared infusional combination chemotherapy (EPOCH: etoposide, vincristine, doxorubicin, cyclophosphamide, and prednisone) with concurrent or sequential rituximab. Plasma was collected prior to the initiation of therapy (n = 57) and after treatment initiation (n = 55). RESULTS: We found that several biomarkers decreased significantly after treatment, including TNFα, sCD25, LBP, and TARC (CCL17). Moreover, pretreatment plasma levels of BAFF, sCD14, sTNFRII, and CCL2/MCP-1 were univariately associated with overall survival, and pretreatment levels of BAFF, sTNFRII, and CCL2/MCP-1 were also associated with progression-free survival. CONCLUSIONS: Our results suggest that patients with ARL who responded to therapy had lower pretreatment levels of inflammation and microbial translocation as compared with those who did not respond optimally.

Primary pulmonary mucosa-associated lymphoid tissue lymphoma with associated fungal ball in a patient with human immunodeficiency virus infection.

We describe a case of primary mucosa-associated lymphoid tissue lymphoma of the lung in a 44-year-old man with human immunodeficiency virus. Low-grade pulmonary lymphomas in human immunodeficiency virus-positive patients are rare and are described most commonly in pediatric patients. The gross, histologic, and molecular features of this unusual case are described.

High expression of latent membrane protein 1 of Epstein-Barr virus and BCL-2 oncoprotein in acquired immunodeficiency syndrome-related primary brain lymphomas.

Nearly all primary brain lymphomas in acquired immunodeficiency syndrome (AIDS) patients are associated withEpstein-Barr virus (EBV). The role of EBV in lymphomagenesis is not totally elucidated. One possible mechanism is the overexpression of the BCL-2 oncoprotein, because the latent membrane protein 1 (LMP1) has been reported to transactivate the bcl-2 gene in vitro. To study the interrelationship beetween LMP1 and BCL-2 in vivo, we have analyzed and compared their expression in 11 AIDS-related primary brain lymphomas and 57 AIDS-related systemic lymphomas by immunoperoxidase technique on frozen sections. In AIDS-related primary brain lymphoma, LMP1 and BCL-2 were expressed in all cases but 1. All positive cases exhibited morphologic immunoblastic features. In contrast, the only negative case was histologically close to Burkitt's lymphoma. In systemic lymphomas, LMP1 was expressed in 21 cases, whereas BCL-2 was positive in only 3 cases, all of which were extranodal. These results indicate that, in addition to the histologic type, the role of EBV genes and BCL-2 expression in lymphomatous cells differ as a function of their localization. In AIDS-related primary brain lymphomas, this correlation between LMP1 and BCL-2 overexpression may have a major implication in lymphomagenesis.

Epstein-Barr virus (EBV) replicative gene expression in tumour cells of AIDS-related non-Hodgkin's lymphoma in relation to CD4 cell number and antibody titres to EBV.

OBJECTIVE: To determine whether activation of Epstein-Barr virus (EBV) replication in tumour cells of AIDS-related non-Hodgkin's lymphoma (ARNHL) is correlated with CD4+ cell counts and influences antibody response to EBV [anti-Z Epstein-Barr replicative activator (ZEBRA), anti-early antigen (EA), anti-viral capsid antigen (VCA)]. DESIGN: Retrospective study based on immunohistochemistry and in situ hybridization to detect EBV replicative gene products in tissue samples from patients affected by ARNHL and correlation with CD4+ cell counts and results of EBV serology (including anti-ZEBRA activity) in sera from the same patients. METHODS: Seventeen out of 22 cases of ARNHL were selected for the presence of EBV [Epstein-Barr early region (EBER) RNA-positive]. Immunohistochemistry was performed with anti-ZEBRA, anti-EA-restricted, anti-VCA antibodies and in situ hybridization with BHLF1/NotI oligoprobes on tumour samples. Results were statistically correlated with those of CD4+ cell counts (17 out of 17) and with anti-EBV antibody titres (13 out of 17) assessed using standard immunofluorescence method and enzyme-linked immunosorbent assay procedure using recombinant ZEBRA protein and synthetic peptides as antigens. RESULTS: BZLF1 (ZEBRA) or early gene products (EA-R and EA-D/BHLF1/NotI) were detected in a small proportion (< 0.01-5%) of tumour cells in eight of these 17 cases by immunohistochemistry and in situ hybridization. Demonstration of replicative gene expression did not correlate with either low CD4+ cell counts (P > 0.05) or anti-EBV antibody titres (P > 0.05). Anti-ZEBRA activity was not significantly increased in patients affected with ARNHL, the cells of which expressed replicative gene products (P > 0.05). CONCLUSION: The degree of immunodeficiency does not clearly enhance replicative gene expression in tumour cells of ARNHL. EBV serology, including anti-ZEBRA activity, is not a reliable tool for predicting the occurrence of such proliferations.

Identification of a common clonal human immunodeficiency virus integration site in human immunodeficiency virus-associated lymphomas.

Infection with human immunodeficiency virus type 1 (HIV-1) is associated with a high incidence of lymphoma. Typically, the lymphomas are B-cell in origin, and although they occur in the setting of HIV-1 infection, historical studies have found no evidence for the presence of HIV-1 within the transformed B-cells. We describe a new class of large cell lymphoma wherein HIV p24 expression within the tumor specimens was found to be extremely high. In the first case, HIV was expressed in the tumor-associated transformed T-cells. In three other cases, HIV was found to be highly expressed in tumor-associated macrophages. These tumors exhibited a mixed immunophenotype histologically. Analysis by inverse polymerase chain reaction, using HIV long terminal repeat primers, demonstrated monoclonal HIV integration sites for all four tumors. Direct sequencing of the T-cell lymphoma inverse polymerase chain reaction products identified the HIV integration site within the fur gene, just upstream from the c-fes/fps protooncogene. Using segments of the fur gene as a probe, the other three monoclonal integration sites mapped to the same region. Although the integration and up-regulation of c-fes/fps was localized to the tumor cells within the T-cell lymphoma, the cells containing the monoclonal HIV in the other mixed immunophenotype lymphomas are currently unknown. These observations suggest that HIV may contribute directly to lymphomagenesis and identify a common site of HIV integration within a subset of acquired immunodeficiency syndrome lymphoma.

Non-Hodgkin's lymphoma in four children infected with the human immunodeficiency virus. Association with Epstein-Barr Virus and treatment.

BACKGROUND: Reports on lymphoid malignancy and its treatment in children infected with human immunodeficiency virus (HIV) are limited. METHODS: Antibodies to Epstein-Barr virus (EBV) were detected by indirect immunofluorescence. DNA was extracted from peripheral blood lymphocytes or biopsy specimens. Polymerase chain reaction was run using primers for EBV. Reaction products underwent Southern blot analysis to confirm EBV specificity. Tumor clonality was assessed by immunohistochemistry and by Southern blot analysis of immunoglobulin heavy-chain and T-cell receptor beta-gene rearrangement. RESULTS: Within 1 year, non-Hodgkin's lymphoma (NHL) was diagnosed in four children infected with HIV. All four were EBV-seropositive and had detectable EBV DNA in peripheral blood lymphocytes. The EBV-linked disorders lymphoid interstitial pneumonia and recurrent parotid enlargement preceded NHL in three and two of the children, respectively. In all four patients, NHL involved at one time the central nervous system (CNS). All three tested NHL tissues were positive for EBV DNA: A 12-week course of chemotherapy given to two children resulted in rapid tumor regression. One of these children experienced meningeal relapse and died 16 months after diagnosis. The other child, who in addition received local irradiation of the affected eye and who underwent surgical removal of the involved ovaries, has been in continuous remission for 20 months. CONCLUSIONS: EBV-associated NHL may be seen more frequently in pediatric patients with HIV. Treatment protocols taking into account NHL propensity for the CNS in this age group need to be developed.

Expression of Epstein-Barr virus proteins in primary CNS lymphoma in AIDS patients.

We examined the expression of the Epstein-Barr virus (EBV)-induced proteins (LMP [latent membrane protein], EBNA-2, and CD23) and a lytic protein, viral capsid antigen (VCA), in five acquired immune deficiency syndrome (AIDS)-related primary CNS lymphomas (PCNSLs). We compared that expression with the expression of the same proteins in PCNSL from six immunocompetent patients and severe combined immune deficiency (SCID) mouse brains injected with EBV-infected lymphoblastoid cell lines (LCLs). Brain biopsy tissue from an AIDS patient with progressive multifocal leukoencephalopathy (PML) and a normal brain was also studied. Three of the AIDS PCNSLs expressed both human immunoglobulin kappa and lambda light chains and two expressed lambda light chain only. All non-AIDS-related PCNSLs expressed a single light-chain isotype. All five AIDS-related PCNSLs expressed LMP-1 (> 40%), EBNA-2 (> 60%), and VCA (1 to 5%) of tumor cells. These proteins were similarly expressed in the SCID/human chimeras. None of the PCNSLs from immunocompetent subjects, the normal brain, or the brain of the patient with PML expressed these proteins. PCNSL in AIDS patients bears greater similarity to EBV-infected LCLs than to PCNSL from immunocompetent patients.

CD30 (Ki-1) positive anaplastic large cell lymphomas in individuals infected with the human immunodeficiency virus.

BACKGROUND: CD30 (Ki-1) positive anaplastic large cell lymphoma (ALCL) has been only rarely described in HIV-positive patients. METHODS: The clinicopathologic features of eight ALCLs occurring in four AIDS and four HIV-positive patients were investigated. The phenotype of each neoplasm was determined by immunohistochemical methods. In three cases fresh tissue was available for molecular analysis. RESULTS: The ALCLs are a clinically heterogeneous group of T (4), B (1) and indeterminate (3) cell malignant lymphomas which presented in the skin (4), liver (1), lung (1), nasal cavity (1; also with bone marrow involvement) and peritoneal fluid (1). While most of the patients had aggressive disease, dying in a median of three months, two patients had either localized or regressing skin lesions. Molecular studies showed that two ALCLs, one of B cell and one of indeterminate cell lineage, contained clonal Epstein-Barr virus sequences. None of the ALCLs examined contained evidence of HTLV-1 or HIV integration nor did they exhibit c-myc or bcl-2 proto-oncogene rearrangements. No mutations or deletions of the p53 tumor suppressor gene were identified in the three cases studied. CONCLUSIONS: HIV-related ALCL represents a clinically heterogeneous group of T cell, B cell and null cell malignant lymphomas, distinct from the previously described categories of AIDS-associated NHL, that may expand the spectrum of lymphoid neoplasms associated with HIV-infection. Identification and investigation of other cases of HIV-associated ALCL is important to determine the nature of the relationship between HIV infection and the development of ALCL.

In vitro establishment of AIDS-related lymphoma cell lines: phenotypic characterization, oncogene and tumor suppressor gene lesions, and heterogeneity in Epstein-Barr virus infection.

Lymphoma represents a major source of morbidity and mortality among AIDS patients. AIDS-associated non-Hodgkin lymphomas (AIDS-NHL) are almost invariably B-cell derived, are classified as high or intermediate grade lymphomas, and display three main histologic types: namely, small non-cleaved cell lymphoma (SNCCL), large cell immunoblastic plasmacytoid lymphoma (LC-IBPL), and large cell lymphoma (LCL). Here we report the in vitro establishment of three new AIDS-NHL cell lines (termed HBL-1, HBL-2, and HBL-3) derived from three AIDS-SNCCL patients differing in primary tumor sites and risk factors for HIV infection. The derivation of the cell lines from the original tumor clones was established by immunophenotypic and molecular genetic analysis. These cell lines display clonal immunoglobulin gene rearrangement, express surface immunoglobulin and B-cell restricted markers, and exhibit a phenotype consistent with SNCCL. Monoclonal Epstein-Barr virus infection was found in only one of the cell lines (HBL-1). Cytogenetic analysis demonstrated the presence of a chromosomal translocation involving the c-myc proto-oncogene and an immunoglobulin locus in all three cell lines. The pattern of genetic lesions detected in HBL-1, HBL-2, and HBL-3 reflects that found in primary AIDS-SNCCL and includes activation of the c-myc oncogene as well as inactivation of the p53 tumor suppressor gene. These cell lines should prove useful in studies of the biological, immunological, and viral factors involved in AIDS-associated lymphomagenesis.

Human immunodeficiency virus-associated systemic lymphomas may be subdivided into two main groups according to Epstein-Barr viral latent gene expression.

PURPOSE: We report a pathologic characterization of human immunodeficiency virus (HIV)-associated systemic lymphomas, including the association of Epstein-Barr virus (EBV) in different categories. PATIENTS AND METHODS: Eighty-seven HIV-associated non-Hodgkin's lymphoma (NHL) were classified according to classic NHL classification and a recent description of morphologic variants of high-grade B-cell NHL. Seventy-one cases were immunophenotypically-genotypically characterized, whereas, in 49 representative cases, the association of EBV was assessed by nonisotopic in situ hybridization (ISH) and the immunohistochemical demonstration of latent membrane protein-1 (LMP-1). In addition, 14 Hodgkin's disease (HD) cases, occurring in patients with HIV infection, were investigated for the frequency of LMP-1 expression. RESULTS: Most lymphomas were of B-cell derivation and showed a blastic cell morphology, with (1) small noncleaved cells (SNCCs; 36 cases), (2) large noncleaved cells (10 cases), and (3) immunoblasts, usually polymorphic (12 cases). Moreover, 12 cases were classified as anaplastic large-cell (ALC) Ki-1-positive (Ki-1+) lymphoma. Combined ISH studies (for viral DNA and EBV RNA [EBER]) and immunohistologic demonstration of LMP-1 suggested that there were differences in viral latent gene expression between ALC Ki-1+ or immunoblastic lymphomas (usually EBV+, LMP-1+), and EBV-infected cells of SNCC lymphomas, which did not show LMP-1 expression. A high proportion (10 of 14) of LMP-1+ HD cases was found. CONCLUSION: Differences in EBV association and LMP-1 expression were found between a major group of HIV-associated systemic NHL with blastic cell morphology, including SNCC lymphoma and its variants, and anaplastic cell lymphomas. A proportion of immunoblastic (polymorphic) lymphomas was different in viral latent gene expression from other blastic cell systemic lymphomas. It is concluded that only a group of these lymphomas (most ALC Ki-1+ and HD cases, along with a nonnegligible fraction of immunoblastic lymphomas) seems to be linked etiopathologically to EBV.

Biologic aspects of AIDS-associated non-Hodgkin's lymphoma.

Acquired immunodeficiency syndrome-associated non-Hodgkin's lymphomas represent a significant and formidable clinical problem. They also represent an important biologic model for investigating the development and progression of high-grade malignant lymphomas and for studying lymphomas that develop in the setting of immune deficiency. A vast majority of non-Hodgkin's lymphomas exhibit clonal immunoglobulin gene rearrangements and, hence, are B-cell neoplasms. Most express B-cell phenotypes, but a minority, predominantly body cavity-based tumors, express indeterminate phenotypes. AIDS-associated non-Hodgkin's lymphomas do not contain HIV. However, approximately 40% of systemic non-Hodgkin's lymphomas, predominantly those with immunoblastic plasmacytoid morphology, and essentially 100% of primary central nervous system AIDS-associated non-Hodgkin's lymphomas contain Epstein-Barr virus. The c-myc protooncogene is rearranged in approximately 80% of systemic cases, predominantly in those with Burkitt's and Burkitt's-like morphology. Point mutations of the ras gene are detectable in approximately 15% of systemic cases. The p53 tumor-suppressor gene is mutated in approximately two thirds of systemic AIDS-associated Burkitt's and Burkitt's-like non-Hodgkin's lymphomas. The retinoblastoma tumor-suppressor gene does not appear to be mutated or deleted in AIDS-associated non-Hodgkin's lymphomas. In summary, various genetic lesions occur in AIDS-associated non-Hodgkin's lymphomas, which appear to vary according to the anatomic site of disease (systemic vs central nervous system vs body cavity) and the histopathology (Burkitt's vs immunoblastic vs large cell). Further active investigation is necessary to determine the role of these and possibly other genetic lesions in AIDS lymphomagenesis.

Human herpesvirus-6 and Epstein-Barr virus genome in primary cerebral lymphomas.

Using dot blotting, we found Epstein-Barr virus genome in three AIDS-related primary cerebral lymphomas (PCLs), but in none of 39 sporadic PCLs. Human herpesvirus-6 sequences were present only in one sporadic PCL, as revealed by polymerase chain reaction and Southern analysis. We conclude that these viruses do not appear to play a major role in PCL pathogenesis in immunocompetent subjects.

Incidence of Epstein-Barr virus in AIDS-related lymphoma specimens.

We used the polymerase chain reaction, in situ hybridization, and immunohistochemical stains against latent membrane protein, CD23, and Epstein-Barr viral nuclear antigens 1 and 2 to identify Epstein-Barr virus (EBV) in fixed and unfixed (cryopreserved) AIDS-related lymphoma (ARL) specimens. The study included 17 cases of large-cell (immunoblastic) lymphoma, 11 cases of small non-cleaved cell lymphoma, and two cases of Hodgkin's disease. The EBV DNA was more frequently detected by polymerase chain reaction in cryopreserved specimens (94%) than in fixed specimens (17%). Significantly, the immunohistochemical and in situ hybridization studies detected evidence of EBV in only a small (< 10%) subset of the cells in 27 of 30 ARL specimens. We conclude that tissue fixation reduced the ability to detect EBV in ARL by polymerase chain reaction and that EBV was detectable in only a minority of cells in most ARL tissues.

Variable morphology of human immunodeficiency virus-associated lymphomas with c-myc rearrangements. The French Study Group of Pathology for Human Immunodeficiency Virus-Associated Tumors, I.

Burkitt lymphoma (BL) and immunoblastic lymphoma (IL) are the most frequent lymphoid tumors encountered in human immunodeficiency virus (HIV)-infected patients. Tumors with a morphology intermediate between BL and IL, and the existence of Burkitt's type translocations in some IL cases makes the classification of these tumors sometimes unclear. We have studied 14 cases of BL and IL in HIV-seropositive individuals with regard to clonality, Epstein-Barr virus (EBV) association, and the presence of c-myc rearrangement. Of seven tumors with morphology of BL, all were monoclonal, six showed a c-myc rearrangement and four were associated with EBV. Five tumors with morphology of IL were associated with EBV and devoid of c-myc rearrangement. Three were polyclonal representing EBV-driven lymphoproliferations similar to those observed in transplant recipients. Two tumors, one with a morphology of IL and the other intermediate between IL and BL were monoclonal, associated with EBV, and harbored a c-myc rearrangement. We propose that these last two tumors represent cases of BL that have adopted an immunoblastic morphotype in the context of acquired immunodeficiency syndrome (AIDS), reflecting the morphologic evolution of Burkitt lymphoma cells observed in culture.

In situ demonstration of Epstein-Barr virus small RNAs (EBER 1) in acquired immunodeficiency syndrome-related lymphomas: correlation with tumor morphology and primary site.

Some acquired immunodeficiency syndrome (AIDS)-related lymphomas (ARLs) are infected with Epstein-Barr virus (EBV), although the frequency and importance of this association is disputed. Using paraffin section RNA in situ hybridization (ISH) with digoxigenin-labeled riboprobes, we screened 16 central nervous system (CNS) non-Hodgkin's lymphomas (NHLs), 101 systemic NHLs, and 11 Hodgkin's disease cases arising in human immunodeficiency virus-seropositive individuals for EBV-encoded small RNA (EBER 1) expression, an EBV gene product transcribed in abundance during latent infection. Tumor cells contained EBV in 85 of 128 ARLs (66%), but infection rates differed with lymphoma type. EBER 1 was expressed in tumor cells in 11 of 11 Hodgkin's disease cases (100%), 15 of 16 CNS NHLs (94%), and 46 of 60 systemic immunoblast-rich/large-cell lymphomas (77%), but in only 12 of 35 Burkitt-type (small noncleaved cell) (34%) and 1 of 6 monomorphic centroblastic (diffuse large noncleaved cell) (17%) lymphomas. In most EBV-positive ARLs, all recognizable viable tumor cells expressed EBER 1. We conclude that (1) EBV infects tumor cells in all AIDS-related Hodgkin's disease cases, in virtually all primary CNS ARLs, and in most systemic immunoblast-rich/large-cell ARLs; (2) only a minority of Burkitt-type and monomorphic centroblastic lymphomas are associated with EBV; and (3) EBER-ISH is ideal for the histopathologic detection of latent EBV in routine tissue specimens.

T-cell lymphoma associated with periodontal disease and HIV infection. A case report.

A case is described of gingival non-Hodgkins lymphoma presenting in a site previously diagnosed as HIV-periodontitis. The lymphoma presented along with other signs of HIV infection and AIDS, which taken together were compatible with increased immunosuppression and reactivation of Epstein-Barr virus. The implications of these findings are discussed, and it is suggested that areas of gingiva which show rapid localised recession, associated with HIV seropositivity, should be monitored closely and considered for biopsy if abnormal signs are detected.

The role of Epstein-Barr virus subtypes in human immunodeficiency virus-associated lymphoma.

High grade B cell non-Hodgkin's lymphoma (NHL) is an increasingly important problem in individuals infected with the human immunodeficiency virus (HIV). Fifty percent of these tumours harbour the Epstein-Barr virus (EBV) and there is an equal frequency of EBV A and B-subtypes in the tumours. This contrasts with the recent report that only A-type EBV is associated with Hodgkin's disease. Such studies are challenging the traditional models of EBV-associated lymphomagenesis and showing the way for further studies in this field. This article reviews the studies of EBV subtypes in HIV-associated NHL and uses this new knowledge to discuss the role of EBV in lymphomagenesis.

Epstein-Barr virus-associated non-Hodgkin's lymphoma in patients infected with the human immunodeficiency virus.

Lymphoproliferations associated with Epstein-Barr virus (EBV) commonly arise in settings of immune dysfunction, including human immunodeficiency virus (HIV) infection. In this study, EBV was associated with 39 of 59 (66%) HIV-related systemic lymphomas. Unlike the lymphoproliferations that arise in the setting of transplantation, the HIV-related lymphomas were monoclonal, as evaluated by Ig heavy chain rearrangements and EBV termini analysis, and associated (40%) with c-MYC rearrangements. Furthermore, analysis of multiple lymphoma tissues from one autopsy showed evidence that a single lymphoma clone was responsible for dissemination. The latent EBV nuclear antigen (EBNA-1) transcripts detected in the HIV-related lymphomas were characteristic of the pattern found in Burkitt lymphoma (g1 EBNA1) and not in transplant-related lymphoproliferations. However, unlike Burkitt lymphoma, EBV latent membrane-associated protein (LMP) transcripts were also detected, thereby constituting an EBV expression pattern (g1 EBNA1+, LMP+) not previously observed in B-cell lymphomas. These findings demonstrate a high frequency of EBV-associated lymphomas in the setting of HIV infection that are distinct from the lymphoproliferations that arise during iatrogenic transplant-associated immuno-suppression or in the general population. However, it is also apparent that HIV-related lymphomas are biologically heterogeneous, which may reflect the multiple mechanisms or steps necessary for eventual malignant transformation.

Replicative viral DNA in Epstein-Barr virus associated Burkitt's lymphoma biopsies.

Epstein-Barr virus (EBV) is linked to a spectrum of human diseases including epithelial and lymphoid malignancies in which it exists predominantly in a latent state. EBV is capable of establishing replicative infection at oropharyngeal and genital sites. Replicative EBV infection also occurs in oral hairy leukoplakia, in EBV associated lymphoproliferative disorders, and to a minor degree in nasopharyngeal carcinomas. Recent evidence also suggests that EBV replication, also, may be associated with AIDS related lymphomas and Hodgkin's disease. However it is widely believed that virus in circulating B-lymphocytes and in B-cell malignancies is stringently latent. We now show that by Southern blot analysis we can detect replicative forms of virion DNA in 14.5% (8 of 55) of EBV-positive Burkitt's lymphoma biopsies. This may be the explanation for the elevation of the titres of lytic cycle EBV antigens that is associated with presentation and relapse of EBV associated Burkitt's lymphoma.