From www.bloodjournal.org by guest on January 12, 2015. For personal use only. 292 CORRESPONDENCE PATHOGENETIC ROLE OF EPSTEIN-BARR VIRUS IN MALIGNANT LYMPHOMAS THAT DEVELOP IN IMMUNOCOMPROMISED PATIENTS To the Editor: The association of Epstein-Barr virus (EBV) with lymphoid neoplasms has become a current topic in hematopathology, especially those that develop in immunodepressed patients, and several reports have recently suggested a pathogenetic role of this virus in these malignancies. However, the majority of these studies are addressed to identify the virus within the tumor cells but not to investigate its effects. Therefore, the putative etiopathogenetic role of EBV remains speculative. Neri et al, in a remarkable work published in Blood, propose a causative role of EBV in endemic Burkitt’s lymphoma as well as in some cases of the sporadic variant and in B-cell lymphomas associated with acquired immunodeficiency syndrome (AIDS). The investigators base their assumption on their finding that these B-cell malignancies contain multiple copies of monomorphic EBV genomes, assessed by Southern blot hybridization of the tumor samples with a DNA probe specific for the fused termini of the EBV genome: thus concluding that EBV infection occurred before the clonal expansion in these tumors. However, the fact that EBV entered the malignant or premalignant cells before its clonal expansion, although necessary, is not sufficient to discriminate between a coincidental event and a contributory role of EBV in these lymphomas. This phenomenon only confirms that malignant lymphomas are clonal in origin, in the same way as Ig gene rearrangement or K/ A light chain restricted expression do. In our opinion, several points should be tested to support EBV pathogenetic contribution. First of all, it would be indispensable to check that all tumor cells are actually infected by the virus. In this regard, in situ hybridization, performed in parallel to Southern blot, can verify whether all or the vast majority of the cells carry EBV DNA, otherwise, EBV could have infected a single cell of an already expanded population, either at an early stage of the lymphomagenesis or during a distant invasion, and EBV DNA would be transmitted to the clone generated by this particular cell but not to the whole neoplastic population. Southern blot hybridization alone, a sensitive technique as it is, would only show the presence of a population of malignant cells containing a unique form of fused EBV termini. In addition, the in vitro cultivation of some of these tumors, followed by the obtention of several cloned ’ lines by limiting dilution, would also help to confirm the presence of the virus in all tumor cells and would also open a new field of investigation regarding the expression and effects of the virus. Alternatively, because of the high rate of EBV chronic carriers among general population, the neoplastic transformation of an EBV latently infected cell may occur without implying a significant contribution of the virus in the neoplasm. Therefore, it would be interesting to prove, by in situ hybridization or polymerase chain reaction of uninvolved lymphoid tissue, that other nonmalignant cells of the same individual do not harbor EBV DNA. Although the presence of non-neoplasic EBV-infected lymphocytes would not preclude completely an oncogenetic effect of the virus in the tumor cells, the opposite finding would strongly support it. Other useful assay could be the identification of viral RNA in the tumor, either by in situ hybridization or Northern blot. The presence of EBV RNA would directly imply viral expression, which presumably is essential to assign an oncogenetic function to EBV. In this sense, the analysis of viral proteins and B-cell activation markers in the tumor cells would also evidence the intensity and type of viral expression. Unfortunately, the investigators do not mention which proteins were expressed by each subtype of the malignant lymphomas analyzed. At this point, it is important to emphasize that, according to the kind of proteins identified thus far in EBV-related lymphomas, the putative etiologic effect of the virus must differ widely in AIDSassociated malignancies from Burkitt’s lymphoma. Thus, EBV nuclear protein 2 and latent membrane protein, plus some B-cell activation factors, are the usual markers of the former? while they are absent in Burkitt’s cells, in which neoplastic transformation depends on the chromosomal translocation and c-myc oncogene expression in 100% of the cases4 Because EBV is associated with only 10% to 20% of sporadic Burkitt’s lymphoma, and according to the results of the mentioned report,’ the infection can take place before or after c-myc activation, it seems that the important fact in the development of this lymphoma is the chromosomal abnormality, being less relevant whether EBV entered the cell before or after its occurrence. As things stand, a solid etiologic role to EBV can only be assessed in the African variant in Burkitt’s lymphoma, although in this case, epidemiologic data provide much stronger evidence than molecular genetics. On the other hand, regarding EBV-associated lymphomas of From www.bloodjournal.org by guest on January 12, 2015. For personal use only. CORRESPONDENCE 293 immunocompromisedpatients, it has to be considered that some of them are oligoclonal or polyclonal in n a t ~ r e .This ~ , ~ fact suggests that the oncogeneticpotential of EBV relies merely on its ability as a lymphoid activator, and that this mechanism of action is similar regardless of the monoclonality or polyclonality of the resultant population. Obviously, in polyclonal lymphoproliferationscontaining EBV DNA, it would be impossible to distinguish a coincidental infection from an active influence of EBV on the basis of genomic termini homogeneity. Therefore, unless further molecular studies are performed, there is not strong evidence that EBV has a direct pathogenetic effect on lymphomas (with the noticeable exception of endemic Burkitt’s type) other than the well-known lymphoid activation which, rise in immunodepressed patients, can give rise to uncontrolled proliferations either monoclonal or polyclonal. PILAR LARDELLI ISABEL ANTON RAMON CISTERNA Department of Microbiology and Immunology School of Medicine University of the Basque County Bilbao, Spain RAIMUNDO GARCIA DEL MORAL Department of Pathology School of Medicine of Granada Granada, Spain REFERENCES 1. Neri A, Barriga F, Inghirami G, Knowles D, Neequaye J, Magrath I, Dalla-Favera R: Epstein-Barr virus infection precedes clonal expansion in Burkitt’s and acquired immunideficiency syndrome-associatedlymphoma. Blood 77:1092,1991 2. Raab-Traub N, Flynn K: The structure of the termini of the Epstein-Barr virus as a marker of clonal cellular proliferation. Cell 42883,1986 3. Young L, Alfieri C, Hennessy K, Evans H, OHara C, Anderson KC, Ritz J, Shapiro RS, Rickinson A, Kieff E, Cohen JI: Expression of Epstein-Barr virus transformation-associatedgenes in tissues of patients with EBV lymphoproliferativedisease. N Engl J Med 321:1080,1989 4. Dalla-Favera R, Neri A, Cesarman E, Lombardi L: Advances on the molecular pathogenesis of Brukitt’s lymphoma, in Gale RP, ~and Lymphoma. ~ k Golde DW (eds): R~~~~~Advances in ~ New York, NY, Liss, 1987, p 165 5. Katz BZ, Raab-Traub N, Miller G Latent and replicating forms of Epstein-Barr virus DNA in lymphomas and lymphoproliferative disease. J Infect Dis 160589,1989 RESPONSE Our data published in Blood have stimulated Lardelli et a1 to raise a number of critical issues on the role of Epstein-Barr virus (EBV) in the pathogenesisof acquired immunodeficiencysyndromeassociated non-Hodgkin lymphoma (AIDS-NHL). For sake of clarity, we would identify the following as the main issues. (1) Is EBV-terminiclonality sufficient to discriminatea coincidental versus pathogenetic role of EBV? We fully agree with Lardelli et a1 that the answer is no and that our data can only suggest a pathogenetic role (see Abstract). However, the goal of our report was to investigate whether clonality (and, therefore, infection before clonal expansion) was present in AIDS-NHL as a feature necessary for a pathogenetic role for EBV in these malignancies.In other terms, if we had found polyclonal EBV forms we would have excluded a pathogenetic role for EBV. We do not agree with the notion that EBV clonality “only confirmsthat malignant lymphoma are clonal in origin in the same way as immunoglobulin gene rearrangement.. .”. In fact, if EBV infection had occurred after clonal expansion and had no significant biologic effect, one would find monoclonality of Ig gene rearrangements contrasting with polyclonality of EBV-termini. (2) Southern blot analysis cannot demonstrate the presence of EBV in all tumor cells. We agree with this point and, in fact, we had analyzed all of our tumor samples for EBV protein expression by immunoistochemistry (see first sentence of Results, Materials and Methods, and ref 4). (3) “c-myc translocation is the real important fact” in lymphoma development because this lesion is present in 100% of cases whereas EBV infection is present only in 30%. In general, there is no real rationale supporting the assumption that lesions present in only a fraction of cases of a given tumor type may be relatively less important. In fact, tumorigenesis is a multistep process that requires multiple lesions and, in the case of Burkitt’s lymphoma and AIDS-NHL, EBV infection, c-myc activation, p53 inactivation, and several additional specific lesions are found associated in different combinations (submitted). It is difficult, and perhaps conceptually disputable, to define which lesion is the important one in each case. In summary, once some important misunderstandings are clarified, we agree with our colleagues that substantial additional work must be performed before a pathogenetic role of EBV in these tumors can be established or excluded. RICCARDO DALLA-FAVERA Division of Oncology Department of Pathology Columbia University New York, NY ~ From www.bloodjournal.org by guest on January 12, 2015. For personal use only. 1992 80: 292-293 Pathogenetic role of Epstein-Barr virus in malignant lymphomas that develop in immunocompromised patients [letter; comment] P Lardelli, I Anton, R Cisterna and RG Del Moral Updated information and services can be found at: http://www.bloodjournal.org/content/80/1/292.citation.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. 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