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Blood, Vol. 93 No. 1 (January 1), 1999:
pp. 284-292
BCR/ABL CD34+HLA-DR
Progenitor Cells in Early Chronic Phase, But Not in More Advanced
Phases, of Chronic Myelogenous Leukemia Are Polyclonal
By
Michel Delforge,
Marc A. Boogaerts,
Philip B. McGlave, and
Catherine M. Verfaillie
From the Division of Hematology, the Department of Medicine,
University Hospital Gasthuisberg, Leuven, Belgium; and the
Division of Hematology, the Department of Medicine, University of
Minnesota, Minneapolis.
 |
ABSTRACT |
Chronic myelogenous leukemia (CML) is characterized by the
Philadelphia (Ph) translocation and BCR/ABL gene rearrangement which
occur in a pluripotent hematopoietic progenitor cell. Ph-negative (Ph ) hematopoiesis can be restored in vivo after
treatment with  -interferon or intensive chemotherapy, suggesting
that normal stem and progenitor cells coexist with the
Ph+ clone. We have previously shown that
Ph progenitors are highly enriched in the
CD34+HLA-DR fraction from early chronic
phase (ECP) CML patients. Previous studies have suggested that the
Ph-translocation represents a secondary clonal hit occurring in an
already clonally mutated Ph progenitor or stem cells,
leaving the unanswered question whether Ph
CD34+HLA-DR- progenitors are normal. To show
the clonal nature of Ph
CD34+HLA-DR CML progenitors, we have
compared the expression of BCR/ABL mRNA with X-chromosome inactivation
patterns (HUMARA) in mononuclear cells and in
CD34+HLA-DR+ and
CD34+HLA-DR progenitors in marrow and
blood obtained from 11 female CML patients (8 in chronic phase and 3 in
accelerated phase [AP] disease). Steady-state marrow-derived BCR/ABL
mRNA, CD34+HLA-DR
progenitors had polyclonal X-chromosome inactivation patterns in 2 of 2 patients. The same polyclonal pattern was found in the progeny of
CD34+HLA-DR derived long-term
culture-initiating cells. Mobilization with intensive
chemotherapy induced a Ph, BCR/ABL mRNA
and polyclonal state in the CD34+HLA-DR
and CD34+HLA-DR+ progenitors from 2 ECP
patients. In a third ECP patient, polyclonal CD34+ cells
could only be found in the first peripheral blood collection. In
contrast to ECP CML, steady-state marrow progenitors in late chronic
phase and AP disease were mostly Ph+, BCR/ABL
mRNA+, and clonal. Further, in the majority of these
patients, a Ph, polyclonal state could not be restored
despite mobilization with intensive chemotherapy. We conclude from
these studies that CD34+HLA-DR cells that
are Ph and BCR/ABL mRNA are polyclonal
and therefore benign. This population is suitable for autografting in
CML.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
CHRONIC MYELOGENOUS leukemia (CML) is a
clonal myeloproliferative disease characterized in more than 90% of
patients by the Philadelphia (Ph) chromosome which involves a
reciprocal translocation between the c-abl gene on chromosome 9 and the
breakpoint cluster region (bcr) locus on chromosome 22 [t(9;22) (q34.11;q11.1)].1 The BCR/ABL mRNA gives rise to
a 210-kD (p210BCR/ABL) tyrosine kinase. Compared with the
endogenous c-abl, p210BCR/ABL has increased kinase activity
and binds significantly more to the actin cytoskeleton. This affects
the regulation of cell adhesion, cell survival, and
proliferation,2-4 all of which are thought to play a
crucial role in the pathogenesis of CML.
The clonal Ph-positive (Ph+) pattern can be found in cells
of the myelomonocytic, erythroid, and megakaryocytic
lineages,5,6 indicating that the BCR/ABL mutation occurs in
an early hematopoietic progenitor. In the chronic phase of the disease,
most T lymphocytes7-9 and some of the B
lymphocytes10 are polyclonal and Ph-negative (Ph). At diagnosis, Ph
progenitors can be detected in steady-state marrow from some CML
patients.11 Further, a Ph state can be
restored, at least temporarily, in marrow or blood cells after in vivo
treatment with either -interferon (IFN-)12 or
intensive chemotherapy,13,14 or after ex vivo
"purging" by growth in long-term cultures,15 exposure
to IFN,16 cytotoxic drugs,17 or antisense
oligonucleotides.18 Finally, we and others have shown that
BCR/ABL progenitors are enriched in an immature
precursor fraction expressing the CD34 antigen, but lacking HLA-DR
antigens (CD34+HLA-DR), but not in
CD34+ cells coexpressing the HLA-DR antigen
(CD34+HLA-DR+),19,20 or other
immature phenotypes.21 All of these studies thus indicate
that Ph progenitors coexist with the
Ph+, malignant progenitor pool.
Previous studies using glucose-6-phosphate dehydrogenase (G6PD)
polymorphisms as a marker for clonality have shown that clonal Ph B cells could be detected in some Ph+
patients,22-24 suggesting that a clonal
Ph stage may precede the Ph+ state in
some CML patients. Because autografts using Ph
progenitors, obtained either by in vivo administration of
chemotherapy13,14,25,26 or by ex vivo
selection27,28 are currently being done or are contemplated, it is crucial to determine the clonal nature of Ph CML progenitors. We compared the presence of
BCR/ABL mRNA in mononuclear cells,
CD34+HLA-DR, and
CD34+HLA-DR+ cells from female CML patients in
chronic and AP with their respective X-chromosome
inactivation (XCI) patterns. XCI analysis is based on the Lyon
hypothesis that inactivation of one of both X-chromosomes occurs in
each female somatic cell early in embryogenesis.29 This
inactivation pattern is stably inherited by all daughter cells.30 Therefore, all cells in a clonal cell population
have the same X-chromosome inactivated, whereas polyclonal tissues are
mosaics for X-linked loci. We have used the trimeric CAG polymorphism in the 5 -region of the human androgen-receptor (HUMARA)
gene31 to determine the clonal nature of
BCR/ABL and BCR/ABL+ cell fractions from
CML patients with chronic phase and accelerated phase (AP) disease.
Because the HUMARA gene has a high heterozygosity frequency,
stable methylation patterns, and polymerase chain reaction (PCR)
accessibility, this assay is applicable to low cell numbers such as
sorted progenitor fractions and individual cell colonies.32
We show that BCR/ABL subpopulations in steady-state
marrow or in mobilized peripheral blood (PB) collections are
polyclonal. In contrast, marrow or blood populations that express the
BCR/ABL mRNA are almost always clonal. We conclude from these studies that Ph, BCR/ABL progenitors
present in CML blood or marrow are polyclonal and, therefore, more than
likely benign.
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MATERIALS AND METHODS |
Sample Characteristics
Bone marrow (BM), steady-state PB, and mobilized PB (PBPC) specimens
were sampled from female CML patients. Diagnosis of CML and
determination of disease stages was performed according to standard
criteria.12 Patients with early chronic phase (ECP) disease
had CML diagnosed less than 1 year before analysis and no signs of AP
disease. AP was diagnosed when 2 or more of the following
characteristics were present: systemic symptoms previously well
controlled with single-agent chemotherapy, splenomegaly, leukocytosis,
thrombocytopenia and/or anemia, greater than 10% basophils in
the PB, cytogenetic evolution, and/or severe reticulin fibrosis
of the marrow. Patients' characteristics are listed in Table 1. Therapy before enrollment in this
study was IFN- alone (n = 4), hydroxyurea alone (n = 4), or a
combination of both (n = 3). Unique patient numbers (UPNs) 6, 7, 8, and
11 had received IFN- for at least 12 months before enrollment was
done, whereas UPNs 3 and 9 had been treated with IFN- for 3 months
and UPN 2 for 6 months. Mobilization was done using the MAC-G protocol: mitoxantrone (8 mg/m2 on days 1 and 2),
cytosine-arabinoside (1,000 mg/m2 twice daily on days 1 and
2), and cyclophosphamide (4 g/m2 on day 1). Granulocyte
colony-stimulating factor (G-CSF) was started on day +5 at 5 µg/kg/d
until the last day of PBPC collection. Alternatively, PBPCs were
mobilized by administration of 1 dose of cyclophosphamide (4 g/m2) followed by G-CSF (5 µg/kg/d) on day +5 after
chemotherapy. PBPC harvesting was started when the absolute neutrophil
counts reached 0.8 to 1.0 × 109/L.
In addition, between 50 and 100 mL of BM was collected from 11 healthy
female volunteer donors (mean age, 23 years; range, 22 to 25)
heterozygous for the HUMARA polymorphism. All samples were obtained
with informed consent according to guidelines of the Human Subjects
Committee at the University of Minnesota and Leuven, Belgium. Samples
were collected in preservative-free heparin for further processing.
Cell Purification
BM and PB mononuclear cells (MNC) were recovered after centrifugation
for 30 minutes at 1,650 rpm over a Ficoll gradient (1.077 g/mL; Nycomed Pharma AS, Oslo, Norway). Cells were washed twice in
phosphate-buffered saline (PBS) supplemented with 0.3% bovine serum
albumin (BSA; Sigma Chemical Co, St Louis, MO) and kept on ice for
subsequent CD34 selection. Enrichment of CD34+ progenitors
from MNC was performed using either CeprateLC columns
(CellPro Inc, Bothell, WA) or the MiniMACS CD34 isolation kit (Miltenyi
Biotec, Bergisch Gladbach, Germany) according to the manufacturer's
instructions. CD34+ fractions were then labeled with
fluorescein isothiocyanate (FITC)-conjugated anti-CD34 antibody
(HPCA-2) and phycoerythrin (PE)-conjugated anti-HLA-DR (Becton
Dickinson Immunocytometry Systems, San Jose, CA). After labeling, cells
were washed, resuspended in Iscove's medium supplemented with 20%
fetal bovine serum (Hyclone, Logan, UT), and sorted on a FACStar-Plus
flow cytometer supplemented with Lysis II software (Becton Dickinson).
Sort gates were drawn around cell populations with intermediate forward
scatter (FSC), low to intermediate side scatter (SSC), CD34-positivity
and high (CD34+HLA-DR+) or absent
(CD34+HLA-DR) levels of HLA-DR
coexpression. Isotype-matched conjugated mouse IgG for both CD34 and
HLA-DR (Becton Dickinson) were used as controls. Sorted populations
were at least 97% pure after reanalysis. A median number of
106 MNC, 104
CD34+HLA-DR+, and 4 × 103
CD34+HLA-DR cells were removed for
clonality analysis from each patient sample and kept frozen in liquid
nitrogen in the presence of 20% fetal calf serum (FCS) and 10%
dimethylsulfoxide (DMSO; Merck, Darmstadt, Germany). At time of
analysis, samples were rapidly thawed in a 37°C waterbath, washed
once with ice-cold PBS, and equally split for DNA and RNA analysis. For
controls, 2 × 104 MNC,
CD34+HLA-DR+, and
CD34+HLA-DR cells were isolated and
freshly processed.
Reverse Transcriptase (RT)-PCR for BCR/ABL and
 -Actin
RNA was extracted using RNeasy spin columns (Qiagen, Chatsworth,
CA) and diluted in 30 µL of RNAse-free H2O. cDNA was
subsequently synthesized from 12 µL of this RNA in a 30-µL reaction
mixture containing 400 U of Moloney murine leukemia virus
(M-MLV) reverse transcriptase, 0.1 mmol/L dithiothreitol
(DTT), 6 µL of 5× First Strand Buffer (all from GIBCO BRL-Life
Technologies, Gaithersburg, MD), 40 U of RNAsin (Promega, Madison, WI),
2 mmol/L of dNTPs (Pharmacia Biotech, Uppsala, Sweden), and 20 pmol/L
of primer A (5-GGAGCTGCAGATGCTGACCAAC-3) for BCR-ABL or
primer BA (5-TACCTCATGAAGATCCTCA-3) for -actin. After
incubation for 60 minutes at 42°C, the RT was inactivated by
heating for 10 minutes at 80°C. Nested PCR for BCR/ABL was
performed as previously described.11 For -actin, a
single-step PCR was performed: 7.5 µL of cDNA was added to 42.5 µL
of PCR mix containing 5 µL of 10× PCR buffer, 1.5 mmol/L
MgCl2, 2.5 U Taq Polymerase (all from Promega), 200 µmol/L dNTPs, and 20 pmol/L of primer BA and BB
(5-TTCGTGGATGCCACAGGAC-3). Denaturing was performed for
45 minutes at 95°C, annealing for 30 minutes at
55°C, and extension for 45 minutes at 72°C for 44 cycles in a
Perkin Elmer Thermal Cycler (Perkin Elmer Cetus Corp, Emeryville, CA).
Several precautions were taken to avoid false-positive results. Each
RT-PCR reaction included a positive (K562 cell line) and a negative
(Raji cell line) control for BCR/ABL expression as well as another
negative control containing all components of the reaction, except for
the target nucleic acids; finally, all PCR reactions were performed in
duplicate. BCR/ABL and -actin PCR products were loaded on a 3%
Nusieve agarose gel (Life Sciences International Ltd, Cheshire, UK) and
electrophoresis was performed for 2 hours at 150 V. After
ethidium-bromide staining, gels were photographed and subsequently
immersed at room temperature in denaturing buffer (3 mol/L
NaCl, 0.4 mol/L NaOH) on an orbital shaker for 60 minutes. DNA was
blotted onto nylon membranes by downward alkaline transfer for 2 hours
using a Turboblotter (Schleicher & Schuell, Keene, NH). After transfer,
membranes were washed, UV-crosslinked, and prehybridized for 30 minutes
in prewarmed Rapid-hyb buffer (Amersham Life Science, Arlington
Heights, IL). Before hybridization, 10 pmol/L probe B3A2
(5-GCTGAAGGGCTTTTGAACTCTGCTTA-3) , B2A2
(5-GCTGAAGGGCTTCTTCCTTATTGATG-3) for detection of
BCR/ABL, and BAP (5-CCATCTCTTGCTCGAAGTC-3) for detection
of -actin was 5-labeled with 50 µCi of 32P
 ATP (4,500 Ci/mmol) (ICN Pharmaceuticals Inc, Irvine, CA) for 40 minutes at 37°C using 10 U of Polynucleotide kinase (Boehringer Mannheim, Indianapolis, IN). Hybridization was performed for 60 minutes
at 42°C. After hybridization, blots were washed for 20 minutes in
5× SSC, 0.1% (wt/vol) sodium dodecyl sulfate (SDS) at room
temperature and 2 times 15 minutes in 1.0 to
0.1× SSC, 0.1% (wt/vol) SDS at 42°C. Blots were dried,
stored overnight on phosphorscreens, and analyzed on a Phosporimager
(Molecular Dynamics, Sunnyvale, CA).
Clonality Analysis With the HUMARA Assay
Restriction enzyme digest and PCR were performed as previously
described.32 Briefly, DNA was extracted from individual
cell fractions and resuspended in 40 µL of H2O. One half
of this sample was incubated overnight at 37°C with 40 U of
HpaII and 10 U of Cfo I (Hha I) (Boehringer
Mannheim) for the digestion of unmethylated (or active) alleles. The
other half of the DNA was treated similarly but without restriction
enzymes. After restriction enzyme digest, residual DNA was amplified in
a nested PCR procedure with one of the inner primers 5 labeled
with 32P and separated on a 4% denaturing 19:1
acrylamide/bisacrylamide gel for 4 hours at 60 W. Gels were dried and
scored on a phosphorimager (Molecular Dynamics) after overnight
exposition to phosphorscreens. For each sample, a corrected ratio (CrR)
was calculated by dividing the ratio of the predigested sample
(upper/lower allele) by the ratio of the non-predigested sample using
ImageQuant software (Molecular Dynamics). This CrR compensates for
preferential amplification of the shorter allele when the number of PCR
cycles increases.32 A clonal population is defined as a
cell population with greater than 75% expression of one of both
X-linked alleles. This corresponds to CrR values of
<0.33 or >3.33,34
| |
RESULTS |
Steady-State BM
CML patients.
We studied steady-state marrow samples obtained from 10 female patients
with CML in whom a polymorphism for the HUMARA locus could be detected
(Table 2). Three patients had ECP CML, 4 patients late CP CML, and 3 patients AP disease.
Mononuclear cells and fluorescence-activated cell sorting
(FACS)-selected CD34+HLA-DR+ and
CD34+HLA-DR cells were examined by
RT-PCR for BCR/ABL mRNA expression and by the HUMARA assay for
clonality analysis. BCR/ABL RT-PCR of the MNC fraction was positive in
all 10 patients in two independent determinations. In 7 of 10 patients,
BCR/ABL expression was associated with skewing of the HUMARA assay
toward the upper (UPNs 5, 7, 8, and 9) or lower (UPNs 1, 2, and 10)
allele, showing the predominance of one single clone in these cell
populations. In two patients, UPN 6 and UPN 11, the HUMARA assay on MNC
showed a more polyclonal pattern. CD34+HLA-DR+
cells from 9 of 10 patients showed presence of the BCR/ABL mRNA, whereas CD34+HLA-DR+ cells from UPN 3 were
BCR/ABL mRNA negative in two independent determinations despite the
presence of -actin in the RT-PCR reactions. Seven of these 9 BCR/ABL
mRNA+ fractions were examined with the HUMARA
assay to determine their clonal origin. In 4 patients, 2 in late
chronic phase (LCP) and 2 in AP disease,
CD34+HLA-DR+ cells were clonal (CrR of 6.28 [UPN 7], 24.20 [UPN 8], 29.74 [UPN 9], and 0.01 [UPN 10]). In
the other three patients (1 ECP CML [UPN 1], 1 LCP CML
[UPN 6], and 1 AP CML patient treated to a partial
Ph state with IFN- but with cytogenetic evolution
[UPN 11]), CD34+HLA-DR+ cells were nonclonal
(CrR of 0.41, 0.43, and 1.71, respectively) even though RT-PCR analysis
showed the presence of the BCR/ABL mRNA. In the only patient in whom
CD34+HLA-DR+ cells did not contain BCR/ABL
mRNA, the HUMARA assay confirmed polyclonality (CrR = 1.47). We also
studied FACS-selected CD34+HLA-DR cells.
Consistent with previous studies from our group,
CD34+HLA-DR cells from 2 of 3 ECP CML
patients were BCR/ABL mRNA (UPN 1 and UPN 3). These
cells were polyclonal by HUMARA (CrR = 0.63 and 1.34, respectively)
(Fig 1A). In contrast,
CD34+HLA-DR cells from the other ECP CML
patient, 2 LCP CML, and 1 AP CML patient were BCR/ABL
mRNA+. In these patients,
CD34+HLA-DR cells were clonal (CrR of
0.13 [UPN 2], 3.35 [UPN 5], 16.92 [UPN 8], 34.24 [UPN 9])
(Fig 2).
CD34+HLA-DR cells from the AP patient
treated to partial cytogenetic remission with IFN- but with
cytogenetic evolution (UPN 11) were BCR/ABL mRNA and
nonclonal (CrR = 1.41).
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| Fig 1.
(A) X-inactivation patterns with the HUMARA assay and
BCR/ABL mRNA expression in MNC,
CD34+HLA-DR+,
CD34+HLA-DR, and DR derived
LTC-IC in steady-state marrow in ECP CML. HUMARA PCR and BCR/ABL and
-actin RT-PCR reactions were performed as described in Materials and
Methods. Results are shown for one experiment in marrow from UPN 1. HUMARA PCRs are shown without ( ) and with (+) prior exposure to
HpaII and Cfo I restriction endonucleases. Shadow bands
are due to slippage of Taq polymerase. BCR/ABL yielded positive signals
in the MNC and DR+ fraction and in one LTC-IC-derived
colony, after hybridization with a B3A2 probe. No positive signal was
detected in the other samples. The housekeeping gene -actin was used
as an internal control for the presence of mRNA. (B) HUMARA analysis
and BCR/ABL mRNA expression in mobilized PB in ECP CML. Results are
shown for day 3 harvest from UPN 1. HUMARA PCR and BCR/ABL and
-actin RT-PCR reactions were performed as described in Materials and
Methods. HUMARA was performed without () and with (+) predigestion
with the methylation-sensitive endonucleases HpaII and
Cfo I. MNC displayed monoclonal patterns and BCR/ABL mRNA
positivity, whereas CD34+HLA-DR+ and
CD34+HLA-DR progenitors were polyclonal
and BCR/ABL mRNA. The erythroleukemic cell line K562
served as a positive control for BCR/ABL (B3A2) expression. The
housekeeping gene -actin was used as a control for the presence of
mRNA.
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| Fig 2.
X-inactivation patterns with the HUMARA assay and BCR/ABL
mRNA expression in MNC, CD34+HLA-DR+, and
CD34+HLA-DR progenitors in steady-state
marrow in AP CML. Results are shown for marrow from UPN 9. HUMARA PCR
and BCR/ABL and -actin RT-PCR reactions were performed as described
in Materials and Methods. HUMARA was performed without () and with
(+) predigestion with the methylation-sensitive endonucleases
HpaII and Cfo I. All samples show monoclonality.
BCR/ABL results are shown after hybridization with the B3A2 probe. The
housekeeping gene -actin was used as a control for the presence of
mRNA.
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Control population.
CrR values of MNC, CD34+HLA-DR, and
CD34+HLA-DR+ fractions were found to be
polyclonal in 9 of the 11 female marrow donors
(Table 3). CrR values ranged from 0.43 to
2.28. Only 2 of 11 controls (8 and 10) had monoclonal
allelic ratios and were therefore considered as constitutionally
skewed. XCI patterns, expressed as allelic ratios, were highly
correlated between MNC and CD34+HLA-DR+
(r = .96) and CD34+HLA-DR
(r = .97) fractions.
Mobilized PB Progenitors
MNC, CD34+HLA-DR+, and
CD34+HLA-DR cells selected from one or
more of the PB progenitor collections obtained in 7 patients (3 ECP
CML, 3 LCP CML, 1 AP CML) after in vivo mobilization with intensive
chemotherapy and growth factor support, were analyzed by RT-PCR for
BCR/ABL expression and by HUMARA for clonality
(Table 4). Cytogenetics on PBPC collections
showed that a major cytogenetic response was obtained
with the in vivo mobilization regimen in 2 ECP and 1 LCP CML patients
whereas between 80% and 100% of metaphases in 1 ECP, 1 LCP, and the
AP CML patient were still Ph+. RT-PCR analysis of the MNC
fraction of all patients showed presence of the BCR/ABL mRNA. MNC were
clonal in UPNs 1, 5, 6, 7, and 9, but polyclonal in UPN 2 and UPN 4 on
day 1 of PBPC collections. Except for patient 2, showing a shift from
monoclonality to polyclonality in the MNC fraction, these findings were
similar to the observations in steady-state BM.
CD34+HLA-DR+ and
CD34+HLA-DR cells selected from 2 ECP
CML patients (UPNs 1 and 2) did not express detectable amounts of
BCR/ABL mRNA in two independent PCR reactions. This was consistent with
the nonclonal character of these cells as determined by HUMARA (Fig
1B). Patient 4, with a complex Ph translocation involving chromosomes
9, 17, and 22 still had, despite BCR/ABL positivity, residual nonclonal
CD34+HLA-DR+ and
CD34+HLA-DR progenitors at day 1 of the
PBPC harvest. However, by day 3 both the
CD34+HLA-DR and the
CD34+HLA-DR+ compartments had shifted toward
full monoclonality (Fig 3).
CD34+HLA-DR+ and
CD34+HLA-DR cells from mobilized PBPC of
all LCP and the AP CML patient were BCR/ABL mRNA+.
CD34+HLA-DR+ cells were clonal in all 4 patients whereas CD34+HLA-DR cells from
UPNs 5 and 7 (LCP CML) and UPN 9 (AP CML), but not UPN 6 (LCP CML),
were clonal in two independent HUMARA assays (Fig 4). For all patients except UPN 4, we
did not find significant differences in BCR/ABL mRNA expression and
HUMARA ratios between PBPC collections from different days.
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| Fig 3.
Clonal evolution in PBPC collections from a patient with
early chronic phase CML (UPN 4). Results are shown for day 1 and day 3 PBPC harvests. HUMARA, BCR/ABL, and -actin PCRs were performed as
described in Materials and Methods. All cell fractions switch from
polyclonality on day 1 to monoclonality on day 3. (), No prior
digest with HpaII and Cfo I; (+), prior digest with
HpaII and Cfo I.
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| Fig 4.
HUMARA analysis and BCR/ABL mRNA expression in mobilized
PB in LCP CML. Results are shown for day 1 harvest from UPN 5. HUMARA
PCR and BCR/ABL and -actin RT-PCR reactions were performed as
described in Materials and Methods. MNC,
CD34+HLA-DR+, and
CD34+HLA-DR progenitors were monoclonal
and BCR/ABL mRNA+. Shadow bands in the HUMARA PCR are
caused by slippage of Taq polymerase. (), No prior digest with
HpaII and Cfo I; (+), prior digest with HpaII
and Cfo I.
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Clonal or Nonclonal Nature of DR+ and
DR Long-Term Culture-Initiating Cells
(LTC-IC)
To further ascertain that CD34+HLA-DR
cells are polyclonal in patients in whom RT-PCR analysis does not
reveal BCR/ABL mRNA, we examined single LTC-IC-derived colony-forming
cells (CFC) from UPN 1 by RT-PCR and HUMARA.
CD34+HLA-DR cells selected from
steady-state marrow were cultured in limiting dilutions onto M2-10B4
feeders (kindly provided by Dr C. Eaves, Vancouver, Canada) for 5 weeks. Stromal feeders and hematopoietic progeny were then overlaid
with cytokine-containing methylcellulose medium and CFC from positive
wells at the lower cell doses collected 14 days later.35
Four of five DR LTC-IC-derived CFC were BCR/ABL
mRNA and the different colonies had either the upper
or lower allele active, further confirming the polyclonal origin of
these BCR/ABL mRNA,
CD34+HLA-DR cells and LTC-IC (Fig
1A).
| |
DISCUSSION |
In this article we report that CD34+ cells in marrow and
blood from patients with CML, which are BCR/ABL mRNA
and Ph, are polyclonal and therefore presumably
benign. X-chromosome inactivation analysis using the HUMARA assay shows
that highly purified, FACS-selected
CD34+HLA-DR+ or
CD34+HLA-DR cells and LTC-IC within
these populations that do not express BCR/ABL mRNA are nonclonal. In
contrast, BCR/ABL mRNA containing CD34+ subpopulations are
clonal in the majority of samples analyzed.
Previous studies using G6PD polymorphisms22-24 have
suggested that immature progenitors that are clonal but do not have the Ph chromosome may be found in marrow or blood of some patients with
CML. This would suggest that acquisition of the BCR/ABL gene rearrangement is a secondary genetic abnormality and results from subclonal expansion in an already clonal, but Ph,
hematopoietic stem or progenitor cell. However, clinical studies using
DNA-based X-chromosome inactivation assays have indicated that CML
patients treated to cytogenetic remission with IFN- usually have
restoration of polyclonal hematopoiesis.36,37 Likewise,
patients that have regenerated Ph hematopoiesis
after transplantation with peripheral blood progenitors obtained after
in vivo therapy with polychemotherapy and G-CSF have polyclonal mature
cells and progenitors in blood and marrow.38 These and
other studies39 indicate that polyclonal
Ph progenitors coexist with the
BCR/ABL+, Ph+ clone. Moreover, a number of
animal studies suggest that the BCR/ABL gene rearrangement is not only
necessary but also sufficient for the development of the clinical CML
syndrome.2 Although this does not preclude the possibility
that acquisition of the BCR/ABL gene rearrangement is a secondary
genetic event leading to CML, the animal models as well as the clinical
experience in humans make the hypothesis that BCR/ABL is a secondary
hit leading to CML less likely.
Because the only curative therapy for CML, allogeneic transplantation,
is available for less than 50% of patients afflicted with this
disease,12 increasing numbers of patients failing IFN-
treatment are being considered for autologous transplantation with
blood- or marrow-derived progenitors. We have identified the
CD34+HLA-DR cell population, selected
from steady-state marrow or mobilized PB of a fraction of patients with
early chronic phase CML, to be BCR/ABL
mRNA.19 Determination of the monoclonal
or polyclonal nature of CD34+ progenitors which are
BCR/ABL and Ph is important.
Monoclonal patterns would confirm that CD34+ progenitors in
CML originate from a clonally mutated preleukemic cell which does not
yet express BCR/ABL. In contrast, polyclonal patterns, as can be found
in normal hematopoiesis, would suggest that the
BCR/ABL compartment consists of normal progenitors.
We used RT-PCR followed by Southern blot analysis to detect the BCR/ABL
gene rearrangement and HUMARA to evaluate X-chromosome inactivation
patterns. Products of the BCR/ABL RT-PCR reactions were considered
positive if at least one of two independent RT-PCR reactions yielded a
BCR/ABL cDNA and the -actin cDNA was visualized. Only those RT-PCR
reactions in which two independent experiments did not yield BCR/ABL
cDNA, despite visualization of -actin cDNA, were considered
negative. The sensitivity of this RT-PCR reaction is 1/105
cells. Duplicate RT-PCR reactions were performed to decrease the
likelihood of obtaining false-negative results when low copy numbers of
BCR/ABL mRNA are present in immature CD34+ cell
populations.40 The sensitivity of the HUMARA assay,
assessed by mixing MNC from two healthy male volunteers with different numbers of CAG repeats, has been previously reported by our
group.32 Use of phosphorimaging can increase the
sensitivity of detection to values between 90% and 95%. This enables
detection of a cell clone contributing only 5% to 10% to the cell
mixture. CrR values were calculated to semiquantify the X-inactivation
patterns. To ascertain accuracy, all HUMARA assays were performed in
duplicate. This yielded a high reproducibility (r2 > .90) between two PCR reactions independently performed on one sample.
Using both BCR/ABL RT-PCR and the HUMARA assay on the same cell
fraction, we found that CML progenitors derived from steady-state marrow or mobilized PB with no detectable BCR/ABL mRNA transcripts were
polyclonal, because their CrR values were within the range found in
normal control subjects. These polyclonal BCR/ABL
mRNA progenitors are particularly enriched in the
CD34+HLA-DR fractions. Further, LTC-IC
present in this CD34+HLA-DR population
of one ECP CML patient were also polyclonal. Because "steady-state" patients with polyclonal progenitors were already being treated with hydroxyurea and/or IFN-, the precise role of these treatment regimens on clonality of CML progenitors could not
be evaluated. However, previous reports have shown restoration of
polyclonality in CML by IFN-.36 Nevertheless, our
results confirm previous studies from our group and other
investigators19,20 that
CD34+HLA-DR cells are enriched for
BCR/ABL primitive progenitors in some patients with
ECP CML. In addition, our HUMARA studies support the concept that
BCR/ABL progenitors in CML are "benign" and
that the Ph translocation is likely the initial genetic abnormality
occuring in a very primitive progenitor. If a clonal event would
precede the acquisition of the Ph translocation, then BCR/ABL
mRNA CD34+HLA-DR
cells and their LTC-IC would be derived from a single clone, a
hypothesis that is not supported by our studies. Although our current
methodology of X-inactivation analysis does not permit detection of a
minor monoclonal Ph population that coexists with
polyclonal Ph progenitors, its contribution to the
total progenitor compartment is low and it does not seem to have any
proliferation advantage.
Despite the presence of clonal Ph+ progenitors in
steady-state marrow, treatment with intensive chemotherapy can restore
a Ph, BCR/ABL mRNA and polyclonal
progenitor compartment as has been shown in UPNs 1 and 2. However, this
residual pool of Ph, polyclonal progenitors
decreases over time and with acceleration of the disease, as a
polyclonal population could no longer be mobilized in patients with LCP
and AP disease. Moreover, as can be learned from the clonality studies
on the PBPC collections from UPN 4, these polyclonal progenitors are
preferentially mobilized during early recovery after chemotherapy. This
observation clearly shows the coexistence of a polyclonal progenitor
compartment together with clonal BCR/ABL mRNA+ precursors,
where the clonal Ph+ population rapidly suppresses the
residual polyclonal hematopoiesis. Although transplantation with
nonclonal progenitor cells may be superior to transplantation with
clonally mutated preleukemic progenitors, future studies will be needed
to demonstrate a survival benefit.
The observation that some of the cell fractions have a polyclonal
nature despite the presence of detectable amounts of BCR/ABL mRNA
transcripts is not a contradiction, but can be explained by taking the
sensitivity of both the BCR/ABL RT-PCR and HUMARA PCR into account.
Because BCR/ABL RT-PCR reactions are up to 5,000-fold more sensitive as
the HUMARA assay, the apparent discrepancy between the HUMARA and
RT-PCR assays indicates that the degree of contamination of these
CD34+HLA-DR+ cell populations with
BCR/ABL+ cells is relatively low. This implies that
polyclonal BCR/ABL mRNA+ progenitor cell fractions are
composed of a mixture of polyclonal BCR/ABL mRNA
precursors and a smaller fraction of clonal BCR/ABL mRNA+
leukemic precursors. Additionally, nonclonal allelic ratios of the MNC
might be attributable to the presence of a sizable population of
nonclonal, nonmyeloid cells, such as B cells and T cells, which are
usually not part of the Ph+ clone in chronic phase
CML.7-10
Are our results in contradiction with previously reported studies in
which the clonal origin of Ph cells in CML was
examined with G6PD-based clonality assays? As for the DNA-based HUMARA
assay, the isoenzyme G6PD-based clonality studies have reported a
sensitivity of 95%. This makes the nondetection of a residual
nonclonal population in the G6PD-based clonality studies unlikely. The
major difference between the studies reported by Fialkow et
al22,24 and our studies are the cell
populations analyzed. Fialkow et al have examined the clonal origin of
Ph Epstein-Barr virus (EBV)-derived lymphoblastoid
cell lines obtained from blood of patients with Ph+ chronic
phase CML. In chronic phase CML, both T and B lymphocytes, which are
long-lived, are usually Ph.7-10
Therefore, it seems reasonable to examine the clonal origin of these
lymphoid cells to determine if a clonal, but still
Ph, population exists in CML. Although EBV is a
polyclonal stimulator of B lymphocytes, there are no data available on
the comparison of clonal patterns of freshly sorted B lymphocytes and
their progeny after EBV-transformation. Additionally, T lymphocytes are
also reported to be Ph. But in contrast to B
lymphocytes, they seem to display more often polyclonal X-inactivation
patterns.9 Finally, Fialkow et al have tried to correct for
excessive constitutional skewing or "extreme Lyonization" in the
clonal Ph fractions by comparing the clonal patterns
of B lymphocytes with these of fibroblasts and skin. However, it has
been recently shown that hematopoietic tissue is more prone to acquired
skewing compared with other somatic tissues.33,34 We have
studied BCR/ABL expression and X-inactivation patterns on highly
purified immature CD34+HLA-DR
progenitors and their more committed
CD34+HLA-DR+ counterparts. We did not have
access to nonhematopoietic tissues to exclude excessive skewing.
Therefore, we cannot discriminate between monoclonality and
constitutional or acquired skewing in patients with more extreme
allelic ratios (CrR <0.3 or >3). However, because
Ph BCR/ABL progenitors were
polyclonal, skewing does not influence the interpretation of these data
nor does it bias our conclusions. We show here that BCR/ABLCD34+HLA-DR
cells are polyclonal even when recovered from steady-state marrow of
patients whose MNC are 100% Ph+, BCR/ABL
mRNA+, and monoclonal. In addition, in those patients in
whom CD34+HLA-DR or
CD34+HLA-DR+ cells were monoclonal in
steady-state marrow, polyclonal cell populations can be recovered at
the time of cytogenetic remissions after in vivo chemotherapy
mobilization. Finally, the rapid suppression of polyclonal progenitors
by clonal precursor cells during PBPC harvesting in one patient
confirms the coexistence of clonal and polyclonal progenitors. These
data allow the conclusion that a polyclonal Ph,
BCR/ABL mRNA population exists in the marrow and
blood of some patients with CML. Therefore, such
CD34+HLA-DR cells should be suitable for
autografting in CML.
| |
ACKNOWLEDGMENT |
The authors thank Scott Wissink, Brad Anderson, and Victor Van Duppen
for excellent technical help.
| |
FOOTNOTES |
Submitted April 24, 1998;
accepted August 28, 1998.
M.D. is a Research Fellow of the Flander's Fund for Scientific
Research (FWO). C.M.V. is a Scholar of the Leukemia Society of America.
Supported in part by National Institutes of Health Grants No.
PO1-CA-65493 and PO1-CA-21737, Leukemia Society of America
Translational Research Grant No. 6377-96, and the University of
Minnesota Hospitals and Clinics.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Michel Delforge, MD, Department of
Hematology, University Hospital Gasthuisberg, Herestraat 49, B-3000
Leuven, Belgium; e-mail: Michel.Delforge{at}uz.kuleuven.ac.be.
| |
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Abstract
Introduction