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Cytogenetically Aberrant Cells in the Stem Cell Compartment (CD34+lin-)
in Acute Myeloid Leukemia
By Bhoomi Mehrotra, Tracy I. George, Kris Kavanau, Herve Avet-Loiseau, Dan Moore II, Cheryl L. Willman,
Marilyn L. Slovak, Susan Atwater, David R. Head, and Maria G. Pallavicini
Leukemia may be viewed
as a clonal expansion of blastcells;
however, the role of primitive
cells and/or stem cells in disease etiology and progression is unclear. We investigated
in leukemia using fluorescence in situ
stem cell involvement
hybridization (FISH), immunofluorescence labelingof hematopoietic subpopulations, and flow cytometric
analysis/sorti n g t o discriminate and quantify cytogenetically aberrant
stem cells in 12 acute myeloid leukemia (AML) and three
myelodysplastic (MDS) specimens. Flow cytometricanalysis
and sorting were
used t o discriminate and collect a primitive
subpopulation enriched in stem cells expressing CD34+ and
lacking CD33 and CD38 (CD34+lin-). A subpopulation containingprogenitorsanddifferentiatingmyeloid
cells expressed CD34, CD33, and CD38 (CD34'lin'). Nine specimens
contained less than 10% CD34+ cells and, thus, were considered to be CD34- leukemias. Mature lymphoid, myeloid, and
erythroid subpopulations were sorted on thebasis of antigen-linked immunofluorescence. Cytogenetically aberrant
cells in sorted subpopulations were identified using FISH
with enumerator probes selected on thebasis of diagnosis
karyotype. Cytogeneticallyaberrant CD34+lin- cells were
present at frequencies between 9% and 99% in all specimens. CD34+lin- cytogenetically aberrant cells comprised
specimens.
between 0.05% and 11.9% of the marrowfblood
Cytogenetically aberrant CD34+lin+ cells constituted 0.01%
t o 56% of the marrow/blood
population. These data demon-
strate thataberrant cells are present in primitive CD34+stem
cell compartments, even in CD34- leukemias. Stem cell
involvement wasconfirmed furtherby sorting lymphoid and
erythroid subpopulations from eightspecimens in which the
predominant leukemic population lacked lymphoid/erythroid differentiation markers. In these specimens, 7% t o
76% of the phenotypically defined lymphoid and erythroid
cells were cytogenetically aberrant. The presence of aberrant cells (albeit at varying frequencies) in a primitive compartment in leukemic specimens, as well as in multiple lineages, suggests involvement of a cell(s) with multilineage
capabilities. The ability of aberrant CD34+lin- stem cells t o
contribute t o clonal and compartmentexpansion within immunofluorescently defined subpopulations was evaluated
t o explore the functional phenotype of aberrant CD34'lincells. Analysis of compartment size and aberrant cell frequency suggests that frequency of cytogenetically aberrant
stem cells is uncoupled from compartmentsize. These data
suggest that cytogenetically aberrant cells in the primitive
compartment show varyingabilities t o expand primitive
compartments. Cytogenetically aberrant
CD34+lin- cells precede the blastsubpopulation in hierarchical maturation and
may in some cases be considered preleukemic, requiring
maturation or additional mutations before transformation
(eg, compartmental expansion) occurs.
0 7995 by The American Societyof Hematology.
L
leukemic population suggests that these cells arose from the
same marked cell capable of multilineage differentiation. An
alternative strategy to address stem cell involvement is to
use immunophenotype and genotype measurements to detect
cells carrying characteristic cytogenetic abnormalities in
phenotypically defined stem compartments. Weusedboth
approaches to investigate stem cell involvement. Specifi-
EUKEMIA MAY BE VIEWED as a clonal expansion
of blast cells, and myeloid dysplasia as a disorder of
hematopoiesis frequently leading to subsequent clonal
expansion. The diagnosis of leukemia is based on the presence of excess blasts, with confirmatory evidence provided
by histochemistry, immunophenotype, andor cytogenetics.
Approximately 80% to 90% of acute myeloid leukemia
(AML) and myelodysplastic (MDS) specimens are karyotypically abnormal."' Although leukemia is a disease of blasts,
it has been a matter of controversy whether the genetic
change of a cell or cells that underlies this clonal expansion,
sometimes referred to as leukemic transformation, occurs in
primitive multipotential or totipotential stem cells or in later
progenitors committed to a specific lineage or, possibly, two
lineages (ie, biphenotypic leukemia)!-" Fialkow et all2postulate the existence of two different types of leukemia: one
in which the transforming event arises in a primitive compartment containing multipotential stem cells, and another
in which the abnormality arises in a more mature progenitor
compartment. Although evidence in support of these concepts is not definitive, this distinction has a bearing on our
concepts of leukemias and their classification and may have
clinical implications."
The extent of stem cell involvement may be investigated
using molecular cytogenetic and cellular methodologies.
Stem cell involvement can be assessed indirectly by determining whether molecular abnormalities present in leukemic blasts are present in differentiated cells in multiple line a g e ~ . ~ "The
' presence of differentiated cells in multiple
lineages carrying the same molecular abnormalities as the
Blood, VOI 86,NO 3 (August l), 1995:pp 1139-1147
From the Division of Molecular Cytometry, Department of Luboratory Medicine, University of California, San Francisco, CA; the
California Pacijic Medical Center, San Francisco, CA: the Departmentof Pathology, University of New Mexico, and University of
New Mexico Cancer Center, Albuquerque, NM; the Department of
Cytogenetics, The City of Hope National Medical Center, Duarte,
CA; the Southwest Oncology Group (SWOG) Leukemia Biology Program, San Antonio, 7X:and the Department of Pathology, St Jude
Children's Hospital, Memphis, TN.
Submitted January 5, 1994; accepted March 9, 1995.
Supported by National Cancer Institute Grant No. CA 60417
(M.G.P.),American Cancer Society Grant No. EDT-54 (M.G.P.),
and Cancer Center Support Grants No. CA 33572 (M.L.S.)and CA
32102 (SWOG Leukemia Biology Program).
Address reprint requests to Maria G. Pallavicini, PhD, Division
of Molecular Cytometry, Deparhnent of Laboratory Medicine, University of California, 1855 FolsomSt,Room 230, San Francisco,
CA 94103.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advenisement" in accordance with 18 U.S.C.section 1734 solely to
indicate this fact.
0 1995 by The American Society of Hematology.
0006-4971/95/8603-0008$3.00/0
1139
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MEHROTRA ET AL
1140
cally, we combined immunofluorescence labeling of primitive and differentiated hematopoietic subpopulations, flow
cytometric analysidsorting, and fluorescence in situ hybridization (FISH)'?to quantify cytogenetically aberrant cells in
sorted subpopulations. A populationcontainingprimitive
cells and stem cells was selected on the basis of CD34 expressionwith
concomitant lack of CD33andCD3814.'s
(CD34'lif). CD34' cellsexpressingCD33
and CD38
(CD34'lin') are considered committed to myeloidlymphoid
differentiation. Maturelymphoidand erythroid cells were
labeled with antibodies recognizing differentiation antigens
present on end cells.Cytogeneticallyaberrant
cells in the
discriminated and sorted subpopulations were identified using FISH, with DNA probes selected on the basis of diagnosis karyotype of the leukemic clone(s).
We describe phenotype-genotype analyses of 12 cases of
AML andthree of MDS at diagnosis. We showthat the
characteristic cytogenetic aberrationwaspresent
in stem
cells in all specimens analyzed, as well as in the lymphoid
anderythroidcompartments
in specimens with sufficient
numbers of cells to allow multiple cell sorts. Furthermore,
we
evaluated
the
functional
phenotype of aberrant
CD34'lin- cells by measuring their ability t o clonally expand and increase stem compartment size. Our data suggest
that, in some cases,cytogenetically aberrant cells in the primitive compartment, which precede the blast subpopulation,
do not contribute to stem compartment expansion.
MATERIALS AND METHODS
Patienrs. Leukemicand normakontrol bonemarrowandperipheral blood specimens from patients with AML
or MDS were
obtained under approval of the Committee on Human Subjects at
University of California, San Francisco (UCSF, San Francisco, CA)
or throughtheSouthwestOncologyCooperativeGroup
(SWOG)
Leukemia Repository of the University of New Mexico (Albuquerque, NM). Marrow and blood samples were drawn at the time
of
diagnosis.Diseasediagnosisandclassificationwereaccordingto
French-American-British(FAB)criteria.Blastpercentagesfrom
Wright-Giemsa-stainedaspiratesmearsweredetermined
by performing differential counts of all nucleated marrow cells. Karyotype
analysis of marrow from UCSF patients was performed
by Integrated
Genetics (Albuquerque, NM) as part of routine clinical diagnostic
procedures. Karyotypes of marrow and blood of leukemic patients
SWOG
enrolledon SWOG clinicaltrialswerereviewedbythe
Cytogenetics Committee (Dr Ellen Magenis, Chair).
Cell phenotyping and separation. Marrow and blood subpopulations were separated using density sedimentation before cryopreservation and immunophenotyping. Frozen cells were thawed gradually
at 37°C andwashedinIscove's
modified Dulbecco'smedium
(IMDM) containing bovine fetal calf serum (10%). Nucleated cells
werelabeledwithHoechst
33342 (HO;7.5pmol/Lat
37°C. 60
minutes), washed in Hank's balanced salt solution containing azide
(0.02%; S-HBSS), and incubated at
4°C for 45 minutes with the
following antibodies: anti-CD3 (mature T cells), anti-CD20 (mature
B cells), anti-CD14 (monocytes), anti-CD34 (primitive progenitors
andstemcells),anti-CD33andanti-CD38(myeloidcells),anti1 (erythroid cells). Antibodglycophorin A (anti-GPA), and anti-CD7
ies [Becton Dickinson Immunocytometry Systems (BDIS), San Jose,
CA] were conjugated with either fluorescein isothiocyanate (FITC)
or phycoerythrin (PE). Erythroid cells were consideredto be CD71'
and CD14-CD3-. Isotype-matched fluorescenated antibodies (Simultest; BDIS) were used as controls for nonspecific immunofluores-
cence. After antibody labeling, cells were washed once with phosphate-buffered saline, pH 7.4 (PBS). Dead cells were stained with
propidium iodide (PI; Calbiochem, San Diego. CA:
1 pg/mL for 5
minutes at 20°C).Cellswere
fixed withparaformaldehyde(PF;
0. I ?h) for 10 minutes and washed once in PBS before flow cytometric analysis.
Flow cytometric analysis and sorting of subpopulations were performed at UCSF using a FACStar PLUS (BDIS) equipped with two
argon ion lasers(Coherent,PaloAlto,CA)tuned
to 488 nm and
35 I to 364 nm (UV). Forward light scatter, perpendicular light scatter, and four fluorescence signals were measured for each cell and
saved in list mode data files using LYSIS 11 software (BDIS). Cell
doublets and aggregates were excluded using forward light scatter
pulse processing. Hoechst and PI fluorescence emission (UV excitation) were collected through a 425-nm ( 2 5 0 nm) band pass and a
620-nm long pass filter, respectively. FITC and PE immunofluoreacence emission (488 nm excitation) were collected through 530-nm
( 2 3 0 nm) and 575-nm ( 2 2 6 nm) band pass filters, respectively.
Hematopoietic subpopulations were identified on the basis of fluorescence intensity. PId"""HO' events were considered to be viable
nucleated cells (see Fig IA). The PId'"" subpopulation is shown in
the R2 windowinFigIA.Anisotype-matchedantibodycontrol
defined non-antigen-specific antibody binding and was used to define the immunofluorescence intensity above which cells were considered labeled specifically. Quadrant markers wereused to estimate
compartmentsize,such that thegatedregionscontained
d.OI%
viable cells when the sample was labeled
with the isotype-control
reagents. The proportion of cells in the CD34'lin-, CD34+lin- regions was estimated to be the frequency of cells in quadrant regions
A and B (Fig 1C and D), respectively. Rectangular regions
within
B were used forsorting CD34'lin (R3)
quadrantregionsAand
and CD34'lin' (R4) and are shown in Fig I . For CD34' lin sorting,
the upper sort boundary was set at a PE fluorescenceintensity channel 58% 2 0.03% of the CD33/38 quadrant boundary, whereas the
left sortboundarywasapproximately
I 13% 2 2% of theCD34FITC quadrant A boundary. The boundaries for sorting CD34'lin
cells were defined as follows: lower boundary was 62% 2 3% of the
maximum CD33/38 fluorescence intensity; upper boundary.115% t
3% ofthe CD33/38' quadrant B boundary. Cells with intermediate
CD3308 fluorescence were not sorted.
Cellsgatedonthebasis
of immunofluorescence intensity were
sortedontoglassmicroscopeslidescontainingCarnoy's(methanol:acetic acid, 3: l : vol/vol) fixative. Normal-C sorting mode was
used to achieve maximal purity, which exceeded 98%. Slides containing sorted cells were placed on a slide warmer for 30 minutes
before immersion in fresh Carnoy's fixative for 5 minutes. Slides
were stored in ethanol (80%) at -20°C until further processing.
FISH. Selection of DNA probes was individualized on the basis
of the karyotype of each leukemic specimen. Probes for chromosomes8and X directlylabeled with SpectrumorangeandSpectrumGreen were provided by Vysis (Naperville, IL). A biotinylated
repeat sequence DNA probe for chromosome 6 (D6Z1) and a digoxigenin-labeled probe (D7Z2) for chromosome 7 were obtained from
Oncor (Gaithersburg, MD). A biotinylated chromosome I -specific
satellite 111 probe (pUC I .77) was prepared in the UCSF Division of
Molecular Cytometry core probe facility.
FISH was performed as describedby Pinkel et all' with the following modifications. Briefly, air-dried cells were treated with freshly
S minutes, allowed to air dry, and
prepared Carnoy's fixative for
then baked at 65°C for 15 minutes. Cellular DNA was denatured at
72°C for 2.5 minutes in 70% formamide (Omnisolv; EM Science,
Gibbston, NJ), saline sodium citrate (SSC; 0.30 mol/L NaCl, 0.03
m o l L sodium citrate, pH 7.0). After dehydration in ethanol series
and air-drying at 42"C, DNA probe ( I to 2 pL) was denatured in
hybridization mixture [fomamide, 50%; dextran sulfate (Sigma. St
+
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1141
STEM CELLS IN LEUKEMIA
A
Fig 1. Multivariate flow cytometric distributions of leukemic
marrowlblood. H 0 and PI fluorescence intensity were used to
select nucleated viable cells that
excluded PI (R2; A) and, thus,
showed red fluorescence. (B
through D) The FITC-linked immunofluorescence is shown on
t h e abscissa, and PE-linked fluorescence is on the ordinate. The
bivariate FlTC versus PE distribution of cells labeled with t h e isotype control reagent (B) and t h e
bivariate distributions of specimens 8 (C) and 2 (D) are shown.
Quadrant B contains cells that
express CD34 and CD33lCD38
(CD34'lin'); quadrant A contains
cells that express CD34 but lack
CD33 and CD38 (CD34'Iin.). R4
shows the sort region that contains CD34'lin' events, whereas
sort region R 3 contains cells expressing CD34 but lacking CD33/
CD38.
Louis. MO). 10%; 2 x SSC] at 72°C for S minutes and immediately
applied to cells on slides that were hybridized overnight in a humidified chamber at37°C. Slides were washed three times at 45°C in
50% formamide. 2X SSC, pH7.0: and once each in2X SSC and
0.2x s s c .
Cells hybridized with biotinylated or digoxigenin-labeled probes
were treated further with 4X SSC containing bovine serum albumin
(BSA: 2%) for S minutes at roomtemperature. Hybrids were detected
using FITC-avidin (DCS: Vector, Burlingame, CA; S pglmL) or a
rhodamine-linked antidigoxigenin antibody (Boehringer Mannheim,
Indianapolis, IN: 0.4 pglmL) as described previously." Chromosomal DNA was counterstained with diamino-2-phenyl-indole dihydrochloride (DAPI) in antifade solution, as described by Pinkel et
Microscopic ann!\ais. Fluorescenated hybrids in phenotypically
discriminated subpopulations were detected using fluorescence microscopy. Cell scoring was performed using a 100 X Plan Neofluor
objective mountedonan
Axioscope (Zeiss, Wetzlar, Germany)
equipped with a mercuryarc lamp and a 3.5-mm camera system.
Green and red fluorochromes were visualized simultaneously using
a multiband emission filter (81P101) andbeam splitter (81P100)
with a dual band excitation filter (81P102) from Chroma Technology
(Brattleboro, VT).
At least SO sorted cells were scored on each slide except for
specimens 14and IS, in which fewer cells were present in the
B
D
R4
.~
CD34fITC
(LW1
stem compartment. The number of sorted cells recovered in each
subpopulation sort is listed in Table 3. The frequency of cells showing hybridization domains and the number of domains per cell were
determined. Cells with disrupted nuclear membranes were excluded
from the analysis. Hybridization domains were considered to represent two chromosomes if the fluorescent hybridization signals were
separate and nonoverlapping. In patients carrying monosomies of
the marker chromosome, a second chromosome-specific enumerator
probe was usedto provide a control for hybridization efficiency.
Cells were considered to be monosomic for the marker chromosome
if the cell showed one hybridization domain corresponding to the
marker chromosome and two fluorescenated hybrids corresponding
to the control enumerator probe. The hybridization efficiency (ie,
two hybridization domains) of control DNA probes exceeded 97%.
RESULTS
Patient cell characteristics. Specimens were obtained
from 12 patients with de novo AML and three withMDS
(patients 6, 12, and13; Table l ) . Specimen sources and
characteristics are shown in Tables 1 and 2. AMLFAB
subtypes MO through M6 are represented in the specimen
survey, with the exception of M3. Diagnosis karyotypes included aneusomies, as well as additional complex regional
losses, gains, or rearrangements in the leukemic clones. A
93
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MEHROTRA ET AL
1142
Table 1. Patient Characteristics
Patient
No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Age ( y t /
Sex
68/F
68lM
35/F
57/M
34/F
50/F
66/F
23lM
37lF
61lF
45lM
76/F
45/M
48/M
61/M
Source
Tissue
SWOG Marrow
SWOG Marrow
Blood
SWOG
SWOG Marrow
SWOG Marrow
Marrow
UCSF
Marrow
UCSF
SWOG Marrow
SWOG Marrow
SWOG Blood
SWOG Marrow
Marrow
UCSF
Marrow
UCSF
SWOG Marrow
SWOG Marrow
FAB
Type
Diagnosis
AML
AML
AML
Karyotype
M5
M1
M2
A ML
AML
MDS
A ML
AML
AML
AML
A ML
MDS
MDS
AML
AML
M2
M5
RAEB-T
M6
M6
M1
MO
M4E0
RA
RAEB-T
M1
M1
Abbreviations: RAEB-T, refractory anemia with excess blasts in transition; RA, refractory anemia.
single karyotypically aberrant clone is present in specimens
1 through 9 and 13 through 15, whereas two aberrant clones
are observed in specimens 10 through 12. Morphologically
identifiable blasts in fresh marrow aspirates varied between
5% and 99%. (Table 2). The relative proportions of blasts
(74% to 95%; Table 2 ) in density-sedimented, cryopreserved
specimens were estimated on the basis of cellular light-scattering properties of marrowhlood.
Specimens were immunophenotyped to measure the antigenic profile of the leukemic blast population and nonleukemic cells. Viable nucleated cells were gated (see R2 gate in
Fig 1A) on the basis of H 0 and PI fluorescence intensity.
The frequency of viable cells ranged between 50% and 90%
(data not shown) among specimens, reflecting effects of cry-
opreservation and storage in liquid nitrogen. Measurements
of antigen expression on selected density-sedimented specimens before and after cryopreservation showed similar immunofluorescence profiles (data not shown). Between 6%
and 95% of the population expressed CD33KD38, whereas
fewer than 1 1 % expressed CD3KD20. In each case,
lymphoid cells represented a relatively small fraction of the
tissue specimen. Expression of erythroid antigens occurred
in 0 to 54% of the cells in the leukemic specimens. In some
cases (ie, specimens 10 and 1 l ) , the high frequency of erythroid expression is consistent with coexpression of erythroid
markers by a subset of myeloid leukemic cells. CD34 expression provides an index of cell maturity. Normal marrow and
blood contain between 1 % and 5% and 0.1 % CD34' cells,
Table 2. Blast Frequency and lmmunophenotype
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
91
98
62
99
10
18
35
95
90
80
<5
25
99
99
75
90
90
85
90
74
80
90
95
90
80
95
80
90
95
72.2
83.6
91.6
94.1
94.5
ND
51.2
26.0
66.8
91.o
42.6
6.5
10.6
93.0
11.1
2.6
ND
ND
ND
ND
2.3
7.6
2.2
0.2
1.3
ND
ND
2.42
9.1
6.4
10.8
ND
ND
0
0.9
Abbreviation: ND, not determined.
* Blasts identified morphologically on marrow smears.
t Blast frequency estimated on the basis of cell light scatter.
lmmunophenotyping performed on thawed specimens.
*
5.9
0.9
1.o
0.2
ND
ND
ND
2.0
ND
ND
ND
ND
ND
ND
ND
3.9
0.0
0
0.1
10.4
2.1*
25.0*
54.0
O*
2.1*
1.1
69.9
0.2
5.6
0.1
0.5
18.9
0.5
10.0
37.9
30.2
0.5
0.3
41.6
1.4
0.57
2.90
0.05
0.18
0.02
0.26
0.45
0.42
1.28
0.57
13.73
0.50
0.07
0.23
0.48
0.49
67.1
0.19
5.40
0.06
0.20
18.50
0.06
7.48
37.36
16.48
0.10
0.24
41.40
0.96
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1143
STEM CELLS IN LEUKEMIA
respectively. Six specimens (specimens 2, 7, 9, 10, 11, and
14) contained greater than 10% CD34' cells. Nine specimens
contained less than 10% CD34+ cells and, thus, are considered to be CD34- leukemias. Thus, both CD34+ and CD34leukemias are represented in the analysis set.
The CD34+ subpopulation was subdivided further on the
basis of CD33 and CD38 expression. CD33 and CD38 are
considered markersof myeloid and lymphoid differentiation,
respectively. Thus,CD34TD33-CD38- cells comprise one
of the most primitive hematopoietic subpopulations. Figure
of two
1Cand D showstheCD34/CD33/CD38analysis
leukemic specimens. Quadrant region A in Fig 1C and D
defines the CD34+lin- population in specimens2and3,
respectively. A rectangular sort region (R3; Fig 1C and D)
within quadrant regionAwasused
to collect CD34'lincells for FISH analysis. A sort gate (R4) within quadrant
region B, containing CD34+lin+cells, was used during cell
sorting to obtain maximal purity of the CD34+lin+ cells.
Normal
marrow
contains approximately 1% and 4%
CD34+lin- and CD34+lin+cells, respectively, with approximately 10-fold fewer cells of the same phenotype in blood.
Specimen 2 (Fig lA), a CD34+ leukemia in which approximately 70% of the cells express CD34, contains only 3% of
the CD34+lin- cells. Specimen 3 (Fig 1D)
contains relatively
few (ie, 0.2%) CD34+ cells, of which 0.05% are lin-. The
size of the CD34'lin- compartment is within or below normal values in all samples except specimens 2 and 11. The
frequency of CD34'lin- and CD34+lin+cells in marrow and
blood specimens varies in a 10-fold range (0.02% to 14%)
among specimens (Table 2). Generally, theCD34+lin+cells
comprise similar or higher proportions than the CD34+lincounterparts.
FISH analysis of sorted subpopulations. Cytogenetically
aberrant cells subpopulations were quantified using fluorescence microscopy after hybridization of sorted cells with
enumerator probes selected according by the leukemic cell
karyotype. Background aneusomylevels in five normal control marrows hybridized with the probes used in leukemic
cell analyses (data notshown)weredeterminedtodefine
the levels above which cells were considered anesomic. An
average disomic frequency of97.4% (20.5%; 1 SD) was
observedwithenumeratorprobes
for chromosomes1,6,
and 8. The remaining cells were trisomic (0.4% ? 0.1%),
monosmic (1.5% ? -0.3%),orlackedhybridizationdomains (0.6% ? 0.2%). The frequency of aneusomic cells in
normal marrow was independent of probe source for chromosomes (data not shown). Figure 2 shows representative
photomicrographs of sorted cells from the CD34 (Fig 2A),
CD3 (Fig 2B), and GPA (Fig 2C) compartmentsof a leukemic specimen with trisomy 8. Cells carrying three red hybridizationdomainscorrespondingtochromosome
8 are
clearly visible in the sorted cells.
FISH analysis of sorted subpopulations ineach specimen
are shown in Table 3. Insufficient cells and technical difficulties precluded complete CD34 subset analysis on specimen 7; therefore, onlythe CD34'lin- compartment was analyzed.Cytogeneticallyaberrant
cells were detected in
CD34+lin- and CD34+lin+compartments in all specimens
analyzed. The CD34+lin- population contains between 9%
and 99% aberrantcells, with an average frequencyof 41.9%.
Onaverage, the CD34+lin+ compartmentscontain48.2%
aberrant clones, with an intersample range of 6% to 97%.
The frequency of cytogenetically aberrant cells in the stem
compartment is independent of CD34 statusof the dominant
leukemic clone and FAB status. For example, specimens 1,
3through6,
8, 12,13,and
15 contained less than10%
CD34+ cells, yet the frequencies of aneusomic cells in the
CD34'lin- compartment in these same specimens were 9%,
99%, 66%, 34%, 25%, 52%, lo%, 16%, and 22%, respectively. In the CD34+ leukemic specimens (specimens 2, 7,
11, 12, and14),cytogeneticallyaberrant CD34+lin- cells
87%, lo%,
spanned a similar range
of values (eg, 70%, 31%,
and11%,respectively).Withinindividualspecimens,the
frequency of aberrant cells in the CD34+lin- compartment
waslessthan or similar to aberrant cell fractions of the
CD34+lin+compartment. Aberrantcells in the stem compartment of CD34-, as well as CD34+, leukemias provide evidence for existence of cytogeneticallyabnormal cells in
primitive Compartments.
C
Fig 2. Photomicrograph of cells sorted from phenotypically defined compartments from trisomic 8 leukemic marrow hybridized with a
chromosome 8 enumerator probe. (A) CD34+lin+ cells from specimen9 dually hybridized with a chromosome 8 enumerator probe (red) and
an X chromosome-specific control probe (green). CW-labeled cells
(B)and GPA-labeled cells (Cl from specimen
2 hybridized with chromosome
8 enumerator probe. Three red hybridization domains are presentinin
each
cells
panel, showing
the presence of trisomy 8 in sorted populations.
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MEHROTRA ET AL
1144
The absolute frequency of aberrant CD34+lin"' cells in
marrow or blood was estimated to evaluate the power of
the combined immunophenotype and genotype approach to
detect low-frequency aberrant stem cells. Absolute numbers
of genetically aberrant CD34+lin- and lin' cells in marrow/
blood specimens were estimated by multiplying compartment size by the frequency of aberrant cells in each subpopulation. Data in Table 4 indicate that CD34'lin- aberrant cells
constitute between 0.01% and 11.94% of marrowhlood. Cytogenetically aberrant CD34+lin+ cells comprise 0.01% to
55.7% of the same specimens. The detection sensitivity of
the combined approach is greater than that observed with
either FISH or immunophenotype alone.
Additional confirmation of stem cell involvement was obtained by FISH analysis of differentiated erythroid and
lymphoid cells in specimens with sufficient cell numbers to
allow multiple subpopulation sorts. Cells expressing differentiation antigens associated with lymphoid and erythroid
maturation were discriminated and sorted in eight leukemic
specimens inwhich mature lymphoid and erythroid cells
were present at frequencies below the predominant leukemic
clone. Cytogenetically aberrant cells (7% to 72%) were observed in the CD3 and/or CD20' lymphoid compartment in
specimens analyzed. In these same specimens, cytogenetically aberrant erythroid cells ranged between 7% and 55%.
The frequency of aberrant cells in the lymphoid and erythroid
Table 3. Frequency of Cytogenetically Aberrant Cells
in Sorted Subpopulations
Patient
No.
Chromosome
Analyzed
CD34+1in-
CD34+lin+
28t (180)
83* (31 1)
7# (108)
56# (225)
97 (137)
78
(137)
78t (487) 4011 (120)
60* (487)
25 (826) 43 (775) 147 (968)
721 (loo)
ND
62 (200)
ND
32 (100) 336 (200)
7 (594)
52 (269) 57 (177)
ND
27 (354) 25 (314)
ND
7 (229)
11 (321)
ND
87 (297) 86 (328)
6 (166)
ND
10 (137)
ND
16 (247) 13 (353)
81 (93)
ND
11 (31)
17 (141)
ND
22 (11)
21# (130)
1
2
8
8
9 (115)
65 (66)
3
1
6
1. 8
99 (106)
66 (106)
34 (80)
4
5
6
7
8
9
10
11
12
13
14
15
1, 8
8
8
1, 8
6
1, 8'
7,8
X, 11
18
11
17
Lymphoid
Erythroid
51# (125)
20** (967)
55# (116)
ND
ND
ND
ND
ND
ND
ND
ND
ND
Data are percentages, and values in parentheses represent the number of sorted cells in each region.
* This specimen contained two cytogenetically aberrant clones, one
of which carried +8.
t CD33lCD38.
CD33ICD34lCD38.
5 CD33lCD34ICD38lCD45.
11 CD20.
CD3.
# Glycophorin.
** CD71.
+
Table 4. Frequency of Phenotypically Defined Aberrant
Cells in Leukemic Specimens
Specimen No.
CD34'lin~
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
0.05
1.88
0.05
0.06
0.01
0.16
0.13
0.22
0.35
0.06
11.94
0.05
0.01
0.025
0.1 1
CD34'lin
0.14
55.69
0.18
3.73
0.03
6.1 1
0.03
1.87
2.62
14.17
0.01
0.03
33.53
0.16
Values represent the frequency (%) of cytogenetically aberrant cells
with the designated phenotype in the marrow or blood specimen.
compartments was independent of the level of aberrant cells
in other sorted subpopulations from the same patient. These
data show that cells expressing lymphoid and erythroid maturation markers carry chromosome abnormalities present in
the myeloid leukemic clone.
The relationship between aberrant cells in phenotypically
defined subpopulations and compartment size and clonal
expansion was explored to evaluate the functional phenotype
ofCD34'linaneusomic cells. Figure 3 shows patterns of
compartment size versus aberrant cell content of five leukemic specimens. Increasing cellmaturity is represented on
the abscissa: CD34+lin- to CD34'1in' to CD34 to morphologically recognizable blasts to CD33/38. Itisrecognized
that there is some overlap between the maturational status
of cells in each of these compartments. Figure 3 shows
CD34+lin- and lin+, CD34' blasts and CD33/38-expressing
compartments in normal marrow andblood. As expected, the
size of these phenotypically defined compartments increases
with increasing maturity (Fig 3A). Genotype-phenotype
analysis of MI leukemias are shown in (Fig 3B through E).
In specimens 2, 9, 14, and 15, the CD34+lin" compartment
is relatively small, although aberrant cells comprise approximately 60%, 22%, 80%, and 20%, respectively, of this compartment. These data suggest varying degrees of clonal
expansion within the lin- compartment, whereas compartment size is maintained at normal or below control values.
The size of the CD34+linf compartment is similar to control
levels in specimen 15; however, it is expanded in specimens
2, 9, and 11. Each M1 specimen contains approximately
the same fraction of blasts. Specimen l 1, an M4E0, shows
approximately 80% aberrant cells in both CD34 compartments with concomitant expansion of both the lin- and lin'
subpopulations. Thus, the size of the CD34' subcompartments appears uncoupled with the aberrant cell frequency
within the compartment and independent of blast frequency.
DISCUSSION
Previous investigations have yielded conflicting results
regarding extent of stem cell and lineage involvement in
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
1145
STEM CELLS IN LEUKEMIA
A
Controls
1007
6
x
1
100
80
60
40
20
D Sample 14: MI
Fig 3. Genotype-phenotype relationships ofleul00
kemic specimens. Maturational state is shown on
the abscissa,andcellfrequency
(YO) ison the ordi6o
nate. (A) Relative proportionsof morphologicallyde40
fined blasts and phenotypically-definedsubpopula20
tions in blood ( B ) and marrow ( D ) are shown. (B
o
through F ) Compartment size (46; El) and aberrant
cell frequency(YO; D ) of leukemic specimen subpopulations.
yl
:ae;a
E Sample 15: MI
F Sample 1 1 : M4EO
m
. - L O j ; "
Q
AML. Several experimental approaches have beenused to
infer stem cell involvement in leukemia. Indices of clonality
such as X-linked polymorphism and alternative allele expression,','" Ig gene and T-cell receptor rearrangernent~,"~'~
and,
more recently, in situ hybridization"."'."."' have beenused
to identify progeny arising from immature cells carrying
distinctive molecular or genetic markers. Some studies suggest that the aberrant cells in AML and MDS are restricted
to myeloid and erythroid lineages and, occasionally, megakaryocytic
lineages,"".~~,~'),?'.??
whereas others suggest
involvement of a multipotential stem cell." For example,
Kibbelaar et al" usedpanning, FISH, and immunophenotypic
discrimination of myeloid and lymphoid cells in MDS patients to determine that T cells didnot carry cytogenetic
abnormalities found in the leukemic clone. Similar results
were reported by Anastasi et all" using FISH with a chromosome 8 enumerator probetomeasure
trisomy 8 in MDS
patients with chromosome 8 aneusomy. VanLometal"
reported FISH analysis of AML patients showing that lymphocytes do not carry trisomy X. whereas neutrophils, eosinophils, and monocytes were trisomic. However, other studies
indicate that the cells in the lymphoid, myeloid, and erythroid lineages may carry genetic markers identical to those
in the leukemic blast^.^.".'^^^^ The basis for these conflicting
results is unclear and may reflect different techniques used
to assess clonality and detect genetically marked cells, as
well as variability between patients. We used FISH to detect
cytogenetically aberrant cells in immunophenotypically defined primitive and mature compartments. Sorted cells carrying thecytogenetic abnormality characteristic of the leukemic blast were used to provide evidence for involvement of
primitivektem cells in AML/MDS.
FISH analysis of lineage-restricted differentiated cells in
the leukemic specimens suggests their derivation from a
common cell with multilineage capability. Cells sorted from
lymphoid and erythroid lineages on the basis of antigen expression carried cytogenetic markers present in predominant
myeloid leukemic blast populations. Aberrant cells com-
n
0
prised between 7% and 76% of the CD3 and/or CD20 subpopulations in the sample set analyzed. In each subpopulation sort, 100 to 967 cells were analyzed. Stringent flow
cytometric sorting conditions (ie, multiple phenotypic gates,
doublet discrimination, exclusion of on-viable cells) were
used to minimize potential contamination of the differentiated subpopulation sorts with leukemic blasts. Although we
cannot completely eliminate thepossibility
of aberrant
lymphoid antigen expression by the leukemic blasts, in several cases the frequency of lymphoid cells was almost an
order of magnitude below that of the leukemic clone. These
findings, coupled with the high frequency of aberrant cells,
suggest that in several specimens lymphoid, myeloid, and
erythroid cells may have originated from a multipotential
precursor. Knuutila et aIz7reported recently that trisomy 8
andmonosomy 7 abnormalities maybe present in single
lineages or multiple lineages, including lymphocytes in a
limited number of MDS/AML specimens.
Evaluation of cytogenetically aberrant cells in the
CD34'lin- compartment providedadditional evidence of
stem cell involvement in these same patients. The CD34'linpopulation is believed to represent one of the most primitive
compartments of human hematopoiesis," containing cells
capable of self-renewal and reconstitution and maintenance
of hematopoiesis for extended periods of time.Recently.
Lapidotetal"reportedthat
transplantation of CD34'lincells fromAMLmarrowandblood
specimens engrafted
severe combined immunodeficiency (SCID) mice and produced large numbersof colony-forming progenitors and, ultimately, leukemia. Our molecular cytogenetic analysis demonstrates thatthe CD34' CD38 compartment in 15 AML
and MDS specimens contains cytogenetically aberrant cells
present at frequencies ranging between 9% and 99%. The
frequency of aberrant cells appears dissociated from FAB
type (M-3 and M-7 cells were not included in the analysis
set), blast frequency, and CD34 expression by the leukemic
blasts. These data. coupled with findings of aberrant cells in
low-frequency lymphoid subpopulations and in erythroid as
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
MEHROTRA ETAL
1146
well myeloid lineages, are consistent with origin of leukemias carrying aneusomies in the stem compartment, even in
CD34- leukemias.
The functional phenotype of aberrant cells in the lincompartment was evaluated to determine
( l ) whether cytogeneticallyaberrant cells contributed to stem compartment
expansion and (2) if frequency was associated with comparment size. The maturational stage at which malignant transformation (eg, acquisition of the leukemic phenotype as reflected in clonal expansion) occurs is variable. Data in Fig
3 and Tables 2 and 3 show that CD34+lin- compartments
may remain at or below control values, yet contain 9% to
80% aberrant cells. Expansion of the CD34' compartment
in CD34+ leukemias may occur at either the lin- or
lin'
stage. In CD34- leukemias, the lin- and lin+ compartments
remain constant, and compartment expansion occurs
after
progressive maturation of the cytogenetically aberrant clone.
Thus, the presence of cytogenetically aberrant cells in primitive compartments doesnot predict compartment expansion,
even though the clone may have expanded to fill the designated Compartment. These data are compatible
with the speculation that the functional and/or phenotypic consequences
of the cytogeneticabnormalities in CD34+lin-cells, or occasionally even in CD34+lin+ cells, may only be expressed as
the cytogenetically abnormal
cells
mature
past the
CD34+lin- stage. Thus, the cytogenetic aberrations may not
confertheleukemicphenotype
(ie, clonalexpansion,decreased apoptosis, etc) until the cells mature to a stage in
which aberrant genes are expressedor additionaltransforming events occur. Apathogenesis for AML has been
suggested by other investigators,6,'6,iowho used analysis of
clonal remissions to postulate existence of a preleukemic
cell. Fialkow et a1633"postulate that the preleukemic cell is
cytogenetically normal but then develops abnormalities at a
later stage. Ras has been implicated aaslate eventassociated
with transformation.i',3z Lapidot et a129report existence of a
leukemia-initiating cell in AML.Ourdataalso
suggesta
preleukemic cell in AML and MDS and indicate that this
cellcarriesgrosscytogenetic
abnormalitiespresentin
the
predominant leukemicblast population. Variableproportions
of cytogenetically aberrant cells in the CD34+lin- compartment may contribute to heterogeneity observed in levels of
human cells in leukemic stem cell-engrafted SCID mice.29
Furthermore, we suggest thatthese cytogenetically aberrant
preleukemic cells may not show transformation (eg, expansion) until a later maturational stage. Variable sites of clonal
expansion are suggested to occur in hybrid 1e~kemias.j~
Further studies will be needed to explore these concepts.
Our data have importantimplications for treatment monitoring and evaluation of residual disease. Residual disease
detection is limited by sensitivity of the detection strategy.
Morphologicand karyotype-baseddetection assays allow
identification of aberrant cells present at frequencies greater
than 1% to 5%. The sensitivity of aberrant cell detection
using FISH with a single marker probe is about 1% to 3%.
Leukemic blastspresentat
frequencies of approximately
0.1% are detected by flow cytometric analysis of cells labeled with antibodies specific for the leukemic cellsat diagnosis. Molecular-based assays, such as in vitro DNA ampli-
fication using the polymerasechain reaction, allow detection
of aberrant DNA sequences in cells present at frequencies
of lo-' to lo-'; however, the phenotype of cellscarrying
the abnormality is unknown. Our data show that combined
immunophenotype-genotype measurements substantially increase thedetection sensitivity of either technique alone. For
example, flow cytometric discrimination of CD34'lin cells
discriminates a subpopulation present in leukemic marrow/
blood at frequencies between 0.01% and 0.05%. FISH analysis of the sorted cells increases detection sensitivity by an
order of magnitude. Thus, subpopulation sorting and FISH
allows detection of low-frequency cytogenetically aberrant
cells in the CD34+lin- compartment that would not be detected using conventional assays.
The clinical
significance
of aberrant cells the
in
CD34'lin- compartment remains to be defined. Incomplete
eradication of the aberrant cells may ultimately contribute
to relapse after differentiation to progeny that gain ascendancy over the remaining normal cells. The responsiveness
of these genetically aberrant, immature stem cells to chemotherapy is unknown, and it is tempting to speculate thattheir
response to treatment protocols may differ from that of the
leukemic blasts. If so, targeted investigations of the functional characteristics of cells in this compartment may lead
to a better understanding of treatment failures. Furthermore,
our data suggest caution in using CD34-based selection of
peripheral blood or marrow stem cells for autologous transplantation of patientswith AML. Even in the absence of
CD34' expression by the leukemic blasts, a fraction of cells
in the CD34'1in- compartment is cytogenetically abnormal.
ACKNOWLEDGMENT
Wethank Vysis for providing several of the enumerator probes
usedinthesestudies.Flowcytometricanalysiswasperformedat
the Laboratory for Cell Analysis at UCSF. We thank Kathleen Richkind at Integrated Genetics for karyotypes of UCSF
specimens.
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1995 86: 1139-1147
Cytogenetically aberrant cells in the stem cell compartment
(CD34+lin-) in acute myeloid leukemia
B Mehrotra, TI George, K Kavanau, H Avet-Loiseau, D 2nd Moore, CL Willman, ML Slovak, S
Atwater, DR Head and MG Pallavicini
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