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Ambiguous
Phenotypes
and
Characterized
By Wolf-Dieter
Ludwig,
Claus
Jochen
Ambiguous
phenotypes
16 children
with
netic,
molecular
ses
as
studies
well
as
were
used
The
first
mia
whose
while
of
the
comprised
five
cells
globulin
(Ig)
netic
were
t(4;1
of the
populations.
cell
one
heavy-chain
A
Further
was
morphologic
and
gene
are thought
progenitor
cell
by several
separate
studies
leukemic
phoid and
cells
myeloid
blast-cell
of a pluripotent
stem
lymphoid
and myelomonocytic
tion,
gene
aberrant
mogenic
event,
progenitor
antigens,
Various
School;
the
of
the
and
ofUlm,
Submitted
May
Supported
in part
Address
8, 1987;
reprint
©I 988
costs ofthis
This article
in accordance
of Giessen.
Hannover
Medical
findings
leukemia
30, /000
article
must
with
Krebshilfe
Based
We present
Ba 770/2-2.
Berlin
MD,
Inc.
the
or
third
insuffi-
Depart-
lineage
exists
implications
in a small
number
heterogeneity
by application
of acute
of recombinanV
heavy-chain
immunoglobulin
either
the
than
heterogeneity
findings,
lymphoid
of 16 children
comprised
cell
features.
understand
a multimodal
the
observed
acute
leukemia,
or genotypic
fea-
characterized
populations
by
demonstrating
Our
results
approach
emphasize
in order
heterogeneous
in a higher
biclonal
homogeneous
or were
or myeloid
and
with
phenotypic
lineage
biclonal
and
relatively
cell
for
et a12#{176}
proposed
biphenotypic,
of separate
necessity
previously
Andreeff
with
one
is the coex-
lines with different
differ
in their
pheno-
monophenotypic,
subtypes.
either
than
cell
monoclonal
populations
coexistence
genotypes
45, FRG.
these
cells
of more
mine
University
mixed
nomenclature
in acute
myeloid
leukemia
acute lymphoblastic
leukemia
(Tgene rearrangement
in T lineage
the data
blast
to deter-
phenotypes
proportion
of acute
and
leukemias
realized.
were defrayed in part by page
therefore
be hereby marked
/8 U.S.C.
section
1 734 solely
MATERIALS
to
Patients.
& Stratton,
on
monoclonal
leukemia
whose
and by
acute
analyzed
lineage
of genotypic
biphenotypic,
monophenotypic
the
Free
in
and clinical
two or more
leukemic
that may not necessarily
between
Ulm,
13, /988.
and
been
illustrated
discriminating
tunes
Ludwig,
took
pluripotent
expression
accepted
demonstrating
expression
istence
of
karyotypes
morphologic
Steglitz.
the
lymphoma,’5
as well as rearrangement
of the gene coding
for
the /3 chain of the T cell receptor
(/3-TCR)
in AML and B cell
precursor
ALL’#{176}”’9 or -y-TCR
in pre-B cell ALL.’9
of
Klinikum
trans-
Inc.
only
(Ig)
gene
rearrangement
(AML)’#{176}’2 and T cell
ALL),’#{176}’3”4kappa-chain
of
Wolf-Dieter
content.
patients
phenotypes
the incidence
been
methods
University
to
of
one
cytometry
DNA
malignant
to
(Ig
in
flow
group
leukemia,6’7
have
has
DNA
University
project
rearrangefeatures
cell
that
gene
no generally
of such
types.
Steof
January
did
close
aberrant
acute
Furthermore,
Medicine,
this fact.
0006-4971/88/7106-0035$3.00/0
1518
leuke-
Klinikum
the Deutsche
gene
patients
& Stratton,
as yet.
Medicine,
Hindenburgdamm
by Grune
biphenotypic
hybrid
of
accepted
rearin five
specifity.
Another
University
Internal
Gene
rearrangement)
ambiguous
from
by Grune
Transfusion
by a grantfrom
requests
“advertisement”
leuke-
FRG.
ofHematology/Oncology,
The publication
charge payment.
the
Pediatrics,
Forschungsgemeinschaft,
of West-Berlin,
indicate
of
Departments
M#{252}nster, University
the Deutsche
of
whereas
reagent
a 1988
myeloid-linked
of Hematology/Oncology.
Department
with
the
differentia-
of the applied
markers.2’3
to describe
such cases, eg,
M#{252}nster, University
the
malig-
hematopoietic
and
infidelity,4
Departments
or
leukemic
from
resulted
and
leukemia
bigenotypic
second
of patients.8”#{176} Additional
lym-
(1)
cell
of rare
lymphoid-
lineage
the Department
Berlin;
University
ment
resulting
immortalization
coexpressing
promiscuity,2
From
Ulm;
expression
or (4) lack of specifity
terms have been proposed
lineage
glitz,
(3)
cells
of
cell.
group
myeloid
antigens.
and
ontogenetically
leukemia,8”#{176} but
pathques-
blasts
by:
stem
mia,5
involving
findings
a stage
and
be demonstrated
suggest
and
lym-
antigens.
of leukemic
we
group
phenotype.
light-chain
16
last
acute
for
expressing
explained
at
the
first
cell
gene
findings.
in the
place
Ig
ALL.
abnormalities
on these
cient
Ig
of differ-
myeloid
nant
transformation
potential
for both
(2)
Sur-
or lymphoid
fidelity
was
and
been
Ig
from a single
leukemias
These
have
of
oligoclonal
simultaneously
features.2
heterogeneity
early
T
could
/9-TCR
of
cell
acute
of
with
none
formation
commitment
expression
acute
Based
surface
with
to arise
myeloid
lineage
of lymphoid
populations
individual
clonal
rearrangement
in which
reporting
In
disclose
patients.
showed
as monoclonal
patient.
The
or T cell-associated
additional
and
Hell,
typical
pre-B
unequivocal
children
Cytoge-
demonstration
in all five
patients
patients.
in two
(fi-TCR).
early
coexpression
heavy-chain
separate
other
of B cell
by
is restricted
to either
However,
more
recently,
tioned
the
evidence
five
of two
six
Gerhard
otherwise
of Ig heavy-chain
revealed
and
coexistence
provided
LEUKEMIAS
abnormal
entiation
ways.’
the
children.
and
ment
immuno-
patients.
these
of
Molecular
receptor
with
(ALL).
with
rangement
expres-
features.
T cell
of myeloid
children
(AML)
leuke-
lymphoid.
simultaneous
rearrangements
of
as well
CUTE
with
1 6 patients.
myeloid
gene
prisingly.
children
in all
one
patients
heavy-chain
two
groups.
as
Hansj#{243}rg Riehm
cases
leukemia
three
of
the
phoblastic
into
acute
of the
Leukemia
Hiddemann,
and
of four
patients
of heavy-chain
indicated
features.
these
analy-
chain
consisted
coexpression
myeloid
three
with
cytometric
the
cytochemical
rearrangements
in
in
cytoge-
Acute
Wolfgang
V. Teichmann,
and
evidence
available
analyses
pre-B
flow
observed
With
Analysis
Raghavachar,
Johannes
marker.
morphologically
and
showed
1 ). In five
marker
cell
gene
data.
were
disclosed
pre-B
studies
DNA
Anand
Seibt-Jung,
Surface
morphologic
to divide
blast
early
genetic
in
and
J#{246}rg
Ritter,
Hannelore
genotypes
genetic.
immunophenotyping
sion
cell
and
leukemia.
standard
group
R. Bartram,
Harbott,
acute
Genotypes
in 16 Children
by Multiparameter
AML
and
immunophenotype
From
500
April
with
1984
ALL
analysis
AND
METHODS
to September
were
as
part
1986,
referred
of
to
the
AML
80 children
our
laboratory
and
Blood, Vol 71, No 6 (June), 1988:
ALL
with
for
BFM
pp 15 18-1528
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
AMBIGUOUS
PHENOTYPES
(Berlin-Frankfurt
wise
GENOTYPES
MUnster)-trials-
typical
antigens,
AND
AML,
mostly
ten
I cell
IN ACUTE
LEUKEMIA
1983. Of 80 children
showed
antigens
evidence
of
CD7,
CD4,
(ie,
1519
with other-
(Tago,
lymphoid-associated
and
CD2),
whereas
myeloid-related
antigens
were detected
on blasts from 20 patients
with ALL. Five children had two distinct cell populations
with either
myeloid or lymphoid
features.
However,
adequate
cell numbers
for
more extensive
studies of surface
antigen
expression
or molecular
genetic analyses
were available
only from the I 6 patients described
in this
report.
In
administration
14 patients,
samples
of induction
were
investigated
chemotherapy.
Two
before
patients
were
by
the
ied at relapse.
Morphology
and
cytochemistry.
The
morphologic
diagnosis
Schiff
reagent
acetate
(PAS),
esterase
myeloperoxidase
(ANAE),
and
evaluated
by the reference
sity Children’s
Hospitals
diagnoses
were
Ultrastructural
(MPO)
were incubated
independent
Endogenous
the method
in
20
mg
diaminobenzidine
10
mL
of
0.05
microscopy
panel.
mol/L
with
taming
2%
incubated
Immunologic
bovine
(30
serum
minutes,
albumin
4#{176}C)
with
The
to the
cells
in the dark in a
and
below.
752;
Coulter
Electronics,
Hialeah,
(DAB;
Sigma,
St
Tris-HCL,
pH
7.4,
F(ab’)2 fragment
goat anti-mouse
IgG
In the case of VIM-2/TdT
double
cells were first incubated
in suspension
TRITC-conjugated
(Fab’)2
fragment
The
panel
of MoAbs
goat
selected
for this
study
is shown
with
rabbit
smears
ringer
were
sodium
azide,
diluted
cD
of patients
Biochemicals,
Milwaukee,
reactivity
was assessed
immunoperoxidase
staining
DNAs
no.
were
extracted
I through
I 6. Ten
from
included
as normal
controls
and
molecular
markers,
respectively.
Probes
and constant
region (Cia), Ig
Antibodies
Cellular
Used
Specifity
in This
Study
or Antigen
Source
CD 1 9
Pan-B
cell
B. D#{246}rken,Heidelberg
CD2O
Pan-B
cell
Coulter
Immunology
J5
CD 10
CALLA
Coulter
Immunology
Leu-9
CD7
Pan-T
cell
Becton
Dickinson
Leu-1
CD5
OKT1 1
CD2
Pan-T
Pan-T
cell
cell (E rosette
Becton Dickinson
Ortho Diagnostic
Systems
OKT4
CD4
T helper/inducer
Ortho
Systems
VIM-2
NA
Granulocytic/monocytic
VIM-D5
CD 1 5
Granulocytic
My7
CD 1 3
Granulocytic/monocytic
My9
CD33
MZ1 7
CD 1 5
NA
HLA-DR
receptor)
Diagnostic
markers
lineage,
lineage
W. Knapp,
Vienna
monoblasts
W. Knapp,
Vienna
lineage
Coulter
Immunology
Granulocytic/monocytic
lineage
Coulter
Immunology
Granulocytic/monocytic
lineage
F. Herrmann,
Mainz
Other markers
NA, not applicable.
weight
for the Ig heavy-chain
joining (iH)
x light-chain
joining (Jx) and constant
region (CK),
Ig A light-chain
constant
regions
(CX) as well as T
cell receptor
13 chain constant
region (CTfl) were described
elsewhere,’#{176}and kindly provided
by P. Leder and i. Seidman.
After
hybridization
the filters were washed under stringent
conditions
and
exposed
to XAR-5
film (Kodak,
Rochester,
NY) using Dupont
Lightning-Plus
intensifying screens for 12 hours at - 70#{176}C.
Cytogenetic
studies.
Bone marrow
cells were prepared
for chromosome analysis after a 24-hour culture with subsequent
synchronization by methotrexate.
Colcemid
was added ten minutes
before
B1
of di iferentiation;
cryo-
with appropriate
restriction
enzymes (Boehelectrophoresed
on a 0.7% agarose
gel,
as described
previously.U
Human
placenta
markers
CD, cluster
tech-
micrograms
HO 37
Abbreviations:
WI).
in frozen
FRG),
and hybridized
were
indirect
analysis.
were digested
blotted,
and
Major
an
samples
Mannheim,
(P.L.
antibody
elsewhere.27
genetic
cell
and ‘y-DNAs
MoAb.
1 . Monoclonal
by
Molecular
preserved
TdT
further
as described
of DNAs
0.1%
anti-calf
indicated,
nique
of 0.003%. Thereafter,
the
for transmission
electron
Table
OKIal
anti-
of labeled cells were
staining
for TdT as
were
cytospin
+
Myeloid
FL).
I . For intranuclear
TdT staining,
cytospin
preparations
fixed in absolute
methanol
(30 minutes,
4#{176}C)
and incubated
When
After two further
washing
procedures,
the binding of MoAbs was
assessed
by indirect
immunofluorescence
with fluorescein
isothiocyanate
(FITC)-conjugated
goat F(ab’)2 anti-mouse
IgG
1gM
Lymphoid
VIM-2
described
activity
et al.22 Unfixed
appropriately
Antibody
Designation
(Epics
in Table
markers
and
the
cytometry
mouse 1gM and cytocentrifuged
preparations
then subjected
to indirect
immunofluorescent
analyzed
either
on bone marrow
(n = 14) or peripheral
blood cells (n = 2).
Immunofluorescence
assays
were performed
in fresh or cryopreserved samples. Only samples containing
80% leukemic
blasts were
assayed.
Reactivity
with murine
monoclonal
antibodies
(MoAbs)
was determined
as previously
described.24
Briefly, leukemic
blasts
were isolated by standard
Ficoll-Hypaque
density gradient
centrifugation.
For surface
phenotype
determinations,
2 x 106 cells were
pelleted,
washed twice with phosphate-buffered
saline (PBS) conImmunophenotyping.
flow
beling with FITC-conjugated
(Jackson
Immunoresearch).
immunofluorescence,
target
were
according
peroxidase
of Rods
H2O2 at a final concentration
washed and further
processed
as described
elsewhere.23
were
observer
for one hour at room temperature
dissolved
containing
cells
using
containing
Louis)
an
and
due to Fc recep-
ment
naphthyl
(AcP)
binding
goat anti-mouse 1gM (Jackson Immunoresearch
Laboratories,
West Grove, PA), followed by incubation with HD37 and counterla-
of
maracid
of the trials (at the Univerand Hannover,
FRG).
All
ALL were determined
(FAB) classification.2’
studies.
was studied
medium
by
and
alpha
phosphatase
laboratories
in MUnster
confirmed
subtypes
of AML
French-American-British
acid
(MPO),
CA). Nonspecific
Background
fluorescence,
determined
by using nonreactive
MoAbs
of the same isotype
as the test MoAbs, was subtracted.
In selected
cases with adequate
numbers
of cells, double immunofluorescence
analysis
was performed
as described
elsewhere.”’’
Briefly, target
cells were first stained with myeloid MoAb (V!M-2)
and tetramethyl rhodamine
isothiocyanate
(TRITC)
conjugated
F(ab’)2 frag-
stud-
ALL and AML was determined
in Pappenheim-stained
bone
row and blood smears. Slides were stained routinely
for periodic
Inc, Burlingame,
tor was avoided by adding heat inactivated
10% goat serum (Sigma)
in both the first and second
incubations.
Fluorescence
of cells was
evaluated
with an epi-illuminated
fluorescence
Zeiss microscope
or
Ortho
Diagnostic
Systems
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
LUDWIG
1520
harvesting.
After treatment
with a hypotonic
KCL solution
(0.075
mol/L)
the cells were fixed in methanol/acetic
acid (3:1) and
dropped
on a cold wet slide. G-banding
was done after a drying
period of three to seven days at room temperature
with a short
trypsin pretreatment
and staining
with Giemsa solution at a pH of
6.8.
DNA
analysis
ments
blood
by flow
of the cellular
and/or
bone
bromide
cytometry.
Flow
cytometric
DNA content were performed
marrow
cells after staining
and mithramycin
in combination.3”32
revealed
the
leukemic
lymphoid
positive
patients,
in three
positivity
for AcP.
the basis
of lymphoid
approximately
present.
Two
measure-
in ethanol-fixed
with ethidium
blasts
morphologically
M5a)
from
seven
(FAB-Li
one
Patient
of whom
PAS
disclosed
classified
to morphology
and
was
diffuse
as ALL
Cytochemically,
on
however,
10% MPO-positive
lymphoid-like
children
were classified
as AML
according
to be
or L2).
also
1 5 was
no.
morphology.
patients
L1/2,
,
ET AL
blasts were
(FAB-Ml,
cytochemical
staining.
In four of the 16 patients,
bone marrow
smears
were
characterized
by two separate
blast populations
with myeloid
and lymphoid-appeaning
leukemic
cells (no. 2, 4, 12, and 16)
(Fig 1). In patient
no. 9, bone marrow
examination
at
For the identification
of DNA aneuploidies
all samples were mixed with diploid mononuclear cells from normal
blood donors
as reference
cells at two
different
concentrations.
The appearance
of a second G0/G, peak
and its change according
to the ratio between sample and reference
cells was considered
to indicate
an aneuploid
DNA stemline.
The
DNA index of diploid cells is by definition
1 33
diagnosis
showed
bling
(slO%)
tion
RESULTS
approximately
lymphoblasts
with
and
monoblastic
of induction
monocytoid
84%
(FAB-L1)
leukemic
a small
features.
chemotherapy
Three
days
for ALL,
large
(75%) appeared
features
cells
resem-
number
of cells
after
initia-
blasts
with
in the peripheral
blood.
patients
ranged in age from 7 months to 16 years; four were
under 12 months,
and two of them had 1 1q23 translocation-
Morphologic,
cytogenetic,
and clinical
data of this patient
have been previously
reported.
Cytochemical
staining
was
inconclusive
in one of these patients
(no. 9). Two patients
(no. 12 and 16) disclosed
MPO-positivity
at light micro-
associated
scopic
Clinical
logic
data
and
hematologic
of the
16 patients
acute
leukemia.
eight were girls.
109/L; the WBC
5.2 x 109/L to
60% blast cells
Clinical
and
Eight
children
were
1
2. The
boys,
and ultrastructural
cytochemical
in Table
2.
studies.
and
activity.
Study
Age (yr)/Sex
WBC (Blast %)
(x 1O’/L)
Rd
8.2/M
8 1 .9 (90)
2.
FAB
Clinics
In patient
in
Ultrastructural
patient
patients
no.
percentage
showed
strong
studies
performed
of blast
cytochemical
on leukemic
(no. 2 and 4) revealed
4, whereas
leukemic
cells
ANAE
cells
MPO
from
cells
activity
patient
no.
were negative
for MPO (data not shown).
In patient no. 6, the morphologic
appearance
of leukemic
blasts
was equivocal
(FAB-L2
v FAB-M 1 ), and the blast
I and He matologi
cal Data
PAS
MPO
ANAE
AcP
ND
ND
ND
ND
L2
no. 2 a small
features
of two of these
data of the 16 patients
are also
Light
microscopic
examination
Time of
level.
with monocytic
Morpho-
Table
Patient
No.
hemato-
in Table
Eight children
presented
with WBC
100 x
in the remaining
eight patients
ranged from
81.9 x 109/L. Peripheral
blood comprised
in 15 patients.
Light microscopy
logic and
presented
data.
are summarized
clinicel ces
SM rd occurred
48
mo after
Dg of ALL.
died
1
mo later
2
Dg
1 .4/M
1 23.5
(92)
L 1 /M5
65
0
3
0
CR after high-dose
to AML-
Ara-C.
and high-risk
consolidation
ALL-pr,
according
CNS rd after
12
mo
3
Dg
1O.7/M
4
Dg
14. 1/M
1 16.4
(88)
Ml
18.5
(65)
M 1/L 1
0
26
8
0
0
CCR (13+
Ot
0
0
CR after ALL-pr,
mo), AML-pr
maintenance
Th with
rd after
AML-pr,
5
Dg
4.6/F
560
(94)
Li
0
0
0
0
Died before
6
Dg
6.8/M
292
(95)
AUL
0
0
0
0
CR after ALL-th,
1 1 mo, died
1 mo later
th
BM and CNS rel after
10 mo,
died 2.5 mo later
7
Rel
4.4/M
8
Dg
16.7/F
9
Dg
0.7/F
42.4
(90)
L2
49.5(77)
230
(78)
60
0
0
0
CR afterALL/AML-th,
Li/2
0
0
0
0
CCR(2i+mo),ALL-pr
Li
0
0
0
0
CR after AML-pr
(4+
10
Dg
0. 1 i/F
i i
Dg
7.i
149.8
12
Dg
13.8/F
13
Dg
6.3/F
i4
Dg
0.9/F
66 (95)
M5a
i5
Dg
0.7/F
i 85 (94)
M i
i/M
(90)
81 (8i)
37
(70)
and steroids.
55
0
0
0
CR after ALL-pr,
Li
0
0
0
0
CCR(29+
mo), ALL-pr
0
CCR
mo),
9
Li
80
50
20
Dg
i 2.4/M
275
(90)
Li/M
1
(i8+
0
70
CCR (i7+
mo). ALL-pr
0
0
80
0
CCR (i8+
mo). AML-pr
0
iO
0
0
CR after ALL-pr
6
0
0
and high-dose
CR after AML-pr,
BM rel after
Abbreviations:
AUL,
acute
Percent
Rel, relapse;
undifferentiated
positive
tDemonstration
BM, bone marrow;
leukemia;
BMT,
Dg, diagnosis;
bone marrow
blasts.
of MPO positive
blasts
by electron
CR, complete
transplantation;
microscopy.
remission;
ND, not done.
pr, protocol;
2 CNS rel, BMT
failure)
AML-pr
0
7
mo)
died 5 mo later (hepatic
ter 5 mo, SM rd after
i6
CCR (19+
mo)
Li
M4/Li
5.2 (38)
BMT.
Ara-C,
CNS rel af-
7 mo
maintenance
th with
ALL-pr.
6 mo, died 3 mo later
CCR. continuous
complete
remission;
th, therapy;
2
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AMBIGUOUS
Fig
PHENOTYPES
1.
Bone
diagnosis.
Note
marrow
the
myelomonocytoid
(FAB-Li)
AND
aspirate
two
populations
features
(Pappenheim
magnification
GENOTYPES
smear
of
of blast
cells.
(FAB-M4)
stain;
IN ACUTE
and
original
LEUKEMIA
patient
no.
larger
smaller
12
cells
i 521
at
Fig 2.
Bone
marrow
aspirate
smear
of patient
no. 6 at
diagnosis
showing
monomorphic
proliferation
of blasts (FAB-L2
v
FAB-Mi
) (Pappenheim
stain; original
magnification
x 950; current
magnification
x495).
with
lymphoblasts
magnification
x950;
current
x495).
gens
cells
were
undifferentiated
by
cytochemical
stains
(MPO,
ANAE,
PAS, AcP negative)
(Fig 2). Ultrastructural
studies
in order to confirm
the dual-lineage
characteristics
of mdividual leukemic
blasts could not be performed
in this patient
due to insufficient
cell numbers.
Immunophenotyping.
The results
of immunophenotyping and
TdT
analysis
patients
clearly
are
disclosed
10, and 15). They expressed
CDI9
and were negative
antigens
consistent
ingly,
however,
with
all
myeloid-related
these
surface
summarized
in Table
mixed
lineage
features
TdT and the pan-B
for CALLA
an early
(CD1O)
pre-B
patients
phenotype.
additionally
antigens
assays
or immunoperoxidase
ing techniques
were
not
3. Five
in
staining.
applied
in
and
some
T cell
expressed
antigens
marker
81
J5
Leu-9
i
90
85
40
75
5
2
5
70
3
0
3
3
0
0
4
70
55
5
50
6
45
7
8
9A
immunologic
essentially
myeloid
phenotype
the
same
as at
analysis
in cases
with distinct
revealed
populations
that
the
leukemic
not
peripheral
blood
consistent
with
confirmed
patient
reactive
in the
immunologic
seven
AML.
days
later
Double
the existence
no.
bone
marrow
phenotyping
2 (CDI9+,
at diagnosis;
of blast
revealed
cells
in the
a marker
immunofluorescence
profile
staining
of two separate
cell populations
VIM-2-
50%;
cells,
in
CDI9-,
and
Immunophenotypes
Myeloid Markers
0KT4
VIM-2
VIM-D5
My7
My9
MZ17
OKlal
ND
5
ND
ND
40
ND
0
ND
90
4
ND
ND
ND
41
5
ND
2
20t
0
60
ND
10
ND
82
40t
i5
40
10
ND
ND
35t
ND
0
10
4
ND
i2
6
60
ND
55
65
0
0
4
ND
ND
0
60
i5
10
ND
77t
55
60
ND
0
6
ND
ND
ND
5t
3
iO
40
55
75
6
3
6
0
ND
ND
3t
30
0
10
75t
ND
45
84
90
55
4
85
6
50
4
5
2
2
ND
ND
ND
90
45
45
5
0
5
0
5
ND
ND
10
0
2
ND
55
12
ND
0
9
ND
ND
ND
ND
40
4
45
ND
45
95
85
10
95
60
5
ii
75
60
0
12
50
45
0
13
85
80
35
2
OKT1 1
2
0
7
6
50t
75
6
5
80t
50
5
ND
ND
0
0
ND
ND
0
0
ND
ND
ND
25
18
8
45
30
50
80
45
ND
ND
ND
ND
5
0
ND
ND
65
5
80
80
83
60
4
62
70
ND
92
14
0
0
4
0
70
2
4
50
8
70
70
0
0
0
ND
ND
ND
78
68
16
60
50
i
53
2
2
ND
ND
16
1
ND, not done.
given
as percent
positive
tlmmunperoxidase
staining
performed
1A. at diagnosis (bone marrow).
§B, seven days later (peripheral
blood).
22
8
40
15
AII results
populations
both
Leu-1
0
Abbreviation:
was
examination
were
however,
Although
dual stainthese
patients
due to
HD37
The
no. 6 at relapse
cell
possessed
blasts with lymphoid
features
in phase microscopy
expressed
TdT, HLA-DR,
CD19 (no. 2, 9, and 12) as well as CD1O
(no. 4 and 16) compatible
with early pre-B cell phenotype,
whereas
the larger blasts in patients
no. 2, 4, 12, and 16
reacted
with myeloid
markers.
In patient
no. 9, myeloid
immunofluorescence
TdT
blasts
characteristics.
Lymph oid Markers
.
Patient
B
lymphoid
Surface
Surpnis-
3.
homogeneous
at least
in light-microscopic
(no. 5, 6, 7,
cell antigen
Table
morphologically
that
diagnosis.
inadequate
numbers
of cells, the overlap in the percentages
of blasts reactive
with lymphoid
or myeloid-associated
anti-
No.
the
of patient
of the
and
and
indicated
cells.
on cryopreserved
cytospin
preparations.
1
26
29
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i522
LUDWIG
Fig 3.
Patient
no. 12. Phase
contrast
ET AL
micros-
copy and indirect
immunofluorescent
staining
of
bone marrow
at diagnosis.
Note the two distinct
populations.
larger blasts with myelomonocytoid
features
and smaller lymphoblasts.
(A) Blast cell
morphology
as seen
under
phase
contrast
microscopy.
(B) Same field as (A). with the cells
stained
for myeloid
cell surface
antigen
(TRITC
labeled
VIM-2
MoAb).
Note that
intermediate
and large blasts are VIM-2-positive.
(C) Same
field as (A). with the cells stained for nuclear
TdT
(FITC-conjugated
antibody).
Note
that smaller
cells resembling
lymphoblasts
are TdT-positive.
(Original
magnification
x 1 .000; current
magnification
x700.)
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
AMBIGUOUS
PHENOTYPES
AND
GENOTYPES
IN ACUTE
LEUKEMIA
1523
VIM-2+
blasts, 25%) as well as in patient
no. 12 (TdT+,
VIM-2cells, 40%; VIM-2+,
TdTblasts,
20%) (Fig
3A-C).
In patients
no. 1, 8, 1 1, and 13, surface
marker
studies
revealed
an early
pre-B
no. 1 simultaneously
other three patients
blasts
with
T cell
cell
phenotype.
Blast
cells
of patient
(CD7
in no.
1 3, and
CD2
kemic
and
with
morphologically
typical
expressing
widely
lymphoid-
overlapped
sion
on
different
and
in these
individual
myeloid-associated
patients,
cells
of
markers
normally
leu-
investigated,
except
distinct
rearranged
indicating
explained
a restriction
Hind!!!
digests
no.
oligoclonality.
by either
or by
patient
fragments
DNA
enzyme
likewise
The
latter
overdigestion
polymorphism,
revealed
multiple
result
or partial
since
rearranged
c,
Jit
Cs
CX
$-TcR
1
nm
R
R
G
G
G
R
DNA
i.0
lndex
2
nm
R
R
G
G
3
nm
R
R
GG
4
nm
R
R
G
5
nm
R
R
6
46,XY,t(4;ii)
R
7
46,XY,t(4;ii)
8
G
G
1.0
G
G
1.0
G
G
G
1.0
G
G
G
G
1.0
R
G
G
G
G
1.0
R
R
G
G
G
G
1.0
R
R
R
R
G
G
1.0
47,XXX,t(il;i9)
R
R
G
G
G
G
1.0
nd
R
R
G
G
G
G
1.0
ii
nd
R
R
R
R
G
G
1.0
12
nm
R*
R
G
G
G
R
1.0
13
nm
R
R
G
G
G
G
1.0
Four
no.
14
nd
G
G
G
G
G
G
1.0
15
46,XX,t(4;ii)
R
R
G
G
G
G
1.0
not
16
nd
R
R
GG
G
G
on
analysis
patients
14 (Fig 4, Table
4).
were observed
in patient
Ig Light Chain
JH
nm
found
blot
in all
Karyotype
9t
10
coexpres-
lineages.
Ig and fl-TCR
gene analysis.
Southern
revealed
an Ig heavy-chain
rearrangement
I 2,
the
and DNA
Analyses
Chain
Patient
No.
antigens
indicating
Moleculargenetic.
Cytometry
Gene Rearrangement
cells coexpressed
T cell antigens
(CD7 in patient
no. 3,
CD7 as well as CD4 in patient
no. 14). The percentage
of
cells
of Cytogenetic.
lg Heavy
in no. 8
AML,
Results
Flow
and 11).
In two children
4.
.
expressed
the CD15 antigen,
and the
disclosed
reactivity
of early pre-B ALL
antigens
Table
is
digestion
EcoR!
Abbreviations:
or
done;
bands
nm,
R, monoclonal
‘DNA
or no analysable
rearrangement;
aneuploidy
Materials
no mitoses
was
G, germline
quantitated
1.0
mitoses;
ND,
not
configuration.
by the DNA
index
as described
in
and Methods.
tAt diagnosis
(bone marrow).
$Oligoclonal
rearrangement.
§At
shown).
(not
the
relapse
presence
CK rearrangement
(see results).
However,
in the
absence
of multiple
copies
of chromosome
possible
alternative
explanation.
Samples
at primary
diagnosis
from
patients
no. 6 and
16 could
be identified
chain
initial
analysis
Fig 4.
Southern
blot analysis
of DNAs obtained
from leukemic
cells of patients
no. 1 through
1 6. BamHl
digests
were hybridized
to a 1 .3 kB EcoRl
CM probe that detects
a 1 7 kb germline
band.
Note identical
rearranged
fragments
in cell samples
of patient
no.
16 obtained
from initial diagnosis
(A) and in relapse
(B).
emerged
cell sample.
configuration
(Fig
CK and
in patient
of patients
5,
relapse
in relapse
blots
that
Jic sequences
no.
no. 8 and
and
lane
data,
I 4 remains
on both occasions
of leukemic
cells
in Southern
sequences
rearrangement
and
16, respectively,
cally rearranged
Ct fragments
16a/b).
Yet clonal
evolution
light
of cytogenetic
were
available
showed
since
was not present
remained
identi-
(Fig 4, lane
of patient
no.
hybridized
16a/b),
a
to !gic
a Cic
in the
in germline
6 (not
shown).
Southern
1 1 also
showed
rearranged
blot
IgK
Fig 5.
Southern
blot analysis
of DNAs obtained
from leukemic
cell samples
of patients
no. 8. 1 1 . and 1 6 as well as human placenta
(N). BamHI
digests were hybridized
to CK sequences
that detect
a
12 kb germline
band.
Rearranged
fragments
are indicated
by
arrows.
In patient
no. 1 6 a CK recombination
is visible in relapse
(B)
but not in cells from initial diagnosis
(A).
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
LUDWIG
1524
A complete
remission
transplant
was
Only
one of the
logically
distinct
lasting
was
patients
than
I
with
year
AML/ALL-directed
and
a bone
marrow
performed.
populations
longer
occurred
relapsed
achieved,
subsequently
ET AL
morphologically/immuno-
achieved
a complete
in
of
spite
treatment
a
remission
combination
protocols.
of
CNS
relapses
in two of these patients
(no. 2 and 9), and two others
I I months
(no. 4) and 6 months
(no. 16) after
and died of progressive
leukemia.
diagnosis
DISCUSSION
Three
Fig 6.
Configuration
of T cell receptor
$-chain
sequences
in
patients
no. 1 and 12 as well as human
placenta
DNA (N). EcoRl
digests
were hybridized
to a CT/I probe that detects
1 2 kb CT$1
and 4.2 kb CT$2 germline
fragments.
Rearranged
fragments
are
indicated
by arrows.
different
attempt
to
multiple
evaluations
ous
five
patient
5),
(Fig
showed
to all other
in contrast
rearranged
IgX chain
cases
(Table
sequences
simultaneous
patients
genes
(Table
exhibited
them.
investigations.
out
1 5), and
7,
tnisomy
leukemias.
of these
equivocal
was
(no. 9). The
was at the
arm
found
t(1
breakpoint
long arm
was
(no. 6,
in all of
observed
whereas
of
number
index
try were
and
successfully
leukemic
cell
t(4;ll)
Treatment
patients
treated
AML-BFM-83
has
Patients
induction
was
diagnosed,
and
later,
the
In patient
patient
and
1).
then
I month
The
CR
after
patient
protocol,
and
(cytosine
arabinoside
two
with
relapse
after
Lineage
was
cytotoxic
[Ara-C],
drugs
ALL
therapy.
(FAB-M5a)
a relapse
those
the
chemowith
lym-
mation
acute
is of short
the
B
cell
with
early
t(4;1
the
1)
have
disease
B cell
may
lineage,#{176}or a
cell
in-depth
line
with
characteriza-
lymphoid
expression
switch
partial
with
to characterize
that
early
of this
patients
and
myeloid
capabilities.42
lineage
all
of lymphoid-
three
with
to lymphoid)
for
the assumption
and
patients
(myeloid
positivity
work
in
the
MPO
that
three
also
in
in patient
the target
kemias
protocols
and
cell
immunologic
that acute
that
should
the
the
consider
t(4;l
t(4;l
data
2 years
with
important
design
its mixed-lineage
< I 5%
of
from
the
here and from
t(4;! 1) represents
the
subgroup
of
still
a very
therapy
in
is apparent
presented
leukemia
has
to cytotoxic
exceeds
It
1),
1) or
is
1) generally
response
far.43’45
future
with
characteristics
survival
clinically
t(4;I
transfor-
cell.
for patients
with
so
reported
and
progenitor
leukemia
with
malignant
mixed-lineage
course;
and
patients
suggests
chemotherapy
clinical
and
ofothers
against
AML
both
in
duration,
patients
effective
added.
of
A leukemic
antigens
with
Acute
aggressive
biologically
were
of
observed
leukemia
to
ALL
three
and
lineage
the
appropriate
according
the
In addi(CD19),
!g gene
evidence
leukemia
of a pluripotent/bipotent
The
clinical
studies
these
was
inconclusive.
origin.4’
immunophenotype
confirmed
by
revealed
6-Thioguanine)
was
and
A
4 years
induction
cell
I 5 are consistent
no.
unknown.
leukemia
switch
no.
resemble
therapy,
relapse.
cytogenetic
treated
ALL
occurred
AML
diagnosis,
and
by
by
monoblastic
occurred.
analysis,
CNS
treated
years
blasts
immunologic
t(4;1
died
proto-
complete
responses
to
no. 1, a common
ALL
achieved
and
no. 7, acute
Three
phoid-like
was
marrow
was diagnosed
therapy.
I 3 entered
CR
of these
no.
of
diagnosis
for transformation
in t(4; 1 1 ) may be a common
progenitor
for B cells and myeloid
cells. Although
patients
no. 5 and 10
lacked
cytogenetic
data, clinical
features
(ie, young age, poor
prognosis)
and
laboratory
findings
(ie, hyperleukocytosis)
were
studies
elsewhere.35’37
3, 1 2, and
I 4 had
for AML.
In patient
and
bone
combined
The outline
in patient
indicating
of simultaneous
7, and
in detail
detected
in
established,
dual
Our findings
I6
were
of the
characteristics
attempting
acute
markers
suggesting
observed
8, 1 1 , and
no.
therapy
revealed
patient
the
myeloid
pan-B
cell antigen
in the heavy-chain
a very
recently
t(4; 1 1 ), the
of
in
progenitor
was
4),
course
and
patients
6), morphologic
Studies
results,
had DNA
indices
of I .0 (Table
stemline
was detected.
clinical
ambigu-
appearance
further
a myelomonocytic,38’39
cells,
mean
described
in Table
2. All children
to protocols
of the cooperative
reported
no.
The
was
origin
myeloid-associated
The
peak
cell
All
or ALL-BFM-83.
been
patients
GO/G,
was
available
I).
conflicting
multipotent
cytome-
t(4;1
3.2%.
outcome.
is briefly
according
by flow
out in all I 6 patients.
the
for
populations
DNA
no aneuploid
and
carried
of variation
coefficient
cols
blast
7 [i(7q)]
analyses
lymphoid
provided
analyses,
revealed
yielded
have
DNA
ploidy.
cell
of these
staining
patients
five
the
from
commitment.
the
6 at
(no.
cytochemical
in
all
with
pre-B
MPO
demonstration
tion
DNA
the
to
were found in two patients:
(no. 9) and an isochromosome
no.
for
mixed-lineage
In one patient
and
16 patients
Four
on the
the
in
obtained
The first group
comprised
whose
blast cells showed
I 5).
positivety
Cytogenetic
of all chromoin band
I 1q23.
in patient
but
discriminated
findings
early
and
based
supporting
group
relapse.
and
cells,
of
5, 6, 7, 10,
expression
of the
of rearrangements
in three
l;19)(q23;p13)
changes
X chromosome
of the
long
16 children
was
15),
chromosomal
analysis
of the
aberration
and
appeared
only once
some
I I aberrations
Secondary
four
(4;11)(q21;23)
6,
(no.
Chromosomal
in only
a structural
A translocation
of the
I 5,
configuration
be
major
in the present
as ALL
blast
tion
carried
patients
classified
of T/3
while
the
4).
Chromosome
successfully
7, 9, and
a germline
the
expression
(no.
shown).
(not
Two patients
(no. 1 and I 2) showed
a rearrangement
sequences
in BamH!
or EcoRI
digests
(Fig 6),
other
4). No
could
summarize
phenotypes
and genotypes.
patients
with acute
leukemias
antigens
sequences
groups
of acute
chemotherapeutic
features.
a
leu-
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
AMBIGUOUS
PHENOTYPES
The
group
second
whom
analysis
revealed
tions,
one
the
with
features
(no.
three
GENOTYPES
of patients
morphologic
studies
AND
comprised
as well
coexistence
lymphoid
and
the
patients,
myeloid
monocytoid
features
hypothesis
of a closer
relationship
progenitors.6
by recent
results
and
shared
surface
reports
t(9;!
differentiating
into
Molecular
genetic
leukemia
with
three
myeloid
and
in this
Thus,
in
group
patients
analysis
revealed
a single
a monoallelic
(patients
no.
suggest
clonally
related.
kemic
cell
of two
different
deletion
populations
on one
clones,
allele
and
a gene
emerged
phenotypes
The
that
but
also
blot
from
analysis
different
in the
detected
characterized
allele
Southern
blots
analysis
multiple
during
the
subpopu-
their
respective
Since
rearrangement
are
the
similar
cell
(Fig
of the Ig heavy-chain
rearranged
fragments.
6,
lane
rearrangement.
may
have
characterized
by distinct
heavy-chain
and
phenotypic
or morphologic
different
ingly,
a recent
chromosome
genes
14, the
in childhood
a poor
observed
as
study
frequent
that,
presence
of more
ALL
response
in patient
in
suggested
B
of B lineage
that
gene
than
antigens,
cells,
ALL
can
AML6”62
expression
that
only
supported
anti-CD4
anti-CD7
whose
reports
but
easily
of
ALL
been
blast
described
indicating
the
exposable
form
cells
antigen
from
B cell
CD2
(CD2)
could
a subgroup
of
neoplasias,63
on AML
of AML
or CD7
and
blasts
was
patients.TM’65
antigens
on early
cell
determinants
lineage.2
are entirely
This
interpretation
is
by the observed
reactivity
of AML
blast cells
and anti-CD7
MoAbs
(patient
no. 14) or only
MoAbs
(patient
no. 3). Interestingly,
recent
have
demonstrated
cells
cells
All patients
the
expression
of the
of monocyte/macrophage
from
adults
with
with
early
pre-B
of the
antigen
as well
typical
ALL
and
antigens
heavy-chain
CD4
lineage66
otherwise
or T cell-associated
AML.9
coexpression
of
demonstrated
Ig sequences.
rear-
Two
of these
also disclosed
light-chain
Ig gene rearrangement,
a
originally
thought
to permit
a more definitive
assessof B lineage
commitment
T lineage
evidence
bigenotypic
demonstration
functional
recently
Patient
of the $-TCR
otherwise
Another
but
cells.’5
gene
also
no.
detected
I also
without
other
of Ig heavy-chain
AML
typical
or infidelity
interpretation
(patient
of gene
suggests
rearrangements
gene
no.
control
that
such
are a component
in
revealed
phenotypic
for T lineage
ALL. Possible
interpretations
features
have been discussed
above.
abnormality
for such
rearrangement
3) may
reflect
in acute
leukemia.
partial
an
and
of normal
nondiffer-
entiation.67
with
Taken
together,
specifity
our
in
this
unexpected
lineage
To
on
of
few antigenic
cell
neoplasms.’4’5’
AML
Expression
in patients
no. 8, 1 1 and 13, has not
and probably
further
supports
the
to a particular
gene regulation
cell
with
or T
cells,59’#{176}it has been
questioned
be regarded
as evidence
for acute
of the
aberrant
T
view
or chronic
observed
in
with
X-hapten,
in a cryptic
to therapy.5#{176} Bigenotypic
features
as
No. 1 2 appear
to be approximately
twice
as
the
leukemia.2
assumption
The
2 z heavy-chain
may be correlated
clinical
drugs,
markers.
of pan-T
antigen
CD7
in a substantial
proportion
blast
cells
been
yet
the
1 , has recently
no.
In
pre-B ALL, as observed
been previously
reported
as on
in which
chemotherapy,
patients
the
in patient
rearrangement
of
two
especially
on
findings
such
malignant
Interestcopies
in
not
cytotoxic
T cell
with
ment
extra
and
studies.25’56’58
expression
observed
in
rear-
absent
is poor.
children
multi-
gene
reports26’47’48’5355
multiple-agent
recently
identified
rearrangements
case
the E-rosette-associated
be identified
on leukemic
However,
molecularly
feathat
in this study consisted
of four patients
whose blast cells coexpressed
myeloid-
of X-hapten
whether
However,
recent
coexpressed
by desialylation
has
and
patients
as observed
several
12).
features.
without
myeloid
clearly
are
simulta-
was
leukemias
ALL-directed
antigens
cells
rangement
of
Tf3
the
/3-TCR
of patients.
intensive
and
group
ALL
leukemic
patients
finding
of differentiation,
emerged
The last
early pre-B
no.
genes (Fig 4, lane I 2) revealed
We tend to interpret
these
In the course
pIe subpopulations
of these
myeloid-
I 2.
for acute
simultaneously
despite
prognosis
with
time
and bigenotypic
be emphasized
neoplasms,52
outcome
patients
AML-
on one
conflicting
data as an indication
for the clonal
relationship
the leukemic
cells, which
is suggested
by the common
gene
that,
on neoplastic
one.
first
precedes
T cell
series
our
including
studies
these
both
in patient
to Tfl sequences
population
suggest
with
patterns
fragments
obtained
9)
Southern
of markedly
on the other
results
of
in
probably
coexist
in larger
course
also
4, lane
fragments
lineages
restricted
distinctions.
above.
of Cjs germline
hybridized
a monoclonal
in
treatment
two
However,
by a C
and EcoR!
digests
(not shown),
existence
of two cell populations,
a distinct
surprising
leu-
composed
no. 9 (Fig
intensity.
the
on the other
discussed
rearranged
by a deletion
and
Most
defined
in patient
two
up in Hind!!!
suggest
the
4 are
clone
morphologic
patients
autoradiographic
came
results
2 and
differ
marked
pattern
that
that
it appears
that,
patients,
leukemic
only
show
hybridization
differs
not
The
Likewise,
4). These results
no. 2 and 4 are
the
no. I 2.
patient
of
that
in precursor
“mixed-lineage”
population
rearrangement
appears
to be less likely.
Hence,
differentiation
process
in those
lations
no.
a myeloid
16,
for
an antigen
rangement
4) or biallelic
interpretation
of patients
cell
of
and
reveals
CD7,
presence
are
4,
cell
2 and
no. 16) Ig gene rearrangement
(Fig
that the leukemic
cells in patients
alternative
hyperleucapable
of
2,
case
expression
of both biphenotypic
at the molecular
level.
It should
cell-associated
with
translocacommon
of patients
blot
or
cells.48
no.
exhibiting
The
patients
other
certain
lymphoid
Southern
(patient
t(1 I ;l 9)
I 1q23 translo-
and
have
this
neous
tures
studied
lymphoma
with
knowledge,
the
and
supported
young
age of patients,
of a progenitor
cell
studies
interest.
with
Morphologic,
immunono. 9 are consistent
with
including
such as the
proliferation
pre-
lymphoid
is further
of 11 patients
characteristics,
kocytosis,
and
in
!g gene rearrangements
that both t(4;!!)
I 1q23 breakpoint
both
that,
accordance
hypothesis
leukemia,
myeloid
evidenced
between
acute
study
t(l1;!9),
suggested
tions involving
the
with
in a lymphoblastic
describing
cation-associated
particular
in
tumor
cell line.46
findings
in patient
A recent
popula-
It is noteworthy
identical
antigens
macrophage
logic, and clinical
and
other
This
disclosing
cell
one
in
marker
separate
involvement
dominantly
monocyte
five children
other
16).
1525
LEUKEMIA
as immunologic
of two
2, 4, 9, 12, and
of these
IN ACUTE
group
of
of
phenotypes
patients
and
probably
due to leukemogenesis,4
the
probes
used
(eg,
genotypes
arise
from
insufficient
multispecific
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1526
LUDWIG
MoAbs68),
and abortive
sequences
inappropriate
rarely
detectable
type.67
Data
in the
to
small
acute
the
AML
of these
with
of
the
regarding
of
disclosing
evidence
cannot
patients
in
this
follow-ups
associated
cell
of
Conclusions
findings
long-term
prognosis
the
myeloid-related
antigens.4’9’72
number
studies
prospective
cells
contradictory
expressing
surface
importance
the
determine
with
as to
be drawn
of the patients
series,
are
this
multiple
leukemic
evolution,73
,more
with
malignant
not
of DNA
tiated
types
aneuploidies
of acute
is higher
leukemias
in the
(eg,
AML
more
M4
differenor M5
and
ALL) than in AML MI and null ALL,24’75 these
results further
support
the origin of ambiguous
phenotyps
and genotypes
from very early precursor
cells.
In conclusion,
the application
of expanded
diagnostic
and
needed
to
subgroup
origin
disclose
for
of
diagnosis
or relapse
DNA
and
will
cell
differentiation.
arising
by
clonal
These
phenotypic
can
and
of leukemic
contribute
genotypic
blast
to the
cells
at
identification
of
lead
to further
insights
into
normal
hematopoietic
of two or
results
ACKNOWLEDGMENT
are consis-
analyzed
features.
the
biologically
and clinically
relevant
subgroups
of acute leukemias. Furthermore,
such approaches
will be useful
in clarifying the pathogenesis
of acute
“mixed-lineage”
leukemias,
detect
the existence
analyzing
characteristics
to
of the cases
mixed-lineage
approaches
possible
generally
indicating
clones.74
clonal
their
was
lines,
cases,
a common
despite
it
stem
or in rare
concurrent
study
did flow cytometry
Furthermore,
aneuploidies.
dence
lineage-associated
leukemia.
In none
tent
are
antigens8”#{176}’56’69’7’ or
the prognostic
of Ig or TCR
hematopoietic
common
of ALL
lymphoid-associated
due
rearrangements
in
literature
clinical significance
surface
DNA
ET AL
Since
in this
the
mci-
We are indebted
Barbara
Komischke
to Gudrun
for excellent
Gassner,
technical
Annette
assistance.
Gatzke,
and
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GV,
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1988 71: 1518-1528
Ambiguous phenotypes and genotypes in 16 children with acute leukemia
as characterized by multiparameter analysis
WD Ludwig, CR Bartram, J Ritter, A Raghavachar, W Hiddemann, G Heil, J Harbott, H Seibt-Jung, JV
Teichmann and H Riehm
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