No Involvement of Bovine Leukemia Virus in Childhood Acute

Lymphoblastic Leukemia and Non-Hodgkin's Lymphoma

Alan P. Bender, Leslie L. Robison, S. V. S. Kashmiri, et al.

Cancer Res 1988;48:2919-2922.

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[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.
2 To 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 Msodium
citrate, pH 7.0); HTLV, human T-lymphotropic virus; ALL, acute lymphoblastic
leukemia; NHL, non-Hodgkin's lymphoma.
2919
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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).
 BOVINE LEUKEMIA VIRUS AND CHILDHOOD ALL/NHL

Mortality studies of veterinarians, who may represent a sentinel RESULTS

population with high exposure potential to animal diseases,
have also yielded mixed results (28, 29). Results of a typical Southern blot analysis are presented in
A large case-control study of leukemia in Iowa found a Fig. 1. BLV-bat DNA carries three BLV proviral DNA mole
significant excess of ALL in rural counties and the excess ALL cules, each with a unique site for EcoKl. Digestion of this DNA
was associated with the presence of herds infected with BLV with £coRIgenerates six fragments which specifically hybridize
(30). Other studies found no association between the presence with the probe. These fragments are easily detectable even when
of EBL in cattle and the occurrence of human leukemia (31, 2 /¿gof DNA was loaded on the gel (Lane A). However, no
32). In 10 separate studies, over 2000 people occupationally hybridization was detected when 10 /¿gof £coRIdigested hu
exposed to BLV were evaluated for the presence of antibody to man DNA was loaded on the gel (Lanes C-J). None of the 157
BLV and all were negative (33). An Israeli and a Rumanian case or 136 control DNA samples demonstrated the presence
study, on the other hand, reported finding antibodies to BLV of BLV.
in a significant percentage of a small number of workers occu The significance of these findings must be evaluated in rela
pationally exposed to this virus (34, 35). Occupational associ tion to the ability of a study of this size to detect at least one
ations between exposure to BLV and human lymphoreticular patient with genomic integration of BLV. The statistical power
malignancies in meatworkers have been reported in Maryland to find a reasonably small proportion (P) of the cases with BLV
was quite high (Table 1). The study's power (44) to detect one
and New Zealand (36, 37).
or more subjects with BLV, from a study population (N), for
MATERIALS AND METHODS different hypothesized proportions of cases with genomic inte
gration, can be calculated as 1 —(1 —P)N.
The resolution of the zoonotic potential for BLV requires highly If the true proportion of cases with BLV genomic integration
representative molecular probes (4). To rigorously test the hypothesis was 4.3%, then for 999 out of 1000 replications of a study with
that genomic integration of BLV is not associated with childhood ALL
and NHL, a case-control study was conducted between December 1983 157 cases, at least one patient with BLV integration would be
and February 1986. Cases (<16 years at diagnosis) were derived from expected. If the true proportion was one in a hundred cases,
the patients diagnosed at the Universities of Minnesota, Wisconsin, there would still be more than a 80% chance of finding at least
and Michigan, the Mayo Clinic and their affiliated hospitals and clinics. one patient with genomic integration of BLV.
One-hundred ninety-six newly diagnosed cases (112 males, 84 females)
were identified and matched (age, sex, and race) with regional popula ABCDEFGHIJ
tion controls. Bone marrow and peripheral blood from 157 cases (131
ALL, 26 NHL) and peripheral blood from 136 controls were studied.
Case samples were obtained prior to treatment and samples from all 20.0kb
subjects were shipped within 24 h to a central laboratory.
Nuclei were prepared from a blood and/or bone marrow sample
using a procedure described by L. Kunkel (38). Ten milliliters of blood
collected in a heparinized tube was mixed with 90 ml of ice-cold 0.32 12.3kb -„
M sucrose/10 mM Tris-HCl (pH 7.5)/5 mM MgCl2/l% Triton X-100.
After cell lysis, nuclei were collected by centrifugal ion at 1000 x g for
10 min. The nuclear pellet was suspended in 4.5 ml of 0.075 M NaCl/
0.024 M EDTA (pH 8.0). 0.5 ml of 5% sodium dodecyl sulfate and 9.5kb ^
proteinase K at 0.5 mg/ml final concentration were added and the 8.3kb <e>
mixture was incubated for approximately 16 hours at 55°C.
RNA was removed by digestion with 20 ¿tgRibonuclease A (Koch-
7.2kb
ringer Mannheim) in 10 mM Tris-HCl (pH 7.5) and 15 mM NaCl for 5.6kb
20 min at 37°C.The digest was extracted with phenol/chloroform/
isoamyl alcohol followed by chloroform/isoamyl alcohol extraction and
subsequently extracted with ether. After removing ether, DNA was
ethanol precipitated and pelleted. The DNA pellet was washed with
70% ethanol and finally, it was dissolved in 10 mM Tris, pH 7.5/1 mM
EDTA. DNA was digested with EcoRl (BRL, Bethesda, Maryland)
according to supplier specification. 10 UKof EcoRl digested DNA and
2 and 5 Mg of £coRI-digested DNA from BLV producing BLV-bat Fig. 1. Representative Southern blot analysis of DNA from blood of human
clones were electrophoresed by horizontal gel electrophoresis in 0.6% cases. 10 MEof human lymphocyte DNA from cases and controls (Lanes C-J)
agarose (39). The DNA fragments were transferred to Zetabind mem and 2 ng (Lane A) or 5 /ig (Lane B) of DNA from BLV producing bat cells was
brane (AMF-Ouno; Meriden, CT) by the Southern procedure (40). 32P- cleaved with EcoRl, electrophoresed and subjected to analysis using "P-labeled
cloned BLV DNA probe.
labeled DNA probe was prepared by nick-translating the cloned BLV
DNA insert (41). Prior to hybridization, the filter and 32P-labeled DNA
Table 1 Statistical power to detect at least one case with genomic integration of
probe were prehybridized separately for 15 h in a hybridization mixture BLV for given proportion of case infections in children with ALL/NHL
containing 3x SSC, lOx Denhardt medium, 0.1% sodium dodecyl
(%)Power0.999 Proportion infected x 100
sulfate, and 0.1 mg of denatured sonicated calf thymus DNA per ml
(42). The conditions of filter hybridization and washing have previously of parents who
been described (43). The filter and prehybridized probe (5-7 x 10' with farming consumed raw
cpm/ml) were hybridized for 18-24 h. After hybridization, the filter cases experience dairy
products(N
(N=157)4.3 (JV=78)8.5 29)21.2
=
was washed several times with 3x SSC, with hybridization buffer
containing 0.1 mg of denatured sonicated calf thymus DNA per ml, 0.99 2.9 5.7 14.7
and finally, with 0.1 % sodium dodecyl sulfate in 0.1 x SSC. All of the 0.95 1.9 3.8 9.8
above procedures were performed at 65°C.The filter was then dried 0.90 1.5 2.9 7.6
and exposed at -70"C to Kodak X-ray X-Omat film with DuPont 0.80 1.0 2.0 5.4
0.50All 0.4Cases 0.9Cases 2.4
Lightning Plus intensifying screen.
2920

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 BOVINE LEUKEMIA VIRUS AND CHILDHOOD
Cases may have been at different risk of exposure to BLV
and the reduced number of higher risk children directly would
decrease the study's power. The number of case children directly
exposed to BLV is unknown. However, the prevalence of BLV-
infected animals in the upper midwest is at least as high as the
average national estimates (45). Therefore, parental farming
experience and consumption of raw dairy products are useful
proxies for BLV exposure. There was an 80% chance of finding
at least one genomic integration of BLV if 2% of the cases with
parents who had farming experience were infected with BLV
or if 5.4% of the children who had consumed raw dairy products
were infected. Similar statistical power applied to the control
series as well, but this observation is less relevant since these
subjects were presumably free from ALL or NHL.
DISCUSSION
The exact mechanism of leukemogenesis of BLV in cattle
remains to be determined. It is known, however, that BLV does
not require a helper virus for replication, and there is good
evidence that BLV infection is necessary but not sufficient for
the neoplastic process (46). In addition, all proposed mecha
nisms for BLV leukemogenesis involve some form of proviral
integration into the host's genome (47). Based on this evidence,
it is unlikely that BLV exerts neoplastic influence extrageneti-
cally. Therefore, the appropriate measure of BLV oncogenic
potential is its ability to integrate with the host's DNA.
The Southern analysis is very sensitive, and has been rou
tinely used to detect BLV-induced animal tumors in many
geographical locations. Under the conditions of Southern analy
sis described here, as little as one tenth of a copy of the BLV
genome in human cells would have been detected if it was
present (48,49). Using the same conditions of Southern analy
sis, BLV sequences have been detected in a large number of
bovine tumors carrying a single copy of BLV per haploid
genome (8, 49). Thus, the negative results reported here are
unlikely to be due to the lack of sensitivity of the experiment.
Although the zoonotic potential of BLV has been considered
very limited by some investigators (33), public health concerns
have persisted in light of the descriptive and analytic human
data indicating high leukemia risk in dairy farmers, the high
prevalence of BLV in raw milk of infected animals, the con
sumption of raw milk by more than 75% of farm families (50)
and 25% of all children, the lack of understanding of the
mechanism of transmission, and the similarities of BLV and
the HTLV family. Indeed, if BLV were a chemical agent, there
would already be adequate justification for minimizing human
exposure (51).
Dairy farming confers extensive exposure to many potentially
toxic substances, several of which appear to be associated with
lymphoreticular malignancies (52). These may ultimately pro
vide a satisfactory explanation for the increased risk of leukemia
in rural regions of this country. It is very unlikely, however,
that BLV is one of these exposures. The high statistical power,
sensitivity, and specificity of this study provide the best evidence
to date that genomic integration of BLV is not a factor in
childhood ALL/NHL.
ACKNOWLEDGMENTS
Study samples were received and processed in the laboratory of Dr.
H. Orr of the Human Genetics Institute at the University of Minnesota.
DNA hybridization was performed by Dr. Kashmiri in the laboratory
of Dr. J. F. Ferrer at the University of Pennsylvania who was supported
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ALL/NHL
by a grant from the Kleberg Foundation and a grant from the Wetter-
berg Foundation. Case ascertainment and samples were also provided
by Drs. J. Cich, M. Hiesel, J. Nickerson, J. Priest, R. Roskos, T.
Silberman, L. Singher, and J. Tillisch. Technical assistance was pro
vided by T. Parker, C. Wilkowski, C. LeBlanc, and T. Hickock. R.
Boatman, R.N., was project director assisted by L. Clark, D.V.M. and
M. Anderson.
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