[CANCER RESEARCH 48, 2919-2922, May 15, 1988] No Involvement of Bovine Leukemia Virus in Childhood Acute Lymphoblastic Leukemia and Non-Hodgkin's Lymphoma1 Alan P. Bender,2 Leslie L. Robison,3 S. V. S. Kashmiri,4 Kenneth L. McClain,5 William G. Woods, W. Anthony Smithson, Ruth Heyn, Jonathan Finlay," Leonard M. Schuman, Colleen Renier, and Robert Gibson Minnesota Department of Health, Minneapolis, Minnesota [A. P. B.J; Hematology-Oncology Division, Department of Pediatrics, University of Minnesota, Minneapolis, Minnesota [L. L. R., K. L. M., W. G. W.]; Section of Viral Oncology, university of Pennsylvania, Kennen Sauare, Pennsylvania fS. V. S. K.f; Department of Pediatrics, Mayo Clinic, Rochester, Minnesota [W. A. S.]; Mott Children 's Hospital, Ann Arbor, Michigan [R. H.J; Wisconsin Medical School, University of Wisconsin, Madison, Wisconsin [J. FJ; and Division of Epidemiology, School of Public Health, University of Minnesota, Minneapolis, Minnesota [L. M. S., C. R., R. GJ ABSTRACT Bovine leukemia virus (BLV) is the causative agent of enzootic bovine lymphosarcoma. Much speculation continues to be directed at the role of BLV in human leukemia. To test this hypothesis rigorously, a case- control study of childhood acute lymphoblastic leukemia and non-Hodg- kin's lymphoma was conducted between December 1983 and February 1986. Cases (<16 years at diagnosis) derived from patients diagnosed at the primary institutions and affiliated hospitals were matched (age, sex, and race) with regional population controls. DNA samples from bone marrow or peripheral blood from 157 cases (131 acute lymphoblastic leukemia, 26 non-Hodgkin's lymphoma) and peripheral blood from 136 controls were analyzed by Southern blot technique, under highly stringent conditions, using cloned BLV DNA as a probe. None of the 157 case or 136 control DNA samples hybridized with the probe. The high statistical power and specificity of this study provide the best evidence to date that genomic integration of BLV is not a factor in childhood acute lympho blastic leukemia/non-Hodgkin's lymphoma. INTRODUCTION Lymphosarcoma or lymphoma is a malignancy of lympho- reticular tissues that has several features common to humans, cows, and other animals. In recent years, there has been increas ing interest in bovine lymphosarcoma as an animal model for human malignancy and for its possible associations with certain forms of human cancer (1). EBL is a highly contagious disease in dairy cattle. The etiological agent in EBL is BLV. BLV is an exogenous retrovirus related to the HTLV family of viruses (2, 3). Despite extensive study and expanded knowledge about the epidemiology and biology of BLV and its possible role in human malignancies, the zoonotic potential of BLV continues to be an unresolved question (4). This report is the result of a case- control study to assess the role of BLV in childhood ALL/ NHL. In evaluating the zoonotic potential of this virus, it is impor tant to consider the epidemiology of EBL, susceptibility of other animal species to BLV, and the evidence for a potential role of BLV in human lymphoreticular malignancies. Received 9/30/87; revised 2/8/88; accepted 2/16/88. 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. 1Support was provided by the National Institute of Environmental Health Sciences (ES03417) of the Department of Health and Human Services and the University of Minnesota Children's Cancer Research Fund. 2To whom requests for reprints should be addressed at, Chronic Disease, Minnesota Department of Health, 717 Delaware St., SE, P.O. Box 9441, Minneapolis, MN 55440. 3 Recipient of a Leukemia Society of America fellowship award. 4 Present address: Laboratory of Tumor Immunology and Biology, National Cancer Institute, Bethesda, Maryland. ' Present address: Pediatrie Hematology/Oncology, Baylor College of Medi cine, Houston, Texas. 6 Present address: Division of Oncology, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania. 7The abbreviations used are: EBL, enzootic bovine lymphosarcoma; BLV, bovine leukemia virus; SSC, standard saline citrate (0.15 M NaCl/0.015 M sodium citrate, pH 7.0); HTLV, human T-lymphotropic virus; ALL, acute lymphoblastic leukemia; NHL, non-Hodgkin's lymphoma. BLV infection is very common in dairy herds in the U. S. The prevalence of BLV infection in individual dairy animals is estimated to range from 20% to almost 50% (5, 6). Estimates of herds with at least one BLV-positive animal are as high as 60% (7). In approximately two-thirds of the cases, the infection is clinically asymptomatic, while one-third of infected animals develop persistent lymphocytosis, which is characterized by a permanent increase in the number of B-lymphocytes in periph eral blood (7). Unlike HTLVs, which are T-cell tropic, the natural target cell of BLV is the B-lymphocyte, where it resides in a repressed state (8). BLV infected cattle are not viremic. Viral particles, viral antigens, and viral RNA have not been detected in BLV- infected lymphocytes before cultivation. Short-term cultivation of infected lymphocytes results in the synthesis of viral RNA, viral antigens, and virus particles. The molecular basis for the covert nature of viral infection remains obscure. The virus is horizontally transmitted among cattle and the infection is usually persistent as evident by continuous high titers of antiviral antibodies (9, 10). Close physical contact between infected and susceptible animals is the most important method of horizontal transmission (11). However, the mecha nism of transmission through direct contact remains to be understood (12). Milk transmission of the virus to newborn calves has also been convincingly demonstrated (13). Under natural conditions, this form of transmission is much less frequent than transmission by direct contact (14). Pasteuriza tion inactivates the virus (15). BLV is infectious to other species (16). Newborn sheep innoculated with lymphocytes of BLV-infected cattle became persistently infected with BLV and 50% died with histologically confirmed lymphosarcomas (17). A newborn goat given cul tured lymphocytes developed an active infection ( 18). BLV can also grow in human, canine, monkey, goat, and sheep tissue cultures (19). Other experiments have provided more direct proof of primate susceptibility to BLV (20, 21). Eight chimpan zees innoculated with large doses of cultured lymphocytes con taining BLV, developed antibody to several viral antigens. Unpasteurized milk has also been implicated in the develop ment of erythroleukemia in two of six chimpanzees fed milk from cows naturally infected with BLV (22). However, antibody to BLV was not detected and since chimpanzees usually develop titers when infected with BLV, the significance of this obser vation is not clear. Previous epidemiológica! investigations have yielded evi dence to both support and refute an association between human lymphoreticular malignancies and BLV. Several mortality stud ies have indicated an excess risk of human leukemia associated with farming and rural residence (23). Geographic correlation studies have consistently found elevated leukemia mortality in rural areas of the U. S. (particularly Minnesota, North Dakota, Nebraska, Kansas, Oklahoma, and Texas) (24). However, sev eral mortality studies have not found this association (25-27). 2919