From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
Phenotypic
Similarities
and Differences
Between
CALLA-Positive
Lymphoblastic
Leukemia
Cells
and Normal
Marrow
CALLA-Positive
B Cell Precursors
H. Ryan,
By Daniel
Expression
of
differentiation
lymphoblastic
with
antigen
cells
In cBML,
with
CD34
expression
cyte
antigen
(CIA).
(Cs)
expression.
correlated
lated
with
cBML,
and
CD2O
sion
of CD1O.
CD2O
inversely
CIA.
ARLY
with
B CELL
bone
closely
most
is acute
lymphoblastic
Cells
easily
small
previously
TdT-,
whereas
related
was
to
to CD1 0
positively
expression
as
of
in
CD34
cBMI.
most
and
but
likely
and
may
posi-
cells,
originate
e 1987
in the
also
of
by
as TdT
and
phenotype
is CDIO+/
found
in 75% of patients
phenotype
are
in childrens’
marrow,
and can be found
in
(1% to 5%)
in adult
bone
marrow.
We
two
subpopulations
of normal
lymphoid
cells (cBMLs).6
weakly
CDIO+/CD2O+/CLA
‘-.12%
of the
cells
The
are
CD 10-
majority
+ /CD34-/
strongly
From
the
Departments
Pediatrics,
of
University
of
Pathology
and
Rochester
of
CDIO+/
Laboratory
Medical
School
of
Medicine
and
Dentistry.
NY:
and
608,
601
Elm-
wood
The
publication
costs
ofthis
charge payment.
This article
advertisement
“ in accordance
indicate this fact.
© 1987 by Grune
& Stratton.
0006-4971/87/7003-0044$3.OO/O
814
article
must
with
Inc.
were
Grant No.
Department
defrayed
therefore
18
U.S.C.
likely
with
by Grune
Cs
to
express
in a coordinated
differences
to
counterparts.
the
The
or Ct positive.
in
marrow
cAll
expression
cells
of CD1
suggesting
a phenotypically
less
CD34-negative
cAll.
does
different
patient
cells
respect
increased
to be CIA
than
from
the
0
that
differentiated
& Stratton, Inc.
The
subpopulation
suggesting
more closely
that this is the
Both CD1O-positive
CD 19 and lack
CD5
The
cALL
aim
of this
cells
with
study
the
marrow
(Leu
lineage.
was
two
phenotype
resembles
that
more differentiated
of
the
of mature
B cell
lymphoid
popula1 ), suggesting
that
A
recent
to compare
the
subpopulations
study
has
phenotype
of
of
cBML.
The
phenotypic
resemblance
of cALL
to the total population
of
cBMLs8
suggests
that on more detailed
examination,
cALL
subpopulations
like those in normal
marrow
may be present.
Multiparameter
demonstrates
that
analysis
ofcells
from 25 patients
with cALL
expression
of CDIO,
CD34,
CLA, and C.z
are correlated
with
each
other
in the same
fashion
observed
in cBML.
In contrast,
TdT and CD2O expression
cALL
are unrelated
to expression
of the other differentiation
markers.
These
data
cell lineage
cALL
when
cALL
suggest
that
are present,
cells
CD1O-positive
counterparts.
differentiation
but that
are
compared
lymphoid
cells
stages
differences
in detail
believed
as
in
of B
are
with
the
to be their
Medicine
Center,
Box
Ave. Rochester,
NY 14642.
Supported
by US Public Health
Service
awarded
by the National
Cancer Institute,
and Human Services,
Bethesda,
MD.
demonstrate
they
belong
to the B cell
confirmed
these observations.7
observed
the Department
of Genetics,
Roswell
Park Memorial
Institute,
Buffalo.
Submitted
November
25, 1986: accepted
May 16, 1987.
Address
reprint requests
to Daniel H. Ryan, MD, University
of
Rochester
and
but
or
with
from
cAll
CD1O,
show
represent
population.
tions express
marrow
normal
and
positive
cells
that
CD2O-/CLA-/CD34+/TdT+.
former
B cells,
that
such
cBMIs.
positive
consistent
patients.
of
no
surface
markers
specific
to the B cell
(B4) and CD2O,”4
as well as markers
ALL
ALL),
less
of cAll
a neoplasm
lymphoid
are
form
showed
CIA.
to
CD34
they
leukemia
(ALL),
are
was
cALL
individual
CD34.
similar
of
in
Analysis
were
0 was
indicate
expression
of TdT and CD2O
cells considered
their
normal
that
correlated
in
CD1O+/TdT+/CDI9+
described
positive
marrow
these cells are
contrast
that
phenotypically
of B cell maturation
frequent
(common
the
detected
numbers
not
markers
fashion
corre-
in the bone
marrow.
The
B cell origin
from most patients
with ALL is suggested
of immunoglobulin
gene
rearrangements.2
most
with
In
the
inversely
expression
subpopulations
that
comparison
within
CD2O
in sorted
or CD1
results
J. Cohen
in cBMLs.
patients
CD2O.
by
and
or
found
Cs expression
CD34,
These
Harvey
intensity
individual
obtained
patient
and
relationship
and
from
patients.
s chain
TdT
the
for
data
(TdT)
The
malignancy
these early stages
of immature
CDIO.
The
TdT+/CDI9+
with ALL.’
cBML.
expression
CIA
These cells also express
lineage,
such as CD19
characteristic
but
precursors
marrow.’
resembles
usually
arises
leukemic
cells
the
presence
in
cells
negative
leuko-
strongly
with
to
intensity
of
corre-
was
and
were
Cs
with
TdT
cALL
TdT
intensity
meawas not related
to expres-
or CD34.
expression.
correlated
density
as
expression
correlated
tively
CD1 0
ALL
lymphoid
ctyoplasmic
Cz expression
Furthermore.
immunoassay
by enzyme
with
). and
expression
in cALL.
sured
and
common
common
A. Sandberg,
association
contrast
transferase
with
Avery
was
is positively
terminal
A. Kossover,
acute
marrow
intensity
(Bi
cALL.
CIA
CD34
TdT
density
E
In
with
normal
correlated
CD2O
Stuart
patients
25
bone
and
inversely
from
of
CD1O
(MY1O)
and
Chapple,
in common
cells
(CD1O)-positive
(cBMLs).
lated
(cALL)
subpopulations
(CALLA)
W.
markers
leukemia
compared
Charles
Acute
CA 39569.
of Health
in part
be hereby
§1 734
by page
marked
solely
to
MATERIALS
AND
METHODS
Patients.
Bone marrow aspiration
from the posterior
iliac crest
was performed
at the time ofdiagnosis
or relapse in 25 patients with
cALL.
The leukemic
cells were peroxidase
negative
and CDIO
positive with lymphoid
morphology
in all patients.
In some patients,
peripheral
blood samples at the time of diagnosis
were obtained
if
insufficent
cells were obtained
from the bone marrow aspiration
for
multiple
marker
studies and if the peripheral
blood mononuclear
fraction
contained
>80% lymphoblasts.
Bone marrow
aspiration
from the posterior
iliac crest was also performed
on healthy adult
donors after informed
consent was obtained
(n - 8). Samples
from
diagnostic
bone marrow aspirates
were obtained
from children
with
the following diagnoses:
immune thrombocytopenia
(1), anemia (1),
neuroblastoma
not involving
the bone marrow
(I ), and ALL in
complete
clinical remission
(2). Collection
ofsamples
was performed
Blood,
Vol
70,
No 3 (September),
1987:
pp 814-82
1
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
PHENOTYPE
OF NEOPLASTIC
V
NORMAL
CD1O+
CELLS
after approval
by the institutional
Committee
on Investigations
Involving
Human
Subjects
and in accord with an assurance
filed
with and approved
by the Department
of Health
and Human
Services.
Cell preparation.
Bone marrow
was collected
in I 0-mL sterile
siliconized
tubes containing
5 mL RPMI
1640 tissue
culture
medium,
10% fetal calf serum (FCS) and 100 U of preservative-free
heparin. Peripheral
blood was collected
in sterile heparinized
tubes.
Ficoll/Hypaque
separation
ofthese cells was performed
as described
previously.9
Cells from patients
with cALL were frozen in liquid
nitrogen9 for later analysis.
Antibodies.
Monoclonal
antibodies
were obtained
from Becton
Dickinson
Monoclonal
Center (Mountain
View, CA) (anti-CALLA
[CD1OJ
FITC, anti-HLA-DR
FITC,
MY1O [CD34],
avidin PE,
FITCand PE-conjugated
irrelevant
murine
IgGI,
and FITCconjugated
irrelevant
murine IgG2), Coulter Immunology
(Hialeah,
FL) (Bl [CD2O] FITC, B2 [CD21J FITC, B4 [CDI9],
J5 [CD1O]
biotin, biotin-conjugated
irrelevant
murine
IgG2), PL Biologicals
(anti-TdT),
Southern
Biotechnology
(isotype
specific
goat antimouse IgG1/FITC),
and Vector Biochemicals
(peanut
agglutinin
[PNA]
FITC).
GAP 8.3 (anti-common
leukocyte
antibody)
was
obtained
from the American
Type Culture
Collection
and conjugated to PE as previously
described.9
Immunofluorescence
staining.
The
choice
of anti-CD
I0
reagents
(anti-CALLA
FITC or iS biotin followed
by avidin PE)
was based on the availability
of FITC or PE conjugates
of the second
marker
to be studied.
All incubations
with antibody
were at 4#{176}C,
followed
by three washes in phosphate-buffered
saline (PBS).
In
each case, cells were washed three times in PBS and resuspended
in
300 L PBS at 4#{176}C
for analysis on the flow cytometer.
For two-color
immunofluorescence
(IF) of CDIO v HLA-DR,
common leukocyte
antibody
(CLA),
Bl (CD2O), B2 (CD21),
PNA,
B4 (CDI9),
and MYIO (CD34),
cells were stained with monoclonal
antibodies
and control reagents
as previously
described.6
For combined
Bl (CD2O)
and MYIO
(CD34)
staining
of
leukemic cells, the cells were incubated
with appropriate
dilutions of
biotin-conjugated
CD2O plus MY1O (CD34),
followed
by three
washes and incubation
with avidin PE plus isotype specific goat
anti-mouse
IgGl/FITC.
Cells were stained with biotin-conjugated
irrelevant
murine IgG2 plus MY1O (CD34),
followed by avidin PE
plus isotype-specific
goat anti-mouse
IgGl/FITC
to control for the
Bi (CD2O) staining.
Cells were also stained with biotin-conjugated
BI (CD2O) plus irrelevant
murine
IgGl (MOPC
21) followed by
avidin PE plus isotype
specific goat anti-mouse
IgG1/FITC
to
control for MY1O (CD34)
staining.
Flow cytometry
analysis.
Samples were analyzed
either fresh or
after fixation with 1% paraformaldehyde.
Stained
cells were analyzed on an EPICS C flow cytometer
using instrument
settings
as
previously
described.6
Forward-angle
light scatter
(FALS)
gates
were set to exclude RBCs, dead cells, platelets,
or debris as well as
large aggregates.
Ninety-degree
light scatter
(L9OLS)
gates were
set to exclude a distinct myeloid population
with high L9OLS signal
when normal
bone marrow
was analyzed
but were left open for
analysis ofcALL
cells. Fifty thousand
cells were counted per sample.
A two-parameter
64 x 64-channel
histogram
of log green fluorescence (LGFL)
v log red fluorescence
(LRFL)
gated on FALS (or
FALS and L9OLS) was collected.
Data analysis.
Data were analyzed
using the EPICS
C data
analysis
software
(projection
analysis
software),
which
enables
graphic
analysis
of the green fluorescence
of cells showing a given
range of red fluorescence
and vice versa. Thus, cells positive for one
antibody
(eg, CD1O) can be analyzed
for staining
with another
antibody.
For determination
of the percentage
of cells positive for a
differentiation
marker, only cells positive for the CDIO antigen were
analyzed.
A cutoff point was set at a point above which only 5% of
815
the CDIO-positive
cells were stained
with the appropriate
control
antibody.
The percentage
of cells positive for each differentiation
marker
was determined
as the proportion
of CDIO positive cells
above this cutoff.
To determine
the fluorescence
intensity
of a stained
population,
the logarithmic
fluorescence
channel
intensity
was converted
to
arbitrary
linear units based on determination
that an increase of 6.25
log channels
in the 64-channel
two-parameter
histogram
is equivalent to doubling
of the fluorescence
intensity.
This factor
was
determined
using microspheres
of calibrated
fluorescence
intensity.’#{176}
The linear intensity
thus determined
was adjusted
for instrument variability
by comparison
with the fluorescence
intensity
of
standard
fluorescent
microspheres
analyzed
on that day.
CDIO density on the cell surface was estimated
using the FALS
signal, which is proportional
to cell size in cells ofsimilar
structure.”
Since surface area is proj,ortional
to the two-thirds
power of volume,
the CDIO
intensity
was divided
by the FALS
intensity
to the
two-thirds
power to estimate
CD I 0 density. The correlation
of CD I 0
with other markers expressed
on cALL was the same whether
CDIO
intensity or density was used. CDIO density was estimated
primarily
to compare
CDIO expression
ofcALL
and cBML.
Cell sorting.
For cell-sorting
experiments,
only fresh cells were
used. Three-droplet
sorting without coincidence
correction
at a flow
rate of 800 cells/s was performed
at a 32-kHz
droplet
formation
rate. The sorting logic was based on FALS, 90#{176}
light scatter
(for
sorting experiments
with normal bone marrow
to gate out weakly
CDIO-positive
neutrophils)
and red and green fluorescence.
Cells
were sorted into a I .5-mL conical polypropylene
vial containing
0.5
mL McCoy’s tissue culture medium with 10% FCS. The sorted cells
were divided
into aliquots
for immunoperoxidase
staining,
TdT
immunofluorescence,
cell cycle analysis,
and TdT enzyme
immunoassay.
Immunoperoxidase
staining.
Cytocentrifuged
cells were fixed
in formol-buffered
acetone
(200
mg/L
NA,HPO4
and 200 mg/L
KH2PO4
in 45% acetone
and 25% concentrated
formalin)
for 30
seconds at room temperature.
The fixed slides were incubated
for 30
minutes at room temperature
with goat serum to reduce nonspecific
binding. After being washed with PBS, slides were stained with an
appropriate
dilution
of peroxidase
conjugated
goat anti-human
heavy chain or goat anti-rabbit
-y heavy chain
(Tago Diagnostics,
Burlingame,
CA) as control for 30 minutes
at room temperature,
followed by washing in PBS and a 30-minute
incubation
with freshly
prepared
developing
solution
(270 mg/L 3-amino-9-ethylcarbazole
and 0.03% hydrogen
peroxide
in 0.1 mol/L acetate
buffer-pH
5.2,
AEC). After being washed in PBS, slides were mounted
in Aquamount,
kept in the dark at 4#{176}C,
and viewed under a microscope
within 4 hours of staining.
The control preparation
showed
<1%
positively stained cells.
TdT immunofluorescence.
Detection
of TdT antigen
in single
cells by indirect
immunofluorescence
was performed
on methanolfixed cytocentrifuge
slides as described.’
TdT enzyme immunoassay.
TdT antigen
was quantitated
by
enzyme immunoassay
(Abbott
TdT-EIA
kit, Abbott
Laboratories,
North Chicago)
as described.’2
Cells (35,000)
were frozen for 12
hours at - 10#{176}C
in phosphate
buffer and added to a microwell
containing
a microbead
coated with goat anti-calf
TdT antibody.
After 90-minute
incubation
at 30#{176}C,
the beads were washed
in
distilled
water,
and rabbit
anti-calf
TdT-horseradish
peroxidase
conjugate
was added to each well. After 90-minute
incubation
at
30#{176}C,
the beads were washed
and incubated
with O-phenylene
diamine/H,02
substrate
solution for 30 minutes
at room temperature. The enzymatic
reaction
was terminated
by addition
of 1.0 mL
5% HSO4,
and the absorbance
was read at 492 nm on a Quantum
1
spectrophotometer
(Abbott
Laboratories).
The TdT antigen concentration in the sample was calculated
from a standard
curve gener-
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
816
RYANETAL
ated by analysis
of standards
containing
0, 5, 10, and I 5 ng
TdT/mL.
The TdT antigen content per cell was calculated
based on
the initial cell count in the analyzed
sample prior to freezing.
Cell cycle analysis.
Cellular
DNA content was determined
by
the method of Krishan.”
Sorted cells were centrifuged
at 400 g and
vigorously
resuspended
in Tris-PI
solution
[1 .21 g/L Trizma
base,
480 mg/L MgCl,, 0.1% Triton X-lOO, 50 mg/mL
propidium
iodide,
and 2 mg/mL
RNAse A (Sigma, St Louis)]. After at least I hour of
incubation
at 4#{176}C,
the cells were vigorously
resuspended
by 25 to 40
aspirations
in a Pasteur
pipette and analyzed
on an EPICS C flow
cytometer.
The percentage
of cells in S+G2/M
phases of the cell
cycle was calculated
by integrating
the region of the DNA histogram
(linear
red fluorescence
reflected
from a 560 dichroic
filter and
collected
through
a 575-band
pass filter) above the G0/G, peak. At
least 5,000 to 10,000 nuclei were counted for each sample. Cell cycle
analysis was performed
on sorted cALL cells; hence, no admixture
of
normal cells was present.
Combined
immunofluorescence
and immunoperoxidase
stain-
ing.
For detection
of cytoplasmic
heavy chain (Cs) in TdTpositive cells, a combined
immunofluorescence/immunoperoxidase
procedure
was performed.
Cells were cytocentrifuged
and fixed in
formol-buffered
acetone as described
above, then stained with rabbit
anti-calf
TdT followed
by FITC-conjugated
goat anti-rabbit
1g.
After the final wash, cells were incubated
with normal goat serum
for 30 minutes and stained with peroxidase-conjugated
goat anti-s
heavy chain as described
above for immunoperoxidase
staining.
The
percentages
of TdT-positive
and Cs-positive
cells were similar
to
those observed with separate
staining
for these antigens.
Table
CD1O
Patient
No.
.
Expression
cells
(n
10) from
from
with
liquid
nitrogen
anti-CD1O
FITC
=
a single
patient
with
cALL
CD34
PNA
CLA
100
thawed
days
mean
calculated
CD1O intensity
was 1 1 3 (arbitrary
linear
fluorescence intensity
units)
with an SE of 6.3. Similarly,
precision
of FALS
measurement
was determined
by measuring
FALS
of aliquots
(n = 1 0) of cells from a single patient
with cALL
over the course
of 3 months.
The mean
FALS
intensity
was
357 (arbitrary
linear
intensity
units)
with an SE of I 1.7.
Staining
for CD I 0 was performed
multiple
times
on each
cALL
sample,
yielding
consistent
results.
The percentage
of cells positive
for differentiation
antigens,
CD1 0 density,
and TdT
intensity
(as measured
by
enzyme
immunoassay)
for leukemic
cells from each patient
are listed in Table
1 . Adult
patients
with cALL
had significantly
higher
numbers
of CD34-positive
cells
(mean
Antigens
in cALL
TdT
. .
CD1O
were
storage
and stained
on different
over the course of 3 months.
The
Cells Positive (%)
.
(MFI)
Phenotype
of cALL
cells.
Ficoll/Hypaque
separated
cells from bone marrow
or blood of 25 patients
with cALL
were frozen
in liquid
nitrogen
for phenotypic
analysis.
The
proportion
of leukemic
cells in the samples
was at least 80%,
and usually
>90%.
Two-color
immunofluorescence
was used
to identify
differentiation
antigens
specifically
on CD 10positive
cells. To evaluate
the precision
of CD1O
intensity
measurement
by immunofluorescence,
multiple
aliquots
of
of Differentiation
Dens,ty
Ci
.
CD2O
TdT
CD21
CD19
Ia
Intensity
(fg/cell)
1
106
95
21
21
1
100
60
3
100
100
35
2
20
94
0
98
99
5
6
94
0
79
98
17
3
37
81
5
60
35
84
22
69
15
100
96
1
4
5
74
0
0
95
89
0
0
0
100
88
0
27
5
12
96
0
93
6
83
35
70
8
99
98
6
79
97
70
10
5
0
60
95
8
92
89
12
7
106
85
98
99
18
6
100
58
49
96
97
45
8
39
91
85
0
70
44
40
83
0
95
97
26
9
12
83
4
97
3
2
2
90
2
99
97
i8
10
92
84
74
46
0
4
7
97
0
100
11
130
80
99
0
7
0
18
83
1
99
12
125
86
72
30
10
69
95
95
17
99
ND
69
13
56
84
48
0
5
0
89
85
40
95
ND
31
14
61
85
30
12
3
ND
10
ND
4
80
ND
15
32
89
7
84
9
0
7
88
0
99
ND
74
99
17
23
ND
25
16
8
86
19
0
7
36
34
77
0
99
ND
12
17
12
96
8
12
2
43
24
79
12
99
ND
12
18
180
40
99
0
2
19
77
2
94
ND
ii
19
13
46
0
1
10
10
62
0
70
ND
26
20
169
93
50
10
8
7
91
44’
91
ND
21
103
63
91
4
0
0
63
72
17
93
96
24
22
22
86
21
85
1
2
58
86
2
90
89
58
23
32
95
3
77
0
6
12
71
43
83
82
13
24
7
50
0
100
95
55
13
56
0
99
78
85
66
78
11
2
69
67
0
99
98
iS
82
42
41
21
24
36
75
ii
94
92
22
100
34
56
59
33
50
18
5
85
90
25
121
cALLMean
63
cBMLMean
16
Abbreviations:
MFI.
i
RESULTS
P NA,
mean fluorescence
Very weakly
positive;
peanut
intensity
cBML
agglutinin;
(arbitrary
data
from
CLA.
comm on leukocyte
linear units).
ref 9.
antigen;
0
37
2
Cz, cytoplasmic
t
heavy
cha in; TdT, terminal
tra nsferase;
15
0.2
ND
N D, not done;
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
OF NEOPLAST1C
PHENOTYPE
72%
n
17%
±
19).
=
these
SEM;
groups
had
n
No other
(mean
was
70%
clinical
higher
17%;
±
n
parameters
6)
=
cells
rather
6)
than
closely
count,
(Table
expressed
marrow).
from data previously
which
was demonstrated
cells
the sensitive
from
cytoge-
antigens
cBML
consistently
in normal
at first
correlaand other
sex,
normal
by
groups.
divide
the
Figure
cALL
1 shows
patients
the
into
expression
CD2O-positive
TdT
likely
more
Weakly
likely
to
to be CD34-positive
CD1O-positive
cALL.
percentage
cBML
TdT.
with
of TdT
two populations
those
observed
A trend
(Fig
1 B), with
cALL
signifi-
positive
of cALL.
in strongly
the notable
cells
in
cALL
is strongly
and
correlated
normal
with
findv weakly
exception
of
previously
marrow.
CDIO
was
These
Figure
1 B is derived
from data
the exception
of Cs expression.
expression
be
or
TdT
intensity
in
marrow
40
Patients
as was
nearly
of
less
strongly
in the
CDIO-positive
technique.
two
difference
expression
significantly
as corn-
cBML.
PNA activity
in weakly
CDIO-positive
statistical
significance.
No statistically
demonstrated
in the
ings are similar
to
published,’
in 32.0%
cALL
CD1O-positive
was
but
than
toward
higher
did not reach
CDIO-positive
v weakly
or Cz-positive
CD2O
and
published,6
previously
done for normal
marrow
CDIO
positive
cells.’ A
level of 50 fluorescence
intensity
units for CD1O density
was
to
v weakly
strongly
cALL
in cALL)
The pheno-
CDJO expression
in cALL
and normal
marrow.
with cALL
were grouped
according
to CD1O intensity
used
CLA-
cant
1), with
bone
immunoperoxidase
by strongly
with
CD1O-positive
cells
studied
markers
pared
at relapse
No significant
phenotype
leukocyte
8%;
±
between
PNA-positive
patients
resembled
CD1O-positive
using
30%
studied
of
did
initial
of cBML
was obtained
the exception
of Cit,
=
(mean
817
difference
group
of patients.
of most differentiation
the exception
of TdT (more
and CLA (higher
expression
(n
CELLS
Patients
numbers
(age,
cALL
sorted
children
(mean
30% ± 8%; n - 19).
observed
between
antigenic
survival)
in this
mean
expression
of
CD1O+
phenotypic
demonstrated.
netics,
The
type
with
than
6)
=
significant
significantly
diagnosis
tion was
v NORMAL
35
3o
25
:‘
equal
differentiation
20
15
:
.
5
100
.
m
90
80
100
strongCALLA
+
-
.
CD34
80
70
Low
A
90
Weak CALLA
+
C1A
CD2O
CLA
CD2O
U
70
#{149}
60
6o
50
40gt
20
80
70
20
10
CD,0
Density
CD4
PNA
CIA
CL
TdT
CD2O
#{149}#{149}
100
-.
100
D
90
90
U
80
.
70
.
.60
50
50
40
U
301
20
10
CDIO
Density
CD4
PNA
HighLow
CDIO
-
CD34
Density
+
.
CIA
Ct
CD2O
TdT
Fig 1.
Comparison
of weakly
(n - i3)
v strongly
(n = i2)
CDi 0-positive
cALL
(A). or weakly
v strongly
CDi 0-positive
cBMLs (n - 6 to 9) (B). Antigen
positivity
(except
TdT and Cit) of
CDiO-positive
cALL or cBMIs
was determined
by two-color
immunofluorescence.
TdT and Ct were
measured
on cytocentrifuged
cAll
cells or cBMls
sorted
on the basis of CDiO intensity.
Data
shown in panel B were previously
published’
and are shown
here
for comparison.
Ordinate
shows
the mean
percentage
of cells
positive
for each antigen
indicated
on the abscissa.
For CDi 0. the
ordinate
represents
the mean
CD1 0 density
of the weakly
or
strongly
CD1O-positive
populations.
Error bars = SEM. Confidence
limits
(P
<
.05;
P < .OOi ) for the difference
between
weakly
and strongly
CDiO-positive
cells were calculated
using the paired t
test (cBMLs)
or the unpaired
t test (cALL).
Fig 2.
(A) TdT expression
in cALL. Ordinate
shows TdT intensity
measured
by enzyme
immunoassay.
Solid columns:
Patients
positive for antigen
indicated
on abscissa
or displaying
high
CDi 0
density.
Stippled
columns:
Patients
negative
for antigen
indicated
on abscissa
or displaying
weak
CD1 0 density.
Patients
were
considered
positive
for an antigen
if at least 30% of the CD1 0positive
cells were positive
for that antigen.
Patients
were considered strongly
CDiO positive
if the Ieukemic
cells displayed
CD1O
density
of at least
50
mean
fluorescence
intensity
units
(MFI).
(B) TdT expression
in sorted
subpopulations
of cBMLs
(n - 5).
TdT positivity
of CDiO.
CD34.
CD2O, and CIA subpopulations
of
CD1O-positive
cells was determined
by sorting
for TdT immunofluorescence
staining
on cytocentrifuge
preparations.
Ordinate
shows
percentage
of TdT-positive
cells. Solid columns:
Sorted
CD1O-positive
cells positive
for antigen
indicated
on abscissa
or
displaying
high CD1O density.
Stippled
columns:
Sorted
CD1Opositive
cells negative
for antigen
indicated
on abscissa
or displaying low CD1O density.
The cutoff for strong
CD1O positivity
was
selected
to best distinguish
the two
subpopulations
evident
in
normal
marrow
and was -25 fluorescence
intensity
units. Error
bars
SEM.
Confidence
limits
( P < .05;
#{149}
P < .001 ) for the
difference
between
TdT expression
of antigen-positive
and antigen-negative
cells were calculated
using the paired
t test
(cBML)
or the unpaired
t test
(cALL).
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
RYAN
818
normal
marrow.’
preparations
were
Examination
revealed
TdT
that
positive,
variable,
as
measured
intensity
previously
by
of TdT
although
of TdT
was
between
no
positive
and
and
intense
TdT
pattern
is
or CD2O
CD34
and
a strong
positivity
in Ct
to
between
Cytoplasmic
row.
As shown
ence
than
contrast
positivity
and
t
and
CD2O-positive
expression
in
correlation
TdT
cALL,
cALL.
did
expression
CD2O-negative
cALL.
show
correlation
a
intensity
cALL
and normal
was no significant
mardiffer-
CD2O-positive
2B).
and
CD2O-
70
60
strongly
However,
expression
with
and
Cj expression
TdT-positive
CLA
and
expression
CD34
positivity
in cALL.
was
The
weakly
was
corre-
inversely
pattern
of CD2O
and TdT reactivity
in cALL
is in contrast
to cBMLs,
which
show a strong
correlation
of both CLA and CD2O positivity
with Cs expression
and a strong
inverse
correlation
of both
CD34
and
relationship
19.4%
were
TdT positivity
with CM expression
(Fig 3B).
of Cs and TdT expression
in normal
marrow
assessed
by combined
staining
CD1O-positive
cells, as described
section.
±
4.3%)
found
Although
and
in sorted
Subpopulations
tients.
Subpopulations
patients
were
both
Cs-positive
of
observed
with
CD34
and
4A
B),
F). These
(mean
±
Ct
were
cells
5.1%)
populations
respect
Figure
4 shows
of leukemic
cells
(Fig
19.0%
(n
=
extremely
5),
rare
cALL
cells
in individual
paof cALL
cells
within
individual
CD1O
positivity.
subpopulations
4E and
TdT-positive
(mean
TdT
and
cells).
The
was
for CM and TdT in
in the Materials
and
CD1O-positive
cells
positive
for both
(0.025%
of CD1O-positive
(Fig
50
C,
correlated
Methods
and
or between
cALL.
with
This
CLA
(Fig
lated
directly
sorted
strong
cALL
TdT-positive
more
between
positivity
between
and
showed
or CDIO
in
there
CLA-
CD34-positive
cALL
positivity
TdT
expression
cALL,
which
expression
in Fig 3A,
to expresin Fig 2A,
TdT
cBMLs,
inverse
was
quantitative
be related
As shown
in
quite
intensity
CD1O-positive
CLA-negative
CD34-negative
TdT
difference
weakly
cells
was
to detect
that might
markers.
significant
strongly
expression
immunoassay
negative
all the cALL
demonstrated)4
enzyme
changes
in TdT expression
sion of other
differentiation
there
immunofluorescence
nearly
ET AL
CD2O
to CD34,
examples
positive
(Fig
4C
subpopulations
CD2O,
and
of patients
and negative
with
for
and
were
D),
studied
and
CDIO
further
to
U
4o
3o
U
20
8B
‘9
2
10
I,
>20
<20
TdT
Intensity
(fg/cell)
+
-
CD34
eBkT
+
+
CLA
CD2O
A
Control
CD34
70
60
ci)
50
.
40
8ri
03O
C
Control
D
___
CD2O
+
TdT
CD34
CLA
CD2O
Fig 3.
(A) Cgs expression
in cALL. Ordinate
shows percentage
of Cs-positive
cells. Solid columns:
Patients
positive
for antigen
indicated
on abscissa
or displaying
strong TdT intensity.
Stippled
columns:
Patients
negative
for antigen
indicated
on abscissa
or
displaying
weak TdT intensity.
Patients
were considered
positive
for an antigen
if at least 30% of the CD1O-positive
cells was
positive
for that antigen.
Patients
were considered
strongly
TdT
positive
if the leukemic
cells displayed
TdT intensity
of at least 20
fg/cell.
(B) Cs expression
in sorted
subpopulations
of cBMIs
(n - 5). Cp positivity
of TdT-positive
and TdT-negative
cells was
determined
by combined
TdT immunofluorescence
and Cs immunoperoxidase
on sorted CDiO positive
cells. Cgs positivity
of CD34.
CD2O. and CLA subpopulations
of CDiO-positive
cells was determined
by sorting
these subpopulations
for Ca-positive
cells. Solid
columns:
Sorted
CD1 0-positive
cells positive
for antigen
indicated
on abscissa.
Stippled
columns:
Sorted
CDiO-positive
cells negative for antigen
indicated
on abscissa.
Error bars - SEM. Confidence limits (#{149}
P < .05;
#{149}
P < .001 ) for difference
between
TdT
expression
of antigen-positive
and antigen-negative
cells were
calculated
using the paired
t test
(cBML)
or the unpaired
t test
(cALL).
!;E2
..
Control
_____
CD19
Fig 4.
Subpopulations
of CD34 (A). CD2O (B). and CDiO
(C)
positive
and negative
cells in three individual
patients
with cAll.
Samples
were stained
with an anti-CD1
0 monoclonal
antibody
(J5
biotin) plus another
marker
or control as described
in the Materials
and Methods
section.
In all histograms,
CDi 0 intensity
is represented
on the ordinate
and the other marker
or control
is represented
on the abscissa.
For each histogram.
50.000
cells were
counted.
The contour
lines indicate
increasing
number
of cells in a
given
channel
of the 64 x 64-channel
histograms.
(A) Control
v
CDiO.
(B) CD34 v CDiO. (C) control
v CDiO.
(D) CD2O v CD1O. (E)
control
v control.
and (F) CD19 v CDiO.
Both CD34-positive
and
CD34-negative
subpopulations
of CD1O-positive
cells are apparent
in B, and both CD2O-positive
and CD2O-negative
subpopulations
of
CDi 0-positive
cells are apparent
in D. Both CDi 0-positive
and
CDi 0-negative
subpopulations
of cells staining
with the B cellspecific
marker
CDi9
are present
in F.
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
PHENOTYPE
OF NEOPLASTIC
determine
if consistent
tiation
antigens
CDiO+
v NORMAL
differences
could
CELLS
in expression
be identified.
CD1O
at
of analysis
819
FALS
CDIO
inten-
were
determined
cytometer,
as described
tion.
Sorted
cells
iodide
for reanalysis
cycle
were
kinetics
time
in the
dium
cell
the
Materials
centrifuged
of
the
flow
were
and
fluorescence.
cells
Finally,
sorted
in each
differences
sorted
subpopulations.
larger
and
lower
were
than
a slightly
showed
compared
no
Significant
differences
TdT-positive
cells
subpopulations
as
well
The
described
cells
or percentage
in kidney,20
to bone marrow.
may
be closely
This suggests
related
to
CD1O
identified
not found
two
phenotype
positive,
in
that these
more
mature
on
B cell
cells
(ie,
positive
positive
ALL
to those
expression
in
degree
ity’5
and
the
whom
of CD34
patients
ity
(55%
1 5%,
and
than
combined
described
the
CD1O-negative
The CD1O-negative
cells demonstrated
2).
64%)
respectively).
In six patients
six
=
than
respectively).
CD1O-/CD34+
(29% and 3%)
CD1O-negative
cells
demonstrated
positivity
(n = 2) as a sign of immatur-
in whom
CD34
negative
(n
with CD1O-/CD34-
the
cells
cells of patients
higher
Cj positiv-
CD1O-positive
cells
(I 1% and
The
CD1O-negative
cells
cells
the
demonstrated
CD1O-positive
lower
Cs
cells (58%
with
both
CD34
and
were
of patients
CD2O
the
CD34-positive
cells
as
Table
FALSt
based
light
on paired
NS, not significant.
of
CD2Othan
if they
strongly
cells
the
The
CD 10suggests
larger
demonstrate
popula-
the
same
v weakly
in expression
and PNA that
marrow.
CD34
Indeed,
higher
in strongly
CD1O-positive
cALL,
of CD2O,
marrow.
quantitative
CLA,
of differenwere similar
CD2O
cALL
differ-
In contrast
to cBML,
cALL
TdT expression
despite
the
or Cz.
and
expression
CD1O-positive
opposite
to the
In normal
marrow,
of CD2O,
CLA,
or
in cALL
was negatively
expression
coexpression,
rare
of Sorted
which
in cBML.
positively
the
Cz
correlated
loss
is nearly
correlated
with
CLA
is common
TdT+/Cs+
in cALL
cells
occurred
but
exceedingly
with
a frequency
c ALL Subpopulations
NS
64
51
785
720
841
774
t test.
recently
CD34-,
TdT-,
CLA+,
from 25 patients
with CD 10-
differences
Ct, CLA,
with acquisition
Cj expression
62
<.02
We
expression,
as in cBML.
However,
in contrast
to cBML,
TdT
and CD2O expression
were unrelated
to the presence
of Cz in
cALL.
This is particularly
striking
in the case of TdT/C.t
be
5
confined
of cBMLs.’
as found in cBML.
cells that were strongly
in normal
in normal
the same
62
3. 1
scatterintensity.
found
retain
CD34+
M (%)
#{149}P
values
ence
cells
P
(fg/cell)
tRelative
Characteristic
are
CD2O-
Cytoplasmiclg(%)
+
to
significantly
in weakly
CD2O+
CDl0density(MFI)
TdT intensity
2.
likely
as they
was
than
with
subpopulations
were
cells
of TdT
complete.
positivity
and 32%,
revealed
CD34,
CD1O+/
are
observed
in cBML.
In contrast,
CD2O
and TdT
were not related
to CD1O
intensity
of the cALL
presence
with
staining
for CD34
and CD2O
was performed,
as
in the Materials
and Methods
section.
In five of the
patients,
S + G2
in whom
cells
into
immature
to determine
normal
to CD 10have been
node,22
(cBML).
population
CLA-,
more
same patients.
We therefore
we sorted
CD1O-negative
subdivided
the patients
and
CD1O-positive
lymph
subpopulations
weakly
CD1O-positive,
cells. We studied
cells
positive
antigens
in
that the cell of origin
in cALL
the normally
occurring
bone
tion of
CD2O+
CDIO
tiation
occurring
phenotype)
lymphocyte
minority
TDT+,
are
and
the cALL
phenotypic
of a
CD34+,
cell
thymus,2’
cells
phenotypic
heterogeneity
Comparison
of cALL
patients
cells. In only one
identify
separate
phenotypic
similarity
CD1O-positive
cells
TdT+/CD19+
marrow
of
that CDIO
is absent
the CD19+/TdT+/
as on
lymphoid
the same
or TdT
intensity
the same
patient.
were
CD1O-positive
neoplasms.
Thus,
the sorted
CD1O-negative
cells may be
either
more or less mature
cells than the CD1O-positive
cells.
These cells did not represent
residual
normal
cells, since they
were TdT positive,
as were the CD1O-positive
cells from the
some
of B cell
of cells.
in CDIO
CD I 0-positive
by immunofluorescence
ALL
subtypes
positive
as were the CD34-negative
did the CD34
and CD2O antigens
bone
marrow
has a close
positive
ALL.”9
Although
and
CD2O-negative
and did demon-
kinetics
of the groups.
Phenotypic
studies
of ALL suggest
the most immature
subtype
of ALL,’
(“null”)
CD19
Pro-B ALL
DISCUSSION
any
CD1O-
CD2O
CLA
CLI
Ia
TdT
ALL
Comnn
of differentiation-related
of
slightly
from
with
larger
expression
cells from
cycle
pairs
density
no difference
intensity.
in cell
I 0#{176}C
for
were
cells
showed
TdT
CD2O
patient
-
CDIO
CD34-negative
lower
Ia
CDio
i9
Common ALL
Fig 5.
Hypothesis
lineage ALL.
using
an
immuno-
the
cells
higher
difference
in Cz
with CD1O-negative
at
between
density
or Cz expression
as compared
cells from the same patient,
but were
strate
propi-
TIlT
CDI9
ALL
null
to evaluate
frozen
parameters
cells
with
Ia
CD19
sec-
-
± CD2O
antigen
by enzyme
immuand significance
levels for
significantly
CD2O-positive
Ia
flow
o
I
Cytocentrifuge
CD34-positive
showed
Cs expression
patient.
stained
cytometer
stained
for Cz
for TdT using
of TdT
the means
of these
the
Methods
subpopulations.
preparations
of sorted
cells
immunoperoxidase
technique
quantitative
measurement
noassay.
Table
2 shows
and
and
on the
on
__
t CD2O
++
C1304
TdT
?CD34
TdT
sity
__
of differen-
and
CD34-
P
<.05
<.01
CD1O+
860
P
CD1O-
-
-
818
NS
3.9
<.05
5.4
4.9
NS
3.2
3. 1
58.4
58.1
NS
14.0
39.7
<.05
29.0
37.6
NS
NS
19.1
20.7
NS
13.7
13.3
NS
22.7
23.8
NS
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
RYAN
820
of I /4,000
marrow
have also demonstrated
CD1O-positive
the rarity
cells.
Previous
of the TdT+
/Ci+
studies
pheno-
explanations
for this observation.
CD2O expression
between
cBML
the
loss
of specific
normal
type.23
potential
A
complicating
lineage
and
ferent
patients
is
patients
with
might
obscure
this
issue,
that
CDIO.
positive
findings
inversely
intensity
to Cz
in cALL
CD2O.
The
evidence
patient
to patient
and
increased
and Ct expression,
phenotypically
more
data
case
unrelated
presented
differences
above
within
that
to
no
between
sorted
subpopufor CDIO,
CD34,
and
suggests
individual
CD34,
CLA,
similar
to cBMLs.
that
both
patients,
cALL
CD1O,
and
CD34-positive
expression
of CD 10 and
suggesting
“immature”
that
they may
form ofcALL
biologically
of CD2O
to
C
than
marrow
CDIO-positive
cells which
their normal
counterparts.24
There
are widely
are several
and
CDIO
appears
CLA,
the observed
cALL
tiation
stages
that
regulation
reasons
The
of
cells,
hypothesis
CLA,
inverse
association
but it is notewor-
the expression
of CD34,
to be coordinated.
cells.
Finally,
of differentiation
CD1O,
modulation
related
and
CD2O
(Figs
among
Cs,
CD2O,
during
potentially
The
biologic
but
ALL
in cALL
features
Prognosis
cells
CD34+
cALL
sample
concerning
with
for
subpopulations
3B and
4B).
CLA,
and
DNA
of
The
TdT
of persistent
is at present
seems
in this
and
is thereB cell.
expression
subtypes
Weakly
to be more
prevalent
is too small
Prognosis
intensity25a
of TdT
shown
to
in
unknown.
study
that appear
to be related
in ALL is not related
strong
expres-
mature
TdT
related
information.
prognosis.
CDIO
synthesis,25
to a proliferating
of differentiation
useful
clinical
the patient
statement
in vitro
detrimental
consequences
proliferating
differenin rare
or down-
occur
sion in cBML
suggests
the possibility
of inhibition
production
by one of these proteins.
TdT has been
fore
Cii,
Alternatively,
by the strong
relationships
demonstrated
between
expression
of TdT
or CM and
CD34,
be mutagenic
pheno-
disorgani-
phenotypes
may reflect
normal
are transient
or found
only
of normal
and
of cALL
CD1Oin adults,
to make
in cALL
C
any
has been
expression,26
two
to each other in this study.
to TdT activity.’4
Compari-
son of antigen
expression
during
in vitro differentiation
of
cALL
and cBML
cells under
conditions
that
permit
cell
growth27
will be important
in determining
the biologic
significance
of the phenotypic
differences
observed
in this study.
certain
with the
considpossible
in cALL
The
a generalized
of certain
cell markers
may
unrelated
to cell differentiation.
correlated
CLA
in cALL.
reflect
in malignant
that
positive,
classification
in this study,
however,
indicate
in cALL
cells as compared
expression
cells
represent
a
than CD34-
merely
regard
Identification
may
provide
Classification
of differentiation
of CD34 or CLA expression
may
relevant
expression.8
ofgene
The differences
in TdT and
and cALL
may result
from
regulatory
signals
as a
differentiation
may
in this
from
in a
cALL
reduced
of aberrant
differences
cBML
is supported
in normal
marrow
The
CD2O
similar
thy
in
to CD34
expression.
TdT
or
typic
subpopulations
unreTdT
described
were not likely
cycle,
since
there
was
distribution
or negative
(Fig
5).
on the basis
therefore
be more
cALL
on the basis
bone
ered
is the
a correlation
presented
express
the markers
coordinated
fashion
The data
phenotypic
the
as
was
differences
in cell
or
CD34 expression
in
with CD1O
intensity
expression,
showed
difference
in cell cycle
lations
of cells positive
negative
cALL
stages
of cALL
CD2O,
consequence
zation
individual
corroborated
subpopulations
in cALL
show
these
expression
in cALL
subpopulations
was
or Cz expression,
in contrast
to cBMLs.
found in cBMLs.
The
be due to differences
cells
from
for CD34,
substantially
dif-
history
that
To address
of cells
or negative
expression
but inversely
related
to CD2O
latter
finding
is the only
occasion
when
expression
from
in
or natural
differences.
of separate
patients.
was correlated
related
cBML.
CD2O
lated to CD1O
of cell
cells
heterogeneity
causation
related
subpopulations
were
The
in investigations
malignant
unrecognized
obtained
from study
cALL
subpopulations
and
using
regard
to
differentiation
we studied
patients
factor
differentiation
ET AL
ACKNOWLEDGMENT
We would like to thank Steven
excellent
technical
assistance.
Mitchell
and Cynthia
Fasano
for
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1987 70: 814-821
Phenotypic similarities and differences between CALLA-positive acute
lymphoblastic leukemia cells and normal marrow CALLA-positive B cell
precursors
DH Ryan, CW Chapple, SA Kossover, AA Sandberg and HJ Cohen
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