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Blood, Vol. 91 No. 1 (January 1), 1998:
pp. 331-339
Lymphoma Cell Burden in Progenitor Cell Grafts Measured by
Competitive Polymerase Chain Reaction: Less Than One Log Difference
Between Bone Marrow and Peripheral Blood Sources
By
Brigitte M. Léonard,
Francis Hétu,
Lambert Busque,
Martin Gyger,
Robert Bélanger,
Claude Perreault, and
Denis-Claude Roy
From the Division of Hematology-Immunology, Maisonneuve-Rosemont
Hospital; and the Department of Medicine, Université de
Montréal, Québec, Canada.
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ABSTRACT |
A controversy persists in autologous transplantation as to which
source of progenitor cells, bone marrow (BM) or peripheral blood (PB),
contains the lowest number of contaminating lymphoma cells, and how
mobilization procedures affect these numbers. To accurately measure the
number of non-Hodgkin's lymphoma (NHL) cells harboring the
bcl-2/immunoglobulin H (IgH) rearrangement in progenitor cell grafts,
we developed a nested quantitative competitive polymerase chain
reaction assay (QC-PCR). DNA from lymph nodes of four patients with NHL
were cloned into the pSK(+) vectors to generate four internal
controls (ICs) (two with major breakpoint region [MBR]
and two with minor cluster region [mcr] rearrangements).
The kinetics of amplification of ICs paralleled those of bcl-2/IgH
rearranged genomic DNA. When used in a QC-PCR assay, these ICs were
accurate at a 0.2-log level and provided reproducible results, as shown
by low intrarun and interrun variability. An excellent correlation
between predicted and observed lymphoma cell content (r =
.99) was observed over a range of at least 5 logs of rearranged cells.
This approach was used to measure involvement by NHL cells at the time
of progenitor cell harvest in 37 autologous transplant patients. The
number of bcl-2/IgH rearranged cells in BM, PB, and mobilized PB (mPB)
was found to vary from 1 to 1.1 × 105 per million cells.
The number of lymphoma cells present in BM was significantly higher
than in PB (P = .0001), with a median difference in lymphoma
cell content between BM and PB of 0.48 log of cells (range,  0.7 to 5
logs). In contrast, we found no difference in the concentration of
bcl-2/IgH rearranged cells present in BM versus PB progenitor cells
mobilized with cyclophosphamide and granulocyte colony-stimulating
factor (G-CSF) (mPB) (P = .57). In conclusion, the QC-PCR
assay described in this study could measure accurately and reproducibly
the number of bcl-2/IgH rearranged cells among normal cells.
Differences in levels of contamination by lymphoma cells between BM and
PB were of less than one log (10-fold), and no differences in lymphoma
cell concentrations were observed between BM and mobilized PB. As more
cells are usually infused with mPB than with BM grafts, mPB progenitor
cell grafts may actually be associated with higher levels of
contamination by lymphoma cells. Furthermore, this QC-PCR assay should
provide an important tool to assess the prognostic impact of lymphoma
cell burden both in progenitor cell grafts and in vivo.
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INTRODUCTION |
THE USE OF peripheral blood (PB) as a
source of progenitor cells has been a major contributor to the rapid
expansion of high-dose therapy with autologous progenitor cell
transplantation (PCT) because it facilitates progenitor cell
collection, decreases the time to neutrophil and platelet engraftment,
and is associated with decreased procedure-related
toxicity.1,2 However, a major concern with autologous PB or
bone marrow (BM) transplantation is the risk associated with the
reinfusion of occult malignant cells present in the progenitor cell
graft.3-5 Indeed, previous studies have shown that
non-Hodgkin's lymphoma (NHL) cells are detectable in the BM of
patients with high-risk disease, even after intensive
therapy.6-8 In addition, in patients with leukemia,
gene-marking studies showed that the progenitor cell graft could
contain cells involved in disease relapse.9,10 Moreover,
purging of NHL cells below levels detectable by the polymerase chain
reaction (PCR) resulted in improved disease-free survival, particularly
in patients with low-grade lymphoma.11,12 Interestingly, a
few studies suggested that PB is less frequently infiltrated by
lymphoma cells than BM, and may thus be a better source of progenitor
cells than BM.13,14 In contrast, other groups showed that
bcl-2/immunoglobulin H (IgH) rearranged cells could be found with
similar frequencies in PB and BM.15-17 In patients with
various solid tumors, it has been shown that mobilized PB (mPB) can
contain neoplastic cells,3,4,18,19 and that mobilization
regimens could enhance the release of tumor cells into the
PB.19
In most previous studies, PCR has been the primary tool used to detect
lymphoma cells by targeting the 14;18 translocation, which is present
in the majority of follicular lymphomas and approximately a third of
NHL with diffuse histologies.20,21 PCR approaches are
extremely sensitive and enable the detection of as few as one lymphoma
cell in a million normal cells.11,15,22 However, these
assays are usually qualitative, and the number of rearranged cells in a
PCR positive sample could vary by more than five logs of lymphoma
cells. Such large differences in the number of PCR-positive cells limit
the interpretation of results and their prognostic and therapeutic
implications.
In this study, we developed a QC-PCR methodology to accurately measure
small differences in the number of bcl-2/IgH rearranged cells
in clinical samples from patients with NHL. Four internal controls
(ICs) generating PCR amplification products of different lengths for
both major breakpoint region (MBR) and minor cluster region (mcr)
translocations were isolated and cloned. The kinetics of amplification,
as well as reproducibility and sensitivity of the test were evaluated
to optimize the assay, and to determine the reliability, and
particularly the precision of this approach. This QC-PCR methodology
was then used to measure the level of contamination by NHL cells in BM,
PB, and mobilized PB collections from patients undergoing autologous
transplantation to determine the optimal source of progenitor cells in
terms of tumor cell contamination.
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MATERIALS AND METHODS |
Patient population.
Clinical protocols were approved by the Human Subjects Protection
Committee of the Maisonneuve-Rosemont Hospital and samples were
obtained with the informed consent of the patients. Eighty-nine
patients with B-cell NHL undergoing autologous BM (n = 50) or PB (n =
39) progenitor cell transplantation (PCT) were evaluated for the
presence of a t(14;18). Overall, 38 patients were retained for the
analysis because they satisfied the following two conditions: (1) the
presence of a bcl-2/IgH rearrangement in BM, PB, or mobilized PB (mPB)
at the time of progenitor cell harvest, and (2) both BM and
corresponding PB samples (28 patients), or BM and mPB samples (17
patients), or samples from all three sources (7 patients) were
available for analysis. All patients had relapsed or failed to achieve
a first complete remission after eight cycles of "curative
intent" chemotherapy and reached either a minimal residual disease
(MRD) status or a partial response (PR). All patients had 5% or less
of the intratrabecular space infiltrated by lymphoma cells on
pathologic examination. Steady-state PB and BM samples were obtained at
the time of marrow harvest or immediately before PB progenitor cell
mobilization. Cyclophosphamide (1.5 g/m2) was administered
on the first day of mobilization, followed by granulocyte
colony-stimulating factor (G-CSF) (10 µg/kg/d) from days 1 to 12, and
leukapheresis was performed on 2 consecutive days when the white blood
cell count was above 8 × 109/L (days 10 to
12).23
Clinical samples.
Lymph node biopsies were obtained from four patients with NHL
presenting a t(14;18). The tissue was stored at 80°C before
DNA extraction. Samples of PB and BM from patients undergoing PCT and
healthy volunteer donors, used as controls, were collected with
preservative-free heparin, and mononuclear cells (MNC) isolated by
ficoll-hypaque density gradient centrifugation (Ficoll-Paque;
Pharmacia, Piscataway, NJ). All cell samples were cryopreserved in 10%
dimethyl sulfoxide (DMSO) using standard techniques and stored in the
vapor phase of liquid nitrogen.24
Lymphoma cell lines.
The cell lines RL and H2 were derived from patients with NHL and
harbored a bcl-2 translocation involving MBR, while DHL-16 cells had an
mcr translocation. RL cells were a kind gift from Dr W. Urba (National
Cancer Institute, Frederick, MD) and H2 and DHL-16 cells were kind
gifts from Dr J.G. Gribben (Dana-Farber Cancer Institute, Boston, MA).
DNA extraction.
DNA was extracted by conventional cell lysis and proteinase K (Sigma,
St Louis, MO) digestion in 100 mmol/L NaCl, 10 mmol/L Tris-HCl pH 7, 25
mmol/L EDTA, and 0.5% sodium dodecyl sulfate (SDS) at 50°C
overnight.22,25 DNA was purified by a phenol-chloroform
extraction and precipitated with 3 mol/L sodium acetate and
ethanol. The concentration and quality of DNA was measured by
spectrophotometry at 260 and 280 nm wavelength.
Construction of internal controls.
Internal controls (ICs) were generated by amplification of DNA from
lymph nodes of patients with NHL. Patient DNA was PCR amplified
with corresponding MBR or mcr outer primers,11,22 and
amplification products were cloned into pSK(+) vectors [pCR-Script
SK(+) Cloning Kits; Stratagene, La Jolla, CA]. Four different plasmids
were obtained: two with a MBR (MACM and GU1M) and two with a mcr (R11
and MD6) rearrangement. Large-scale preparation of the cloned ICs was
performed by lysing bacteria with the alkali method and purifying by
equilibrium centrifugation in cesium chloride-ethidium bromide
gradients (Boehringer-Mannheim, Mannheim, Germany).25
Inserts were sequenced to confirm that inner and outer primer binding
regions were conserved (Sequenase 1.0; United States Biochemical Inc,
Cleveland, OH). The plasmids were then linearized with EcoRI
(LifeTechnologies, Inc, Gaithersburg, MD). Plasmid DNA concentration
was determined spectrophotometrically and the copy number calculated. A
precise series of dilutions of the ICs, ranging from 0.1 ng to 0.05 fg,
were prepared for competition assays.
Nested competitive PCR amplification.
Conditions for the nested competitive PCR reaction at the MBR
translocation site were optimized using the ICs GUIM and MACM so that
one copy of IC in 2.5 × 105 normal MNCs was
reproducibly detectable. Similar results were subsequently obtained
with the other ICs.
For the first amplification, different amounts of IC were added to 1.5
µg of purified sample DNA and were amplified in a 100-µL reaction
volume containing 25 pmol of each oligonucleotide primer, 200 mmol/L
each of deoxyribonucleoside triphosphates (dNTPs), 2.5 U
of Taq DNA polymerase (Boehringer, Mannheim, Germany), 10 mmol/L
Tris-HCl (pH 8.3), 2.5 mmol/L MgCl2, 50 mmol/L KCl 0.1%
gelatin.11,22 Oligonucleotides for the first amplification
included MBR: 5 -CAGCCTTGAAACATTGATGG, or mcr
5-CGTGCTGGTACCACTCCTG, and JH:
5-ACCTGAGGAGACGGTGACC.11,22 DNA was subjected to 27
cycles of amplification in a Perkin Elmer Cetus 9600 thermal cycler
(Cetus, Emeryville, CA). Cycle conditions included a 94°C
denaturation step (15 seconds), a 55°C annealing step (15
seconds), and a 72°C extension step (15 seconds). The initial
denaturation step was prolonged to 2 minutes and the final extension
step to 5 minutes. On completion of the first amplification round, 10
µL of the amplified mixture was then reamplified for another 27
cycles using oligonucleotide primers internal (i) to the original
primers (nested PCR) MBR-i: 5-TATGGTGGTTTGACCTTTAG, or mcr-i:
5-GGACCTTCCTTGGTGTGTTG, and JH-i:
5-ACCAGGGTCCCTTGGCCCCA.11,22 This second PCR
amplification was performed with the same parameters as described
above, except for the addition of 3.5 U of Taq polymerase and 50 pmol
of each inner primer.
Aliquots of 20 µL from the first and the second PCR amplification
were analyzed by electrophoresis in 6% acrylamide-7 mol/l urea or 2%
agarose gels (Boehringer) containing 0.5 mg/mL of ethidium bromide in
Tris-borate buffer and visualized under UV light. The intensity of
fluorescence for each PCR product was measured with a gel documentation
system (Alpha Innotech Corp, San Leandro, CA). Estimations derived from
the QC-PCR assay were multiplied by a correction factor (size of ICs'
PCR product [bp]/size of patient's PCR product
[bp]).26,27
In all instances, at least four aliquots of each sample were evaluated
to determine PCR positivity for bcl-2/IgH rearrangement. With each
experiment, one positive control consisting of IC only and a negative
control sample consisting of water instead of DNA were always used. To
confirm that DNA was amplifiable, negative samples were amplified with
commercial primers for the  -globin gene (Gene Amplimer, Cetus). PCR
procedures included the following measures to avoid false-positive
results due to contamination.22,28 Processing of samples
was accomplished with disposable pipettes in a biological hood never
exposed to post-PCR material. Reagents and samples were mixed in
sterile tubes with dedicated pipettors and filter pipette tips
(Molecular Bio-Products Inc, San Diego, CA) and all reagents aliquoted
for single use. Post-PCR products were manipulated in a different
laboratory.
Kinetics of IC.
To determine the kinetics of amplification of the ICs, JH and JH-i
primers were labeled by direct phosphorylation kinase reaction with
( 32P) ATP using T4 polynucleotide kinase (Boehringer).
Radiolabeled primers were added to unlabeled primers in a proportion of
1 in 10 to the PCR reaction. A total of 30 µL of each PCR product was
migrated in triplicate on a 2.5% agarose gel, each band was excised
and radioactivity measured.26
Statistical analysis.
Although the distributions of log-transformed values were fairly
normal, all data were analyzed using nonparametric techniques because
many comparisons involved small subsamples. Comparisons between various
measures of infiltration in BM, PB, and mobilized PB were performed
using the Wilcoxon match-pairs rank test, with a Bonferroni-corrected
alpha level of .05. Mann-Whitney U tests were used for between-groups
comparisons. All analyses were considered significant when reaching the
.05 alpha level.
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RESULTS |
Generation of internal controls.
To precisely determine the number of target lymphoma cells, the
amplification of target DNA templates is compared with that of known
amounts of ICs in a competitive PCR assay. For optimal competitive PCR
amplification, ICs should ideally be amplified by the same set of
primers as the target template and be of similar size.26,29
To satisfy these objectives, ICs were generated from lymph nodes
heavily infiltrated with lymphoma cells harboring the bcl-2/IgH
rearrangement. Taking advantage of the inherent diversity in the
breakpoint of the t(14,18) between different patients, lymph nodes from
four patients were selected to obtain PCR amplification products of
various sizes. DNA was extracted and amplified with the outer PCR
primers. PCR products were purified, inserted into pSK(+) vectors, and
cloned. Two ICs with MBR translocations were generated: GU1M and MACM,
which on reamplification with internal primers, provided a second PCR
product of 224 bp and 174 bp, respectively (Table
1). Similarly, two ICs with mcr
rearrangements, MD6 and R11, provided second PCR products of 516 bp and
1045 bp, respectively. Sequence analysis at inner and outer primer
homologous binding regions showed that no nucleotide was lost or
modified during the cloning of ICs (data not shown).
Kinetics of amplification of the IC.
The first round of PCR amplification should remain in the exponential
phase to prevent a loss of sensitivity and specificity and to avoid the
formation of hybrid PCR products in the subsequent amplification with
internal primers.26 To define the kinetics of amplification
of the IC during the first round of PCR, 5 × 104
copies of IC (GU1M) were amplified for an increasing number of cycles
with 32P end-labeled JH primers. This high number of
rearranged cells was chosen to ensure that saturation of the
amplification curve would not occur. PCR amplification products were
electrophoresed, cut out of the gel, and levels of radioactivity were
plotted against the number of cycles of amplification (Fig
1A). During the first round of PCR
amplification, the exponential phase occurred between 20 and 30 cycles
of amplification. To determine the kinetics of amplification of the
total nested PCR reaction, 32P end-labeled inner JH primers
were added to the second PCR reaction. In these experiments, the number
of cycles of amplification was maintained identical for both rounds of
PCR amplification. At these high concentrations of DNA target template,
a plateau was observed after 20 cycles of amplification, but a loss of
efficiency occurred only after 35 cycles. Thus, we limited each round
of PCR to 27 cycles. With this approach, one bcl-2/IgH rearranged cell
among 250,000 normal cells was consistently detected and generated a
signal 100-fold above the detection threshold of the gel (data not
shown).22 Using this strategy, no bcl-2/IgH rearrangements
were identified in PBMNCs from 19 normal controls.
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| Fig 1.
Kinetics of amplification of ICs. (A) Amounts of
amplification products (cpm) of the IC MACM (5 × 104
copies per PCR reaction) were measured after the first round ( ) and
second round of nested PCR ( ), and plotted against the number of PCR
cycles. Results are means of two experiments in triplicate. (B)
Products generated after PCR amplification of 5 × 104
copies of plasmid IC (MACM) ( ) were compared with those generated
after amplification of genomic DNA (5 × 104 RL cells)
( ).
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The kinetics of amplification of our plamid IC was then compared with
that of genomic DNA (RL cells) (Fig 1B). The same number of copies (5
× 104) of IC and genomic rearranged DNA were PCR
amplified with 32P-labeled outer primers and the amount of
radioactivity was measured. The shape of the PCR amplification curve,
as well as the level of PCR products measured, were identical for the
IC and genomic DNA, confirming that the kinetics of amplification of
plasmid IC and genomic rearranged DNA were identical.
Nested competitive PCR assay.
We then evaluated the capacity of IC to measure the number of bcl-2/IgH
rearranged cells in a nested competitive PCR assay. Increasing amounts
of ICs were added to aliquots of a simulated patient sample, consisting
of DNA from 500 H2 cells added to one million normal mononuclear PB
cells. Following nested PCR and electrophoresis, the intensity of PCR
products corresponding to GU1M IC and H2 cells was measured (Fig
2). For each concentration of IC, the ratio
of H2/GU1M signals was calculated and plotted as a function of the
number of copies of IC. The point where the ratio of patient and IC
products was equal to 1 corresponded to the number of bcl-2/IgH
rearranged copies in the sample.26,29
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| Fig 2.
ICs were used in a nested competitive PCR assay to
quantitate rearranged NHL cells. (A) The following number of copies of
IC (GU1M): (1) 5,000; (2) 3,200; (3) 1,600; (4) 800; (5) 500; (6) 320;
(7) 160; (8) 50; (9) 0 were added to aliquots of H2 cells
mixed with normal PBMC at a ratio of 5 ×
103/106. Each aliquot, a positive (10: IC only)
and a negative control (11: water only) were amplified by nested PCR,
run on a 2.5% agarose gel, and stained with ethidium bromide. (B)
Amounts of PCR products were measured by multidensitometric analysis.
(C) Fluorescence ratios () were plotted against the concentration of
IC (corrected for differences in molecular weight). The estimated
number of bcl-2/IgH rearranged cells in the sample corresponded to a
ratio of 1 (correlation coefficient = .945).
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Reproducibility.
To assess the reproducibility of this test, we prepared a single sample
of 500 H2 cells (enumerated in a counting chamber) in 106
normal mononuclear PB cells. Six aliquots of the initial mixture were
amplified in the same PCR run using the QC-PCR approach described
above. The mean number of rearranged cells evaluated by QC-PCR was 867
± 90 (± standard deviation [SD]) cells. Different aliquots of
the same original cell mixture were then evaluated by QC-PCR in six
different nested PCR runs, with different master mixes, over 6
different days and using six different preparations of the IC (GU1M).
Even in these extreme conditions, the mean number of cells measured
(829) was almost identical to that obtained in intrarun conditions, and
the SD (303) was only one third of the mean number of copies. Both
agarose and polyacrylamide gels were also used to evaluate PCR
products, and similar estimations were obtained with both types of gels
(data not shown).
Coamplification of IC and NHL DNA.
We then determined the linearity of our QC-PCR assay over the whole
range of bcl-2/IgH cells that can be detected in patient samples. For
this purpose, RL and H2 cells (harboring a MBR rearrangement) and
DHL-16 cells (mcr rearrangement) were mixed with normal mononuclear PB
cells at ratios varying from 5 to 5 ×
104/106, the latter level of infiltration was
selected to correspond to infiltration by 5% lymphoma cells. These
samples were coamplified with the ICs, MACM, GU1M, and MD6,
respectively. When experimental results were plotted against those
predicted, the slope of the regression curve was almost identical to
that of a theoretical line of identity (Fig
3). In addition, for each sample evaluated,
only small variations were observed between different experiments and
for all ICs. Therefore, these experiments show that our ICs can be used
in this QC-PCR assay to measure NHL cell content over a large range of
cell concentrations and with high levels of accuracy.
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| Fig 3.
Correlation between the number of bcl-2/IgH rearranged
cells predicted and observed. H2 (A), RL (B), and DHL-16 (C) cells in
concentrations ranging from 5 to 5 × 104 cells, were
added to 106 normal PBMC and then evaluated with the ICs
(thick line): GU1M, MACM, and MD6, respectively. Results, mean ± SD
of three experiments, are compared with an ideal line of identity (thin
line). Correlation coefficients of observed versus predicted results
were greater than .99 for all three ICs.
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Bcl-2/IgH rearranged cells in BM and PB samples.
We used this QC-PCR approach to measure the number of lymphoma cells
in BM and steady-state PB samples of patients with high-risk NHL before
autologous transplantation at the time of progenitor cell harvest.
Among the 28 patients where both BM and PB cells were available, all
but two patients had at least one site with a bcl-2/IgH rearrangement
detectable by PCR, and within this latter group, lymphoma cells were
detectable in 69% of PB and 92% of BM specimens. When measured by
QC-PCR, 11 patients (42%) had less than 5 bcl-2/IgH rearranged cells
per million BM and PB cells evaluated, 5 patients (19%) had between 5
and 49 rearranged cells, 5 patients (19%) had between 50 and 499
rearranged cells, 3 patients (12%) had between 500 and 4,999
rearranged cells, and 2 patients (8%) had more than 5,000 and 50,000
rearranged cells per million cells (Fig 4).
We found no differences in the number of rearranged cells in patients
with MBR or mcr rearrangements (data not shown).
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| Fig 4.
QC-PCR evaluation of the number of bcl-2/IgH rearranged
cells measured in BM ( ) and PB () from 26 different patients at
the time of progenitor cell harvest. Lymphoma cell numbers were
significantly higher in BM than in PB (P = .0001).
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Overall, the number of lymphoma cells detected in BM was significantly
higher than in PB (P = .0001). The number of lymphoma cells in
BM was higher than in PB in 21 patients, lower in 3 patients, and both
results were equal in 2 patients. In individual patients, differences
in the number of lymphoma cells between BM and PB samples were of 1 log
(10-fold) or less in 23 patients, with a median difference of 0.48 log,
corresponding to a threefold higher number of lymphoma cells in BM
(range, 0.7 to 5 logs). Differences of more than 1 log of NHL
cells (1.2, 2.6, and 5 logs) were observed in only three patients, and
in each instance, lymphoma cells were more prominent in BM than in PB.
Numbers of NHL cells in the BM and mobilized PB grafts.
The widespread use of PB progenitor cell mobilization before autologous
PCT warranted an evaluation of their lymphoma cell content and a
comparison with levels of BM infiltration. At the time of harvest,
bcl-2/IgH rearrangements were documented in 71% of BM samples and 82%
and 88% of mobilized PB samples collected on the first (mPB1) and
second (mPB2) day of leukopheresis, respectively (Fig
5). From 0 to 98,608 rearranged cells per
million cells were measured in BM, while 0 to 68,362 cells per million
were observed in mPB products.
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| Fig 5.
Quantification of lymphoma cells present in the BM ()
and the first () and second () mobilized PB collections from 17
different patients listed according to the number of rearranged cells
detectable before autologous PBPCT. No difference in lymphoma cell
burden was identified (P = .57).
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The distribution of the contamination level in this patient population
(Fig 5) was similar to that observed in the previous group of patients
(Fig 4). However, differences in lymphoma cell content between BM and
mobilized PB were of only 0.06 log (0 log with mPB1 and 0.11 log with
mPB2), without any significant difference (P = .570).
Clinical parameters versus infiltration by NHL cells.
To determine whether patient or disease characteristics were associated
with the degree of infiltration by NHL cells, we compared the numbers
of NHL cells measured by our QC-PCR assay with various clinical
parameters (Table 2). We found no
correlation between patient age, sex, stage of disease, remission
status and number, and levels of rearranged cells. However, patients
with follicular lymphoma had higher levels of infiltration by bcl-2/IgH
rearranged cells in BM and mobilized PB than patients with lymphoma of
diffuse histology (P = .041 and .034, respectively). This
difference was not observed in steady-state PB. In addition, the
presence of bone marrow infiltration by pathologic examination, at any
time before transplantation, was associated with higher levels of NHL
cells by QC-PCR in BM and PB (P = .015 and .012, respectively)
than patients without detectable lymphoma cells on pathology. However,
when only evaluated at the time of harvest, BM biopsy results did not
correlate with the number of lymphoma cells measured by QC-PCR.
Effect of mobilization on NHL cells.
Lymphoma cell content could be evaluated in BM and sequentially in PB
obtained before and after chemotherapy and G-CSF progenitor cell
mobilization in seven patients. The number of bcl-2/IgH rearranged
cells in PB increased following mobilization in six of seven patients
(Fig 6). Although increases were limited in
most patients, the number of NHL cells in two patients' PB more than
tripled after mobilization.
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| Fig 6.
Effect of mobilization on lymphoma cell levels. The
number of bcl-2/IgH rearranged cells detectable in BM and PB obtained
immediately before mobilization and in mobilized PB collections (1 and
2) are plotted for each of seven individuals.
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| |
DISCUSSION |
To accurately measure the number of NHL cells present in minimal
residual disease states, we developed a nonradioactive competitive
quantitative PCR approach. After generating appropriate internal
controls, we defined the kinetics of amplification, as well as
sensitivity, reproducibility, and accuracy of this methodology. To
identify which hematopoietic sampling site contains the least NHL cells
and is the best source of progenitor cells in terms of tumor cell
contamination, we used this QC-PCR to measure the number of
bcl-2/IgH translocated cells in BM, PB, and mobilized PB. Although
extremely variable between patients, the concentration of NHL cells
found in BM was higher than in steady-state PB, but identical to that
in mobilized PB.
Competitive PCR determines the amount of target DNA by comparing its
signal to that of known quantities of an exogenous template that is
amplified in the same PCR reaction and with the same
primers.26,27,29 This approach, which is probably the most
accurate quantitative PCR methodology, necessitates the synthesis of
internal controls satisfying strict size and structure requirements. To
satisfy these requirements, we cloned several bcl-2/IgH rearranged
sequences directly from patient lymphoma cells. In contrast to previous
studies that used cell lines as ICs,30,31 we could take
advantage of the inherent diversity in the translocation sites of
various NHL cells to obtain ICs of various sizes, which can be
discriminated from patient PCR products for both MBR and mcr
rearrangements, while remaining of similar size for accurate
quantitative assessment. Moreover, the generation of such clones of ICs
is simple and, importantly, ensures the preservation of intact
sequences complementary to both outer and inner primers.
In this study, efforts of optimization and standardization were
especially important to develop a quantitative approach capable of
detecting extremely small variations in lymphoma cell content and to
accurately determine the reliability and precision of such measures. We
first ensured, by sequence analysis, that no nucleotides were lost or
modified during the cloning of ICs, particularly in the outer primer
homologous sequences, as the primers used for cloning were the same as
those used for quantification. The first round of amplification also
occurred in the exponential phase, while the second round was stopped
before reaching the saturation phase, even with high levels of initial
target template. In addition, kinetic studies confirmed that
amplification of plasmidic IC paralleled that of genomic DNA. In these
conditions, lymphoma cells could be measured over a large range, at
least 5 logs of cells, and for each IC, this relation was linear,
accurate (within 0.2 log of cells) and reproducible. Because the
competition phenomenon is log-log linear only when DNA content is near
equimolarity,26 we narrowed the number of different
concentrations of ICs within one log of the target value, an approach
that enabled us to monitor differences of less than one log (10-fold)
with unequalled precision and standardization.
In patients with NHL undergoing PCT, it has been postulated that the
level of infiltration of the progenitor cell graft by residual
malignant cells plays an important role in disease
relapse.11,14 A few studies have used sensitive detection
methods to determine whether lymphoma cells were present in PB versus
BM.13-15,30,32 Gribben et al13 evaluated 45
patients with high-risk NHL and found that bone marrow samples from all
of these patients were infiltrated by lymphoma cells, while
corresponding PB samples were positive in only half of these patients.
Similarly, Sharp et al14, using an in vitro culture system,
showed that lymphoma cells grew less frequently from PB than BM
samples. In contrast, other investigators found similar numbers of PB
and BM samples positive for bcl-2/IgH
rearrangements.15-17,30,32 These studies provided
important correlations between the presence of lymphoma cells in BM and
or PB cells, yet only quantitative comparisons could clarify the issue.
To our knowledge, this is the first large scale study to accurately
measure the number of lymphoma cells present at these various sites and
also in mobilized PB.
Using the QC-PCR approach described in this study, we showed wide
variations, ranging from 1 to 107,245 bcl-2/IgH rearranged cells per
million MNCs evaluated, in BM, PB, and mPB of patients in complete or
partial remission. With such large variations, an evaluation of
lymphoma cell content in BM and PB could only be performed by comparing
samples obtained from the same patient and at the same time. We found
that BM samples had a higher number of contaminating lymphoma cells
than PB, but that differences in NHL cell mass between BM and PB were
of one log or less in most (88%) patients. Such small (10-fold)
differences in lymphoma cell numbers may explain why, in most
qualitative PCR studies, similar frequencies of PCR positivity were
detected in BM and PB samples.15-17,30,32 The lower
frequency of infiltration by NHL cells in PB than in BM, identified by
a few groups,13,14 could be attributable to the study of
larger numbers of patients with threshold levels of lymphoma cells,
where the 10-fold lower level of infiltration in PB identified in this
study could result in a negative test in PB and a positive result in
BM. This also suggests that PB should be a reliable sampling site to
quantitatively evaluate lymphoma cell burden in a majority of patients.
In contrast, we found no significant difference in the concentration of
bcl-2/IgH rearranged cells present in BM and mobilized PB. Thus,
although steady-state PB may offer an advantage in terms of the
proportion of lymphoma cells, this difference with BM is very slight
and completely abolished after mobilization with growth factor and
chemotherapy. Moreover, as the total number of cells infused is usually
larger with mPB than BM, mobilized PB grafts could actually contain
higher numbers of lymphoma cells than conventional BM grafts. It is
noteworthy that in a few individuals, differences in the number of NHL
cells found in BM versus PB could reach up to 5 logs
(105-fold differences). Such large differences again
underline the critical importance of measuring the number of NHL cells,
and not only PCR positivity, before stem cell harvesting to identify
the importance of lymphoma cell infiltration.
Immunohistochemical studies in patients with breast and lung carcinoma
showed that the number of malignant cells can increase in PB following
mobilization procedures, and that this phenomenon seems related to BM
involvement.19 We analyzed levels of NHL cells before and
after mobilization with chemotherapy and G-CSF and found that lymphoma
cell content in PB also increased with mobilization in a majority of
patients. These preliminary results, along with results obtained by
comparing lymphoma cell levels in BM and mPB, suggest that in vivo
progenitor cell mobilization may not be appropriate as a purging
strategy unless differences in the kinetics of release of lymphoma and
normal progenitor cells can be identified. This seems unlikely because
in this study, NHL cell levels were increased at the time when
CD34+ cells are usually at optimum levels for collection.
The fact that various types of tumor cells are mobilized into PB
suggest that dissemination probably involves a common pathway related
directly or indirectly to the mobilizing agents used. The monitoring of
this effect should be important in the study of normal stem cell
physiology, as well as of mechanisms implicated in tumor cell release.
In addition, different numbers of tumor cells in progenitor cell grafts
may have a different impact on patient outcome. For example, Sharp et
al5 showed that patients with detectable lymphoma cells in
BM grafts had a lower disease-free survival than patients without such
colony growth. However, this lymphoma colony assay is less sensitive
than nested PCR and possibly identifies patients with higher levels of
tumor cell infiltration. Our results clearly show that major variations
in lymphoma cell content can be observed in different patients and even
in different harvest sites. The definition of clinically significant
threshold levels of infiltration is thus clearly needed. It will be
important to follow our patient population to prospectively determine
whether the amount of lymphoma cells in progenitor cell grafts affects
relapse and if a minimum level of bcl-2/IgH rearranged cells could be
transplanted without impairing patient prognosis.
We found that patients with NHL of follicular histology had higher
levels of infiltration than patient with diffuse NHL, independent of
disease stage. This finding could explain why purging strategies seem
particularly beneficial in patients with follicular
lymphomas.11,12 It also reinforces the importance of
developing strategies to purge NHL cells from mobilized PB grafts, and
not only BM grafts. Fortunately, various strategies using monoclonal
antibodies, either with complement, immunomagnetic beads, or
immunotoxins or other purging agents can eliminate several logs of
malignant cell line cells, while sparing normal hematopoietic
progenitors.11,31,33-35 This QC-PCR assay may be an
appealing and easily reproducible approach to devise purging strategies
to enhance the elimination of lymphoma cells.
The preponderance of lymphoma cells in BM versus PB is compatible with
the existence of a blood-marrow barrier.36,37 In addition,
the almost constant one log difference in lymphoma cells between these
sites strongly suggests that a gradient rather than a threshold
phenomenon is involved. The elimination of such a gradient following
stem cell mobilization with chemotherapy and growth factors may be
attributable to direct toxic effects from cytotoxic agents. Indeed,
Shirota and Tavassoli37 showed that cyclophosphamide
administration altered the permeability of murine sinusoidal cells in
BM endothelium. On the other hand, cytokines were also shown to modify
the proliferation and regulation of cell adhesion molecules in normal
hematopoietic progenitor cells following in vivo mobilization
procedures.38 Such alterations at the level of either
lymphoma cells or marrow stromal cells could result in enhanced
circulation between PB and BM compartments.
In this study, 50% of NHL patients had below 10 lymphoma cells
detectable per million cells. As described in Materials and Methods,
extremely rigorous methods were used to minimize the likelihood of
false positive results. In addition, 19 healthy volunteer donors were
tested at different times with our assay and, in all instances, these
samples were PCR negative. Nevertheless, a few investigators have
reported the presence of bcl-2/IgH rearranged cells in normal
invidividuals.39,40 However, this rearrangement was only
documented at very low levels, mostly in older individuals, and in
highly enriched B-cell populations with a frequency of 1 in
105 or less circulating B cells. These findings raise
numerous questions regarding the biologic significance of low levels of
bcl-2/IgH rearranged cells in normal individuals and NHL patients. The
present QC-PCR assay may be useful to determine the evolution of these
cells over time, their proliferation and clonogenicity, and also help
identify instances where additional oncogenic events are implicated.
In addition to the above applications, accurate measurements of
lymphoma cell numbers should be useful for the evaluation and
optimization of (1) chemotherapy and immunotherapy protocols, (2)
progenitor cell mobilization regimens, and (3) myeloablative regimens.
Precise quantitative monitoring of lymphoma cells in vivo may also help
to resolve one of the most crucial issues in lymphoma management: the
impact of molecular detection of lymphoma cells on disease relapse.
| |
FOOTNOTES |
Submitted April 14, 1997;
accepted August 22, 1997.
Supported by the Cancer Research Society of Canada. L.B. and D.C.R. are
Scholars of the Fonds de la Recherche en Santé du Québec,
Canada.
Address reprint requests to Denis-Claude Roy, MD, Division of
Hematology-Immunology, Maisonneuve-Rosemont Hospital Research Center,
5415, L'Assomption Blvd, Montreal, Quebec, Canada, H1T 2M4.
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.
| |
ACKNOWLEDGMENT |
The authors thank Dr F. Coutlée for helpful discussions, Dr M.
Dumont for statistical analysis, Dr S. Cousineau for his
hematopathologic expertise, C. Le Houillier for technical assistance,
and M.J. Guertin and O. Marchand for their help with patient samples.
| |
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Abstract
Introduction
Abstract