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CONCISE
REPORT
B-Lymphocytic
Hairy
By Thomas
Lion,
Cells
Nighet
Contain
Razvi,
Harvey
HTLV-ll
has been
found
in some
cases
of the rare T-cell
form
of hairy-cell
leukemia
(HCL) and in a leukopenic
chronic
T-cell leukemia
mimicking
HCL. We asked
whether
the virus
is implicated
in the more frequent
B-cell form of
HCL.
DNA
extracted
from the mononuclear
cells derived
from spleen
(eight
cases)
or peripheral
blood (eight cases)
T
HE
hairy
cell
from
leukemia
the
observation
with
correlated
virus
HTLV-II
has been
with a variant
T-cell form of
(HCL).”2
However,
as a malignancy
for HTLV-II
dominantly
tive evidence
from
lymphotropic
two patients
HUMAN
isolated
of cell-surface
T cells
HCL
is seen
pre-
of B-cell lineage.
Some suggesin connection
with HCL
comes
Tac
and selected
antigen,
T-cell
which
is
malignancies,
in B
lymphocytic
cells.3 In adult
T-cell
leukemias,
the expression
of cell-surface
Tac antigen
is strongly
correlated
with the
presence
of human
T-cell
leukemia
virus type-I
(HTLV-I).4
Furthermore,
and
both
and
HTLV-I
transforming
normal
II, whose
host
infects
B-cells
range
has
not
in vitro.5
directly
whether
nant B-cell form
II are
capable
T lymphocytes
been
These
HTLV-II
of HCL.
observations
AND
HTLV-
established,
also
led us to ask
fully
is associated
MATERIALS
of infecting
in vitro.57
with
the
predomi-
METHODS
Mononuclear
cells from 16 patients
with HCL were
Ficoll-Hypaque
gradient
centnifugation
from either
peripheral
blood (PB) of patients
with high leukemic
cell counts
(eight cases) on from splenic
tissue (eight cases).
The number
of
hairy cells in PB ranged
from 70% to 98% as determined
by
morphologic
and immunologic
criteria.
All 16 HCL
cases investigated were of B-cell lineage as determined
by reactivity
to anti-B 1,
by lack of reactivity
to anti-T4,
and by failure to rosette with sheep
Cells.
RBCs.
DNA
extraction
was extracted
by treatment
with
M. Golomb,
with
RNase,
cellular
and
molecular
hybridization.
from
cells
proteinase
phenol,
and ethanol
DNA
From
Chicago
using
Total
precipitation.8
with
alcohol,
cellular
followed
digestion
digests
of human
K digestion
chlorofonm-isoamyl
were made
the
Joint
and
Michael
Submitted
Section
Individual
restriction
endonucleases
of Hematology/Oncology.
Reese
September
Supported
in
No. IN-41-Y-20.
under
Grant
No.
Medical
8, 1987;
part
Grant
S
by Grune
1988
& Stratton.
RESULTS
provide
DNAs
to liberate
HTLV-II
HTLV-II
had
were
by
Centers,
accepted
American
and a grantfrom
2-507-RRO5893from
Chicago.
June
Cancer
the Illinois
the
Cancer
National
Cancer
Council
Insti-
reprint
Genetics
requests
to Bernard
in Medicine,
Box
8031,
H. Brownstein,
Washington
ofMedicine,
St Louis. MO 63110.
The publication
costs ofthis article
were defrayed
charge
therefore
payment.
“advertisement”
indicate
1988
This
article
in accordance
must
with
this fact.
by Grune
& Stratton,
0006-4971/88/7204-0040$3.00/0
Inc.
18 U.S.C.
PhD.
University
after
by
section
Center
As
in part by page
hereby
marked
1 734 solely
to
nonspecific
various
DNA
of the
host
harbor
cell DNA.
a further
control
cases
enzymes
(Fig
was hybridized
taming
HTLV-I
to each
exposure
signals
on the
or HTLV-II
viral
sequences.
fragments
were
In both
liberated
length
genomic
if
DNA.
examined.
blot
human
to
c
Similar
analysis
for
in five cells
1 0 g human
10
(equivaDNA).
cell genome,
we
method,
were
cases,
by
to the film for up to two
were obtained
in digests
cell
probed
known
to
for the presence
lines
of
the predicted
digestion
with
1, lane b for HTLV-II).
When
with an HTLV-I
probe,
only
DNA
Blood,
The
after 48 hours on the autoradiogram,
as
a. At a ratio of 1 :5, a signal could be seen
72 hours. Even with
no HTLV-II-positive
HTLV-I
signals.
any signal
homologous
sequences
(Fig
1, lanes
16 HCL
genome
In
to
endonucleases
cell
found for all 16 samples.
of sensitivity
of our Southern
of one viral
viral
of predictable
the
detect
DNA
HTLV-II.
sequences
restriction
fragments
into
able to
HTLV-I
saw a signal
in Fig 1 , lane
of hairy
School
possible
with
!Lg of DNA/lane
was one viral genome
lent to 2.6 pg viral DNA of 9-kb length/
readily
shown
Institutional
was analyzed
for
probed
for HTLV-I
integrated
e) in any
were
limit
At a ratio
15. 1988.
Society
not
or
through
of
DNA
were
a control
for
were digested
HTLV-II
BamHl,
University
hairy
cell
all samples
B-type
addition,
weeks,
Address
1428
H. Brownstein
EcoRI, and Xhol
for detection
of HTLV-II
DNA sequences,
or with
Sad, EcoRI,
and Smal
for detection
of HTLV-l
DNA sequences;
the latter enzymes were also used to detect HTLV-I
DNA
in
infected
marmoset
cells. The digested
DNA
(10 to 20 g)
was
electrophoresed
in I % agarose gels, transferred
by a rapid alkaline
method9
to Genescreen
Plus membrane
(Dupont),
hybridized
at
42#{176}Cin 1% sodium
dodecyl
sulfate
(SDS)l
mol/L
NaCI/lO%
dextran sulfate, and washed at 65#{176}C
with 2 x SSC/l%
SDS as
recommended
by the manufacturer
(Dupont
catalog no. NEF-976).
HTLV-Ilprobes.
The 3.5-kilobase
(kb) BamH I fragment
of the
HTLV-II
sequence’#{176}”and a 1.9-kb Smal
fragment of the HTLV-I
sequence’2
were obtained
from the laboratory
of I. Chen (UCLA)
and were used as probes. The appropriate
fragments
were isolated
from the plasmids and labeled by 32P-oligolabeling.’3
Limits
ofsensitivity.
Aliquots
of 10 ig human placental
DNA
were mixed with various amounts
of HTLV-II
viral DNA
in ratios of
viral genomes
per human
cell ranging from 20:1 to 1:5. The mixed
DNA was digested with the appropriate
restriction
enzymes, electrophoresed
in agarose
gels, Southern
blotted
to Genescreen
Plus
membrane,
and hybridized
with 32P-labeled
HTLV-II
probe following the exact protocol used for the HC samples.
We
tute.
©
Bernard
Sequences
of 1 6 patients
with the B-cell form of HCL was probed.
No
viral sequences
were
detected
at levels
of sensitivity
as
low as one viral genome
in five cells.
Therefore
HTLV-ll
may not be involved
in the B-cell form of HCL.
results
The
for
and
DNA
by
purified
DNA
no HTLV-H
gave
a positive
signal.
viral
DNA
restriction
a similar
the lane
We
Vol 72, No 4 (October), 1988:
also
blot
con-
probed
PP 1428-1430
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B-LYMPHOCYTIC
HAIRY
CELLS
1429
b
C
e
d
f
DISCUSSION
‘0
I
I
23
Our
results
are in accordance
one study performed
formed
by Rosenblatt
21
HCL
with
the serologic
patients
tested
for
the
presence
HTLV-II
was positive;
the latter
HTLV-II.
study,
none of 21 HCL patients
All these data are consistent
this
result
that neither
HTLV-II
nor
the B-cell form of HCL.
The
of
responsiveness
of antibodies
to
be a false positive.
might
In
tested
positive
for
with the inference
is likely
HTLV-I
per1 of
to play
in
treatment
with
(a-IFN)
hematopoietic
is unique
among
the malignant
system.’6
IFNs have potent
disorantivi-
ral activity
in addition
to antiproliferative
properties,’19
and
treatment
B-lymphocytic
a-IFN
HCL
of
by
its
to
a role
a-interferon
ders of the
of
clinical
findings
by Quesada
et al’4 and another
et al.’5 In the former
study,
only
HCL
differentiating
and
may
classical
mediate
remission
antiviral
effect.
If
HTLV-II
had been found associated
with HCL, infection
of
lymphocytes
and/or
stem
cells
with
the virus
might
be
hypothesized
as the stage susceptible
to a-IFN.
The involve-
$
0.6-
ment
of HTLV-II
is excluded,
HBLV#{176}) or an unidentified
virus
.
.
F
q
f%.t’
‘Is’
4)
form of HCL remains
possible.
may be attributable
to another
t’;f
HTLV-I
and
B lymphocytes
T
cells.57
HTLV-I
malignancies
Fig
1.
Southern
analysis
of hairy
cell
DNA
for
HTLV-ll
sequences.
For this blot, all samples
were digested
with BamHl
and run on the same agarose
gel. Lane a contains
human placental
DNA to which
purified
HTLV-ll
DNA was added at a ratio of one
viral genome
per human
genome;
lane b contains
DNA from a cell
line infected
with HTLV-lI
DNA.
Lanes a and b both show the
expected
genome.
3.5-kb
band
representing
the
3’ end of the
HTLV-ll
Lanes c through
e contain
BamHI
cut DNA samples
from
three
different
HCL patients
and do not show a positive
signal.
DNA samples
from 1 3 other
HCL patients
gave the same result.
Lane f is a control
containing
DNA extracted
from a marmoset
HTLV-l-infected
cell line, and tests for possible
cross-hybridization against
the HTLV-Il
probe; it is also negative
but gave several
bands
between
23 and 9.4-kb
when the blot was reprobed
with
HTLV-l
DNA.
hairy
cell
DNA
ments
to show
that
HCL
patient
DNA
bands
were
seen.
HTLV-II
expected.
contained
been
for
immunoglobulin
properly
DNA
Both
rearranged
lanes.
Thus,
gene
digested
if
the
sequences,
and
patient
DNA
positive
results
such
yet transformation
is associated
as
adult
is reported
exclusively
T-cell
only
with
leukemia
(ATL)
With
pathology,
leukopenic
HTLV-II
chronic
was
T-cell
only in T-cell
HCL,”2
a
mimicking
HCL,25
and,
more
recently,
HTLV-II
lymphoproliferative
HTLV-Il
exposure
T-cell
malignancy
HTLV-II
infection
obvious
malignancy.
has been
Together
these observations
capable
of transforming
HTLV-I
of involvement
known
in other
T-cell
disorders.26’27
Other
reports
describe
in AIDS
patients
without
a coexisting
and
present
serologic
evidence
of
in intravenous
(IV) drug users
with no
vitro,
Therefore,
implicated
to
and
lymphoma2t24.
detected
leukemia
regard
of
T-cell
T-cell
rearrange-
was present
are
Alternatively,
a-IFN
action
effect on malignant
cells.
capable
ofinfecting
both T and
peripheral
range
the
HTLV-II
in vitro,
but a different
virus
(eg,
in the etiology
of the B-cell
with
the
results
obtained
suggest
only
that both viruses
cells of the T lineage
and HTLV-II
may show a T-cell
in lymphoproliferative
in
may be
in vivo.
limited
disorders.25’3#{176}
ACKNOWLEDGMENT
in the
germline
the outstanding
technical
support of
of probe by Irvin Chen, the gift
I-infected
marmoset
DNA
by Phil Andersen,
helpful
with Joe Rosenblatt,
and reading
of the manuscript
Schlessinger and Mark Gurney.
We
acknowledge
Hutchinson,
samples
had
would
have
the gift
Mary Ann
of HTLVdiscussion
by David
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1988 72: 1428-1430
B-lymphocytic hairy cells contain no HTLV-II DNA sequences
T Lion, N Razvi, HM Golomb and RH Brownstein
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