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Journal of Virology, August 1999, p. 7096-7097, Vol. 73, No. 8
0022-538X/99/$04.00+0
LETTERS TO THE EDITOR
EBNA-1 Sequences in Endemic and Sporadic Burkitt's Lymphoma
 |
LETTER |
In their recent manuscript, Habeshaw et al. (4) used
mutations in EBNA-1 to identify Epstein-Barr virus strains in
immortalized cell lines and Burkitt lymphomas (BL) from different
geographic regions. Like us (1, 2), Habeshaw et al. found a
near absence of P-ala in BL from any part of the world but frequently
identified V-leu and P-thr. However, whereas we concluded that some
subtypes present in normal B cells are not found in BL, implying a
differential pathogenicity in the context of BL, Habeshaw et al.
concluded that within any geographic area, the EBNA-1 subtypes present
in BL simply reflect those prevalent in the population. If the data on
BL subtypes are similar in these studies, why are the conclusions different?
One problem is that Habeshaw et al. studied only three tumors from
Europe (including the only P-ala-positive tumor in their series), with
most of their data coming from East African tumors. The lack of
European tumor data makes it impossible to demonstrate a geographical
bias in the distribution of EBNA-1 subtypes in tumors, and the
concordance of EBNA-1 subtypes between tumors and subtypes present in
nonmalignant B cells cannot be proved, except in the context of East
African BL. They will need to study additional tumors from Europe and
show that P-ala is prevalent in such tumors to confirm their
conclusion. If they were able to demonstrate this, it would contrast
with our data, for we have not found P-ala in American BL.
A second problem is that Habeshaw et al. do not think it important that
they used lymphoblastoid cell lines as opposed to peripheral
lymphocytes (PBLs) as controls. It seems probable, from the sum total
of the data, that lymphoblastoid lines do not permit identification of
the complete virus repertoire in an individual. We and others have
observed multiple EBNA-1 subtypes (and multiple subtypes based on other
genetic loci) in single individuals as well as in T-cell nasal
lymphomas and have proposed that some of these subtypes evolve
postinfection (3, 5, 7).
We have also observed differences in the relative proportions of the
major subtypes associated with BL (P-thr and V-leu) in BL from
different regions, as is hinted at by Habeshaw's data from New Guinea,
but neither we nor Habeshaw et al. have observed other subtypes that
can be detected in peripheral blood or saliva, namely V-val and V-pro,
in BL from any of the combined regions we have examined. In contrast,
we and others frequently find V-val in nasopharyngeal carcinomas
(2, 3, 6). Habeshaw et al. did not find either V-pro or
V-val in the lymphoblastoid lines they examined. This does not surprise
us
we have found V-val only in saliva in nonimmunosuppressed
individuals, and neither we nor anyone else, to our knowledge, has
found V-pro in lymphoblastoid lines, suggesting that it is in some way
transformation defective.
In patients from Europe and America, we have found V-leu (along with
multiple other subtypes) in PBLs from individuals with infectious
mononucleosis or with immunosuppression, but we have yet to find V-leu
in PBLs from normal individuals. This could be a quantitative
phenomenon, but it nonetheless suggests that it is not simply geography
(as suggested by Habeshaw et al.) that dictates the distribution of
EBNA-1 subtypes in tumors: host factors and the biology of the tumor
cells themselves may well play a role and, indeed, may be more
important than geography.
| |
REFERENCES |
| 1.
|
Bhatia, K.,
A. Raj,
M. I. Gutierrez,
J. G. Judde,
G. Spangler,
H. Venkatesh, and I. T. Magrath.
1996.
Variation in the sequence of Epstein-Barr virus nuclear antigen 1 in normal peripheral blood lymphocytes and in Burkitt's lymphomas.
Oncogene
13:177-181[Medline].
|
| 2.
|
Gutierrez, M. I.,
A. Raj,
G. Spangler,
A. Sharma,
A. Hussain,
J. G. Judde,
S. W. Tsao,
P. W. Yuen,
I. Joab,
I. T. Magrath, and K. Bhatia.
1997.
Sequence variations in EBNA1 may dictate restriction of tissue distribution of Epstein-Barr virus in normal and tumor cells.
J. Gen. Virol.
78:1663-1670[Abstract].
|
| 3.
|
Gutierrez, M. I.,
G. Spangler,
D. Kingma,
M. Raffeld,
I. Gurrero,
O. Misad,
E. S. Jaffe,
I. T. Magrath, and K. Bhatia.
1998.
Epstein-Barr virus in nasal lymphomas contains multiple on-going mutations in the EBNA-1 gene.
Blood
92:600-606[Abstract/Free Full Text].
|
| 4.
|
Habeshaw, G.,
Q. Y. Yao,
A. I. Bell,
D. Morton, and A. B. Rickinson.
1999.
Epstein-Barr virus nuclear antigen 1 sequences in endemic and sporadic Burkitt's lymphoma reflect virus strains prevalent in different geographic areas.
J. Virol.
73:965-975[Abstract/Free Full Text].
|
| 5.
| MacKenzie, J. Personal communication.
|
| 6.
|
Snudden, D. K.,
P. R. Smith,
D. Lai,
M. H. Ng, and B. Griffin.
1995.
Alterations in the structure of the EBV nuclear antigen, EBNA1, in epithelial cell tumors.
Oncogene
10:1545-1552[Medline].
|
| 7.
|
Triantos, D.,
A. W. Boulter,
J. C. Leao,
L. D. Alberti,
S. R. Porter,
C. M. Scully,
W. Birnbaum,
N. W. Johnson, and C. G. Teo.
1998.
Diversity of naturally occurring Epstein-Barr virus revealed by nucleotide sequence polymorphism in hypervariable domains in the BamHI K and N subgenomic regions.
J. Gen. Virol.
79:2809-2817[Abstract].
|
| | | | |
Kishor Bhatia
Ian Magrath
Lymphoma Biology Section
Pediatric Branch
National Institutes of Health
Building 10
Room 13N240
Bethesda, Maryland 20892
|
| |
AUTHORS' REPLY |
We were cajoled into the study of EBNA-1 sequence polymorphism by the
assertions Bhatia and colleagues made in their original paper
(2). They studied two geographically heterogeneous groups of
Epstein-Barr virus (EBV)-positive Burkitt lymphomas (BLs) comprising 24 cases from North and West Africa (of which only the latter is a
BL-endemic area) and 12 from North and South America. These were
compared with two control groups comprising 6 healthy carriers from
West Africa and 24 carriers from the Americas; a further 21 North
American controls were added in a subsequent study (5). On
this basis, Bhatia and colleagues proposed (i) that a particular EBNA-1
sequence variant, designated V-leu on the basis of the identity of
amino acid 487, was preferentially found in association with BL and not
in healthy carriers and (ii) that another variant, P-ala, was rarely
found in BL yet was prevalent in healthy controls.
Given the increasing evidence for geographically linked polymorphisms
among EBV isolates (1, 7), we determined to compare BL and
control samples within more circumscribed geographic areas (6). Our findings on the above two points are as follow (for consistency we use Bhatia et al.'s nomenclature, though that does oversimplify the actual polymorphisms observed).
(i) V-leu was the most common EBNA-1 sequence variant in endemic BL in
East Africa (29 of 55 tumors). However, it was also found in 18 of 32 control donors from the same area, hence refuting the notion of
preferential association with the tumor. By contrast, only 1 of 32 European (Caucasian) control donor isolates carried a V-leu sequence
and V-leu was not seen in any of the 3 EBV-positive European BLs
available to us for analysis.
(ii) The P-ala variant was not found in any of the 55 East African BLs
examined, but neither was it represented among the 32 East African
control isolates. However, this EBNA-1 sequence was common among
European controls, being seen in 14 of 32 cases, and was also present
in 1 of the 3 European BLs. As Bhatia et al. point out, more European
tumors need to be examined before any firm conclusions can be drawn;
however, two more cases have recently been analyzed in another
laboratory and both also have the P-ala sequence (8).
The evidence to date therefore suggests that, within any one area, the
EBNA-1 sequence variants found in virus-associated BL reflect those
seen within the community at large. Bhatia et al. also rightly point
out that our analysis of virus strains in control donors was based on
EBV isolation in vitro rather than on direct amplification of viral
sequences in blood and/or throat washings. Given the potential
importance of this issue, we recently used direct amplification to
identify resident viral strains in healthy donors from whom in vitro
isolates were also available. In the large majority of cases, the two
assays identified the same resident EBNA-1 sequence. From the findings
so far, therefore, we do not believe that a reliance on in vitro
isolates has grossly distorted our analysis of viral variants present
in control populations.
Bhatia et al. also extend their concept of disease-associated EBNA-1
variants to a second EBV-positive tumor, nasopharyngeal carcinoma, in
which they and others detect a high frequency of the V-val sequence
(5, 9). Noting that most of the tumors studied in that work
were of Chinese origin, we recently analyzed virus isolates rescued
from the blood of healthy Chinese donors and likewise found the V-val
sequence in 7 of 11 cases; we infer that V-val is yet another example
of a polymorphic marker common among Southeast Asian virus strains
(4, 7). A recent independent study of EBV's association
with gastric carcinoma, this time in Japan, leads to an exactly similar
conclusion (3).
A direct effector role for EBNA-1 in EBV-associated oncogenesis remains
an interesting possibility. However, as our paper tried to emphasize,
if one is looking for circumstantial evidence of such a role by
analyzing EBNA-1 sequences in tumors, an equally rigorous analysis of
viral strains in the appropriate control populations is essential.
| |
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EBV strain variation: geographical distribution and relation to disease state.
Virology
190:168-175[Medline].
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| 2.
|
Bhatia, K.,
A. Raj,
M. I. Gutierrez,
J. G. Judde,
G. Spangler,
H. Venkatesh, and I. T. Magrath.
1996.
Variation in the sequence of Epstein-Barr virus nuclear antigen 1 in normal peripheral blood lymphocytes and in Burkitt's lymphomas.
Oncogene
13:177-181.
|
| 3.
|
Chen, Y. Y.,
K. L. Chang,
W. G. Chen,
D. Shibata,
K. Hayashi, and L. M. Weiss.
1998.
Epstein-Barr virus-associated nuclear antigen 1 carboxy-terminal gene sequences in Japanese and American patients with gastric carcinoma.
Lab. Investig.
78:877-882[Medline].
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de Campos-Lima, P. O.,
V. Levitsky,
J. Brooks,
S. P. Lee,
L. F. Hu,
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1994.
T cell responses and virus evolution; loss of HLA A11-restricted CTL epitopes in Epstein-Barr virus isolates from high A11-positive populations by selective mutation of anchor residues.
J. Exp. Med.
179:1297-1305[Abstract/Free Full Text].
|
| 5.
|
Gutierrez, M. I.,
A. Raj,
G. Spangler,
A. Sharma,
A. Hussain,
J. G. Judde,
S. W. Tsao,
P. W. Yuen,
I. Joab,
I. T. Magrath, and K. Bhatia.
1997.
Sequence variations in EBNA1 may dictate restriction of tissue distribution of Epstein-Barr virus in normal and tumour cells.
J. Gen. Virol.
78:1663-1670.
|
| 6.
|
Habeshaw, G.,
Q. Y. Yao,
A. I. Bell,
D. Morton, and A. B. Rickinson.
1999.
Epstein-Barr virus nuclear antigen 1 sequences in endemic and sporadic Burkitt's lymphoma reflect virus strains prevalent in different geographic areas.
J. Virol.
73:965-975.
|
| 7.
|
Khanna, R.,
R. W. Slade,
L. M. Poulsen,
D. J. Moss,
S. R. Burrows,
J. Nicholls, and J. M. Burrows.
1997.
Evolutionary dynamics of genetic variation in Epstein-Barr virus isolates of diverse geographical origins: evidence for immune pressure-independent genetic drift.
J. Virol.
71:8340-8346[Abstract].
|
| 8.
| Mackenzie, J., and R. Jarrett. Personal
communication.
|
| 9.
|
Snudden, D. K.,
P. R. Smith,
D. Lai,
M. H. Ng, and B. Griffin.
1995.
Alterations in the structure of the EBV nuclear antigen, EBNA1, in epithelial cell tumours.
Oncogene
10:1545-1552.
|
| | | | |
G. Habeshaw
Department of Pathology and
CRC Institute for Cancer Studies
University of Birmingham
Birmingham B15 2TA
United Kingdom
|
| | | | |
A. B. Rickinson
CRC Institute for Cancer Studies
University of Birmingham
Birmingham B15 2TA
United Kingdom
|
Journal of Virology, August 1999, p. 7096-7097, Vol. 73, No. 8
0022-538X/99/$04.00+0
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