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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 8 (October 15), 1999:
pp. 2901-2910
Shiga-Like Toxin-1 Receptor on Human Breast Cancer, Lymphoma, and
Myeloma and Absence From CD34+ Hematopoietic Stem Cells:
Implications for Ex Vivo Tumor Purging and Autologous Stem Cell
Transplantation
By
E.C. LaCasse,
M.R. Bray,
B. Patterson,
W.-M. Lim,
S. Perampalam,
L. G. Radvanyi,
A. Keating,
A.K. Stewart,
R. Buckstein,
J.S. Sandhu,
N. Miller,
D. Banerjee,
D. Singh,
A.R. Belch,
L.M. Pilarski, and
J. Gariépy
From the Department of Medical Biophysics, University of Toronto and
the Ontario Cancer Institute, Toronto, Ontario, Canada; the Department
of Oncologic Pathology, Princess Margaret Hospital, Toronto, Ontario,
Canada; the Autologous Blood and Marrow Transplantation Program,
University of Toronto and the Toronto Hospital, Toronto, Ontario,
Canada; the Samuel Lunenfeld Research Institute, Mount Sinai Hospital
(Toronto) and the Department of Pathology, Women's College Hospital,
Toronto, Ontario, Canada; and the Department of Oncology, University of
Alberta and the Cross Cancer Institute, Edmonton, Alberta,
Canada.
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ABSTRACT |
The ribosome-inactivating protein, Shiga-like toxin-1 (SLT-1),
targets cells that express the glycolipid globotriaosylceramide (CD77) on their surface. CD77 and/or SLT-1 binding was detected by flow
cytometry and immunocytochemistry on lymphoma and breast cancer cells
recovered from biopsies of primary human cancers as well as on B cells
or plasma cells present in blood/bone marrow samples of multiple
myeloma patients. Breast cancer cell lines also expressed receptors for
the toxin and were sensitive to SLT-1. Treatment of primary B lymphoma,
B-cell chronic lymphocytic leukemia, and myeloma B or
plasma cells with SLT-1-depleted malignant B cells by 3- to 28-fold,
as measured by flow cytometry. Depletion of myeloma plasma cells was
confirmed using a cellular limiting dilution assay followed by reverse
transcriptase-polymerase chain reaction analysis of
clonotypic IgH transcripts, which showed a greater than 3 log reduction
in clonotypic myeloma cells after SLT-1 treatment. Receptors for the
toxin were not detected on human CD34+ hematopoietic
progenitor cells (HPC). HPC were pretreated with a concentration of
SLT-1 known to purge primary malignant B cells and cultured for 6 days.
The number of HPC was comparable in toxin-treated and untreated
cultures. HPC were functionally intact as well. Colony-forming units
(CFU) were present at an identical frequency in untreated and SLT-1
pretreated cultures, confirming that CFU escape SLT-1 toxicity. The
results suggest the ex vivo use of SLT-1 in purging SLT-1
receptor-expressing malignant cells from autologous stem cell grafts of
breast cancer, lymphoma, and myeloma patients.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
GENE MARKING STUDIES suggest that relapse
observed in cancer patients treated with high-dose chemotherapy and
autologous stem cell transplantation (ASCT) may reflect, at least in
part, the reinfusion of contaminating tumor cells in the stem cell
graft.1,2 In myeloma, recent experiments have demonstrated
that malignant progenitors from granulocyte colony-stimulating factor
(G-CSF)-mobilized blood transferred myeloma to immunodeficient mice as
measured by the presence of lytic bone lesions and polymerase chain
reaction (PCR)-detectable disease (Pilarski et al, manuscript
submitted). Significant improvements in disease-free
survival have been demonstrated for follicle center cell lymphoma
patients transplanted with autologous stem cell grafts purged free of
PCR-detectable tumor cells with anti-B-cell antibodies and rabbit
complement.3,4
A limited number of strategies for purging tumor cells from stem cell
grafts have been developed to date. Most approaches focus on the design
of protein fusion molecules or chemically derived toxin conjugates
incorporating a cytotoxic component derived from a protein toxin
covalently linked to a growth factor, cytokine, or antibody domain,
enabling the resulting constructs to target specific cancer cells. Such
constructs were initially created to cope with the broad receptor
specificity of native protein toxins. The bacterial toxin, Shiga-like
toxin 1 (SLT-1), is internalized by cells that express the glycolipid
receptor globotriaosylceramide (Gb3, CD77) on their
surface.5,6 Our group has recently shown that SLT-1 can
successfully purge a human Burkitt's lymphoma cell line from a murine
bone marrow (BM) graft ex vivo before transplantation in severe
combined immunodeficiency (SCID) mice.7 The potential use
of SLT-1, a ribosome-inactivating protein, is presently limited to an
ex vivo application, because its toxicity includes endothelial cell
damage and hemolytic uremic syndrome observed in patients after
gastrointestinal infections with SLT-1-producing strains of
Escherichia coli or Shiga toxin-producing strains of
Shigella dysenteriae.5,8-14 We now report the
expression of SLT-1 receptors on clinical tumor specimens and cell
lines of cancers that may benefit from autologous stem cell
transplantation and have addressed the survival of CD34+
cells after exposure to SLT-1. Our findings suggest that SLT-1 could be
used as an ex vivo purging agent for autologous grafts from patients
with 3 malignancies commonly treated with high-dose chemotherapy and
autologous stem cell transplantation, namely breast cancer, lymphoma,
and multiple myeloma.15,16
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MATERIALS AND METHODS |
Cell lines, tissue sources, and culture conditions.
Human breast cancer cell lines were obtained from the American Type
Culture Collection (Rockville, MD) and from the laboratories of Dr Ron
Buick (Ontario Cancer Institute, Toronto, Ontario, Canada) and Dr Irene
Andrulis (Lunenfeld Research Institute, Toronto, Ontario, Canada). The
MDA-MB series of cells were cultured at 37°C in Leibovitz L-15
medium in the absence of CO2, whereas the other breast
cancer cell lines were cultured in  -minimal essential medium
(-MEM), Iscove's modification of Dulbecco's modified
Eagle's medium (IMDM), or RPMI 1640 in the presence of
5% CO2 with 10% fetal calf serum (FCS),
except for JS-1,17 which was maintained in 20% FCS.
Clinical specimens were obtained with informed consent according to
protocols approved by ethics committees at Women's College Hospital,
the Toronto General Hospital, the Princess Margaret Hospital, the
University of Toronto, and the Alberta Cancer Board. Lymphoma specimens
(including peripheral blood, biopsies, BM, or fine-needle aspirates)
were obtained from patient samples sent to the flow cytometric unit
(Princess Margaret Hospital, Toronto, Ontario, Canada) for
immunophenotyping. Blood and BM samples from myeloma patients were
obtained from the Cross Cancer Institute (Edmonton, Alberta, Canada) or
the Toronto Hospital. Blood from a B-cell chronic lymphocytic leukemia
(B-CLL) patient or G-CSF-mobilized blood samples from
myeloma, lymphoma, or breast cancer patients were obtained at the Cross
Cancer Institute or the Red Cross Apheresis Unit (Edmonton, Alberta,
Canada). Lymph node biopsies having 30% to 60% malignant B cells were
obtained from 2 patients with B lymphoma (provided by Dr R.G. Coupland,
Cross Cancer Institute). Lymph node cells were dispersed by passage
through a sieve. Mononuclear cells (MNC) from blood, BM, or lymph nodes
were purified on Ficoll Hypaque (Pharmacia, Dorval, Quebec, Canada).
Breast cancer specimens were derived from surgical biopsies of primary
breast cancers at Women's College Hospital (N.M.). All functional
assays were performed on freshly obtained tissues.
Monoclonal antibodies (MoAbs).
Antibodies used for immunocytochemistry were bought from the following
sources: anti-CD10 (Dako, Glostrup, Denmark), biotinylated mouse
antirat IgM, unconjugated rat IgM anti-CD7718 and
anti-B-B4 antibodies (Serotec, Oxford, UK),19 anti-CD13
(Coulter, Burlington, Ontario, Canada), AE1/AE3 anti-cytokeratin
(Zymed, South San Francisco, CA), anti-low molecular weight keratins
(Cam 5.2; Becton Dickinson, Franklin Lakes, NJ). The antiepithelial
cancer antibody Ber-EP420 was purchased from Dako. ONC-M38,
a hybridoma producing a murine antimucin antibody,21 was
from Dr Peter Linsley (Bristol-Myers Squibb, Seattle, WA). M38 antibody
was purified from culture supernatants by protein G affinity
chromatography. MoAb 17G10-phycoerythrin (PE) was used to
detect CD45.22 Anti-CD19 MoAb (FMC63) was coupled to PE or
fluorescein isothiocyanate (FITC) and used to detect B
cells in lymphoma biopsies and myeloma MNC as previously
described.23,24 Plasma cells in myeloma BM were identified
using an anti-CD38-PE conjugate (Leu17; Becton Dickinson) and based on
their light scattering properties.25 Hematopoietic
progenitor cells (HPC) were detected by staining the cell population
with a combination of anti-CD34-PE (8G12-PE; Becton Dickinson) and
anti-CD45-Quantum red (Sigma) conjugates and gating for the
CD34+45lo subset, as previously
described.26
Toxin purification and labeling.
SLT-1 was initially purified to greater than 95% purity by column
chromatography from a transformed E coli strain, as described elsewhere.27 The binding subunit, SLT-B, was purified to
homogeneity, as described previously.28 It was labeled with
FITC, as stated elsewhere.7
SLT-1 cytotoxicity assays for breast cancer cell lines.
The toxicity of SLT-1 towards adherent cell lines was measured using
the sulforhodamine B (SRB) dye binding assay.29 Briefly, cells cultured in 96-well plates were exposed to a range of toxin concentrations for a 1-hour period in phosphate-buffered saline (PBS). The toxin-containing solution was subsequently
diluted with the appropriate medium containing FCS and the treated
cells were cultured for another 48 hours. The medium was removed and the remaining adherent cells were fixed with ice-cold 10%
trichloroacetic acid, air-dried, and stained with 0.4% SRB (Molecular
Probes, Eugene, OR) dissolved in 1% acetic acid. The excess dye was
washed away, and the remaining bound SRB dye was extracted from the
cells with 10 mmol/L Tris base. The absorbance of the dye was read at 540 nm using a plate reader.
Flow cytometry and immunocytochemistry.
The expression of SLT-1 receptors was detected by flow cytometry using
FITC-labeled SLT-B subunit (FITC-SLT-B) and by immunocytochemistry with
an unconjugated anti-CD77 MoAb. Flow cytometry of human lymphoma samples was performed as previously described.7 Flow
cytometric data were acquired from human breast cancer aspirates or
cells recovered from minced tumor samples stained with antibodies to surface antigens (CD77, Ber-EP4, and M38) and viability dyes. A
cocktail of both Ber-EP4 and M38 antibodies was used for flow cytometry
of breast cancer biopsies because of the limited recovery of cells.
Cytospins of lymphoma and breast cancer cells were immunostained with
anticytokeratin antibodies (cam 5.2 [Becton Dickinson] and AE1/AE3
[Zymed]). All immunocytochemistry was accomplished on paraformaldehyde-fixed samples.
Purging of ex vivo malignant B cells.
MNC derived from lymphoma biopsies or from blood/BM samples of myeloma
patients were treated with 5 µg/mL SLT-1 for 60 minutes at 37°C,
washed to remove unbound toxin, and cultured for 6 to 10 days in RPMI
supplemented with 10% FCS. Cultured cells were harvested, and the
total number of viable cells was counted and stained with FITC-labeled
anti-CD19 as well as with propidium iodide (PI) to identify viable
cells. The absolute number of viable CD19+ B cells
remaining in the cultures was calculated as the percentage of viable
CD19+ cells (CD19+PIlow) multiplied
by the total number of viable cells. Plasma cells were stained with
FITC-labeled anti-CD38 MoAb as well as PI.
Quantitation of clonotypic cells in myeloma.
To confirm the purging of malignant plasma cells from myeloma BM cells,
a cellular limiting dilution reverse transcriptase-PCR (RT-PCR) assay24 was performed to detect the
presence of clonotypic IgH VDJ rearrangements using patient-specific
primers. Briefly, graded numbers of untreated or SLT-1-pretreated BM
cells were deposited into PCR tubes containing lysis buffer. RNA from
the lysed cells was reverse transcribed and the resulting cDNA was amplified in the same tube. The patient-specific IgH VDJ sequence was
identified from single plasma cells shown to be specific for this
patient and expressed by greater than 80% of individual plasma cells
recovered for this patient, confirming that the myeloma-specific Ig
sequence had been identified.24
Short-term culture of HPC.
Mononuclear cells from G-CSF-mobilized blood were pretreated with 5 µg/mL of SLT-1 for 60 minutes at 37°C, washed, and cultured in
commercial long-term culture medium (Myelocult plus 10% Hemostim; Stem
Cell Technologies, Vancouver, British Columbia, Canada) for 7 to 10 days. Harvested cells from paired untreated and SLT-1-pretreated cultures were stained with anti-CD34-PE and anti-CD45-QR conjugates. The percentage of HPC
(CD34+45loScatterlow/med) was
determined as previously described.26
Assay for colony-forming units (CFU).
MNC from G-CSF-mobilized blood samples were enriched for HPC by
negative selection according to instructions from the manufacturer (StemSep; Stem Cell Technologies). Enriched HPC (40% to 60% HPC) were
then treated or not with 5 µg/mL of SLT-1 for 60 minutes at 37°C,
washed once, and cultured to monitor the number of CFU. Increasing
numbers of toxin-treated or untreated cells (25 to 200 cells per well)
were added to a mixture of growth factors and methyl cellulose
(Methylcult; Stem Cell Technologies) according to the instructions from
the manufacturer and deposited into culture wells. The number of
colonies was counted under a microscope after 20 days, as recommended
by the manufacturer. CFU values reported represent the average of
colony numbers observed in 6 to 12 replicate wells.
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RESULTS |
Expression of SLT-1 receptor on breast cancer cell lines and SLT-1
cytotoxicity.
The expression of SLT-1 receptors on 18 breast cancer cell lines was
analyzed by flow cytometry using FITC-SLT-B. Two antibodies were used
to label breast cells: Ber-EP4, which recognizes an unknown antigen on
epithelial-derived carcinomas,20 and M38,21 which detects the epitope MUC1 on breast-derived mucin.30
Of the 18 breast cancer cell lines studied, 5 had less than 15%
positivity for FITC-SLT-B, whereas 13 (72%) expressed surface SLT-1
receptors (Table 1). Sixteen of 18 (89%)
breast cancer cell lines were positive for M38 (MUC1) and 12 of 18 (67%) were positive for Ber-EP4. The results demonstrate the frequent
expression of SLT-1 receptors on the surface of breast cancer cell
lines. The level of expression of SLT-1 receptors on breast cancer cell
lines correlates with their sensitivity to killing by SLT-1
(Fig 1 and Table 1, CD50 values).
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| Fig 1.
Effect of SLT-1 on the viability of human breast cancer
cell lines. The percentage of cell viability of 8 breast cancer cell
lines based on the SRB dye binding assay was measured as a function of
toxin concentration. Cell viability curves are shown for CAMA-1 ( ),
JS-1 ( ), MCF-7 ( ), MDA-MB-231 ( ), MDA-MB-468 ( ), MDA-MB-469
( ), SKBR3 ( ), and T47D ( ).
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Expression of SLT-1 receptors on breast cancer biopsies.
The expression of SLT-1 receptors on biopsies of primary human breast
cancers obtained from 10 patients was analyzed by flow cytometry and
immunocytochemistry. Cell suspensions were prepared from fine-needle
aspirates or by mechanical disaggregation of solid tumors. Eight of 10 samples (80%) showed greater than 15% positive staining for
FITC-SLT-B (Table 2) and the intensity of
fluorescence staining was high (Fig
2C). In contrast to the established breast cancer cell
lines, Ber-EP4/M38 was expressed on 4 of 10 samples, all 4 of which
were positive for SLT-1 receptors (SLT-R). Four of 8 SLT-R+
samples were negative for the breast cancer markers (Ber-EP4 and M38).
SLT-1 receptor-positive cells were confirmed as breast cancer cells by
morphological examination and immunocytochemistry with anti-low
molecular weight cytokeratin and anti-CD77 antibodies (Fig 3A and B).
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| Fig 2.
SLT-1 binding by malignant B cells, breast
carcinoma cells and HPC. Representative views from the flow cytometric
detection of SLT-1 receptors on follicle center lymphoma grade I (A),
multiple myeloma plasma cells (B), and breast carcinoma (C). The
binding of FITC-SLT-B is shown in relation to anti-CD19 labeling (a
pan-B-cell marker) for the lymphocyte gate in (A) or against B-B4
(plasma cell marker) for BM cells in (B) and against the combined
expression of Ber-EP4 and M38 (MUC1) for a breast cancer biopsy (C).
Dual-positives are shown in the upper right quadrant (A and C) or as a
boxed area (B).
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| Fig 3.
Immunocytochemical analysis of breast cancer cells (A
through D, immunohistochemical stain shown in red and nuclear
counterstain in blue). Cytospins of cells from a breast cancer biopsy
were stained with anti-LMWK (A, original magnification × 400) and
anti-CD77 (B, original magnification × 400). The metastatic breast
cancer cell line JS1 was grown on plastic slides and stained with
anticytokeratin AE1/AE3 (C, original magnification × 400) and
anti-CD77 (D).
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Expression of SLT-1 receptor on lymphoma and myeloma.
Clinical samples from patients with hematological cancers were
routinely screened with FITC-SLT-B to monitor the expression of SLT-1
receptors. Flow cytometry data were collected on 134 sequential samples
(Fig 4) classified according to the REAL
system.31 On average, 3% cells (±4%) recovered from
nonmalignant samples stained with the fluorescent probe (results not
shown). Clinical samples were scored as positive when at least 15% of
cells (3 SD above the mean background) bound FITC-SLT-B.7
SLT-1 receptors were frequently expressed on follicle center cell
lymphoma grades I, II, and III (Fig 4), with 31 of 43 (72%)
patient samples positive. These results agree with the expression of
CD77 on normal follicle center cells.32-37 Thirty-three
percent (15 of 46 samples) of small lymphocytic lymphomas with or
without chronic lymphocytic leukemia were positive for FITC-SLT-B
staining, as were 42% (5 of 12 samples) of large B-cell lymphomas.
FITC-SLT-B staining was not observed on mantle cell lymphomas, which
are thought to arise from the normally CD77 B cells
in the follicular mantle zone. Marginal zone lymphomas were also
CD77. The B-cell acute lymphocytic leukemias were
essentially CD77. Flow cytometry (2 samples)
and immunohistochemistry (5 samples) were performed on BM aspirates
collected from multiple myeloma patients. The marker syndecan-1,
identified by the antibody B-B4, was used to detect normal plasma cells
as well as multiple myeloma cells.19,38 Normal plasma cells
were found to be syndecan-1+, CD77,
whereas plasma cells from multiple myeloma BM were
syndecan-1+, CD77+ (results not shown).
Multiple myeloma includes circulating CD19+ B-cell
components of the malignant clone as well as the BM-localized plasma
cells.23-25,39 On average, 66% of B cells in myeloma
peripheral blood mononuclear cells (PBMC) are
clonotypic24 and approximately 30% are
polyclonal.25 Figure 5 shows
that the set of B cells shown elsewhere to be clonotypic24
express SLT-1 receptors (arrowhead), whereas the set of residual normal
B cells in myeloma patients does not bind SLT-1 (arrow).
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| Fig 4.
SLT-1 receptor (CD77) expression on lymphoma. The
expression of SLT-1 receptors on malignant lymphoma was detected using
a fluoresceinated binding subunit of SLT-1 (FITC-SLT-B). Cell
suspensions recovered from patient samples (peripheral blood,
fine-needle aspirates, lymph node, and BM biopsies) were sent to a flow
cytometry unit (Ontario Cancer Institute) for immunophenotyping. A
lymphocyte population in which 15% of the cells stained positively
with FITC-SLT-B (dashed line) was defined as positive for the presence
of SLT-1 receptors. This percentage value (15%) was calculated from
the average percentage of FITC-SLT-B-positive cells (3% ± 4%)
observed in samples of noncancerous patients, plus 3 SD. Abbreviations
used: B-LL/ALL, B-cell lymphoblastic lymphoma/acute lymphoblastic
leukemia; ML, LG, malignant lymphoma, low grade (nonclassifiable);
B-CLL/SL, small lymphocytic lymphoma with or without chronic
lymphocytic leukemia; HCL, hairy cell leukemia; LPL, lymphoplasmacytoid
lymphoma; FCC I, follicle center cell lymphoma, follicular, grade I;
FCC II, follicle center cell lymphoma, follicular, grade II; FCC III,
follicle center cell lymphoma, follicular, grade III; MCL, mantle cell
lymphoma; MZL, marginal zone lymphoma; D-M, diffuse-mixed small- and
large-cell lymphoma; B-LC, diffuse large B-cell lymphoma; T-PLL, T-cell
chronic lymphocytic lymphoma, prolymphocytic leukemia; AILD,
angioimmunoblastic T-cell lymphoma; ALCL, anaplastic large-cell
lymphoma; ATL/L, adult T-cell lymphoma/leukemia; T-LL/ALL, T-cell
lymphoblastic lymphoma/acute lymphoblastic leukemia; T-LC, T-cell
intermediate-grade large-cell lymphoma. Patients with diffuse large
B-cell lymphoma who had a documented history of follicle center cell
lymphoma were included in the FCC-III category.
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| Fig 5.
SLT-1 receptors are present on a B-cell subset of myeloma
patients and absent on HPC. Cells from peripheral blood of myeloma
patients (A and B) were stained with anti-CD19-PE and FITC-SLT-B.
Mobilized blood mononuclear cells (C) were stained with
anti-CD34-PE/anti-CD45-QR and FITC-SLT-B and files were gated for the
HPC subset (D), as described in Materials and Methods. For (C) and (D),
the arrows indicate the HPC subset. For myeloma, the peripheral blood
includes both polyclonal and monoclonal B cells.24 The
arrowhead indicates the monoclonal subset of B cells and the arrow
highlights the polyclonal subset, presumptively normal B cells that
lack SLT-1 receptors.
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Hematopoietic progenitor cells do not bind SLT.
HPC are phenotypically defined as CD34+45lo
Scatterlow/medium cells.26,40,41 Hematopoietic
progenitor cells from G-CSF-mobilized blood MNC were stained in
multicolor immunofluorescence to determine SLT-1 binding. A
representative flow cytometry plot is shown in Fig 5 for unfractionated
G-CSF-mobilized MNC (Fig 5C) and the same mobilized blood
MNC gated for the HPC subset, showing that normal HPC (indicated by
arrows in Fig 5C and D) do not bind SLT-1. Similar results were
obtained for HPC from 4 different mobilized blood samples.
SLT-1 purging depletes malignant cells from lymphoma, B-CLL, and
myeloma tissues.
MNC from lymphoma node biopsies or blood/BM from myeloma patients were
treated in vitro with SLT-1 to evaluate its ability to purge malignant
cells recovered from these tissues. Unlike cell lines that, after
exposure to 1 µg/mL or less of toxin, die within 2 to 3 days,7 the toxic effects of SLT-1 on freshly cultured cells
required relatively high concentrations of toxin (5 µg/mL) and were
detectable only after 6 to 10 days in culture (not shown). For 2 lymphoma biopsies, MNC were pretreated with 5 µg/mL SLT-1 and
cultured, and the number of phenotypically defined B cells was
enumerated using flow cytometry. Figure 6
(top panel) shows that the absolute number of lymphoma B cells was
reduced by 3- to 6-fold after pretreatment with SLT-1. For 1 patient
with B-CLL, B cells were reduced 28× after pretreatment with
SLT-1 (Fig 6, top panel). For B cells from myeloma blood, a similar reduction in total B cells was detected after SLT-1 pretreatment for 2 patients with the high numbers of B cells characteristic of myeloma
patients (Fig 6, bottom panel). However, for 1 myeloma patient who had
recovered from autologous transplant and had no detectable circulating
clonotypic cells (not shown; MMB 3, Fig 6, bottom panel), there was
only a marginal reduction in B cells after SLT-1 pretreatment,
suggesting that only malignant B cells are targeted by this strategy.
For myeloma BM, pretreatment with SLT-1 efficiently depleted the
majority of plasma cells by 10- to 23-fold as measured phenotypically.
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| Fig 6.
Pretreatment with SLT-1 depletes malignant B-lymphoma
cells, B-CLL, and B or plasma cells from myeloma patients. MNC derived
from lymphoma biopsies or PBMC from B-CLL (top panel) or from myeloma
blood or BM (bottom panel) were pretreated with 5 µg/mL of SLT-1 for
60 minutes, washed, and cultured for 6 to 11 days. Viable cells were
then enumerated and analyzed using anti-CD19 MoAb to detect B cells and
anti-CD38 MoAb to detect plasma cells, together with PI staining to
identify live versus dead cells. The absolute number of B or plasma
cells remaining in cultures was calculated as the percentage of viable
CD19+ or CD38+ cells times the number of
viable cells per culture. The numerical values indicate the extent of
B-cell or plasma cell depletion by SLT-1 as compared with the untreated
control cultures. Patient MMB3 was posttransplant and had no detectable
clonotypic B cells before harvest of the blood sample used here (not
shown).
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Molecular analysis confirms a greater than 3 log reduction in
clonotypic myeloma cells after SLT-1 pretreatment.
One complicating factor in evaluating the extent of purging in the
experiments cited above is the presence of residual normal B lineage
cells that may resist the action of the toxin. Because phenotypic
analysis does not reliably distinguish between normal and malignant B
or plasma cells, we evaluated the extent of purging using a novel
molecular assay for clonotypic transcripts. Myeloma cells are uniquely
identified by the IgH VDJ rearrangement that characterizes the
malignant clone in each patient. Untreated or SLT-1 pretreated BM cells
from MM patient no. 4 were deposited at limiting dilution into lysis
buffer in PCR tubes, followed by RT-PCR using patient-specific primers.
Figure 7 shows that, for the untreated
cultures, by day 6, essentially all cells were clonotypic (3 of 3 tubes
were positive at the 1 cell/tube concentration). However, for the
SLT-1-treated cultures, clonotypic transcripts were undetectable even
in the tubes containing 1,000 cells per tube. The frequency of
clonotypic cells remaining after SLT-1 pretreatment was less than
1/3,000 cells in light of the fact that all 3 tube replicates were
negative from the presence of transcripts (for a total of 3,000 cells
analyzed).
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| Fig 7.
SLT-1 pretreatment depletes myeloma plasma cells by more
than 3 log units as measured using a cellular limiting dilution RT-PCR
analysis for clonotypic transcripts. Harvested cells from sample in Fig
5 were further analyzed for the presence of clonotypic cells to
enumerate specifically the depletion of the malignant clone. For
myeloma patient no. 4, untreated or SLT-1-pretreated cultures were
harvested at day 6 and deposited at limiting dilution into PCR tubes
containing lysis buffer, followed by RT-PCR analysis using
patient-specific primers as described in the methods
section.24 The product amplified from cells of the control
cultures was a clonotypic IgH VDJ transcript of the expected size and
sequence. Products amplified from cells that were pretreated with SLT-1
were of an incorrect size and did not have an Ig sequence.
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HPC and CFU from G-CSF-mobilized blood escape SLT-1 toxicity.
To be effective, a purging agent must spare normal hematopoietic
progenitor cells. To evaluate the effects of SLT-1 treatment on
progenitor cells and their functional activity, we analyzed the
survival of HPC in toxin-pretreated cultures and of cells able to give
rise to hematopoietic colonies in vitro (CFU). These 2 approaches
represent surrogate assays for engraftment potential. Table 3A shows that, when MNC or
HPC-enriched MNC are pretreated with 5 µg/mL of SLT-1, a
concentration shown above to effectively purge malignant cells,
phenotypically identified HPC are present in equivalent numbers in
untreated or SLT-1-treated cultures at days 6 to 7 posttreatment. For
the HPC-enriched MNC population derived from mobilized blood, HPC were
more frequent in SLT-1-treated than in untreated cultures (Table 3,
analysis of HPC-enriched CD34+45lo
populations), probably reflecting a further enrichment of the toxin-resistant HPC as SLT-1-sensitive cells died. These experiments indicate that the HPC population as a whole survives SLT-1
pretreatment, but does not address the functional capability of these
HPC.
The functional potential of HPC pretreated with SLT-1 was evaluated in
terms of their ability to form hematopoietic colonies (Table 3,
analysis of CFU). HPC would not be expected to have sufficient
generative potential to form a visible colony if their survival was
compromised by their exposure to the toxin. Enriched HPC were
pretreated with 5 µg/mL of SLT-1 under conditions identical to those
shown to purge lymphoma or myeloma cells and assayed for their
colony-forming ability. These experiments show that the number of
colonies was the same for untreated or SLT-1-pretreated HPC,
indicating that SLT-1 is not toxic to human CD34+
hematopoietic progenitor cells. Similar results were obtained for
mobilized HPC from breast cancer and lymphoma patients (Table 3). To
more accurately confirm that CFU are SLT-1-resistant, a limiting
dilution analysis of CFU numbers was performed in which 25 to 200 enriched HPC, pretreated or not with 5 µg/mL SLT-1, were plated and
resulting colonies were counted 20 days later (Fig 8). The frequency of CFU was identical
for untreated or SLT-1-treated enriched HPC (~1 CFU per 12 enriched
HPC), further confirming with a functional assay that hematopoietic
progenitors escape SLT-1 toxicity.
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| Fig 8.
Toxin treatment of HPC derived from mobilized blood does
not affect the number of CFU observed. Mobilized HPC from a lymphoma
patient were enriched using a negative selection approach described in
Materials and Methods. Increasing numbers of enriched HPC (25 to 200 cells per culture) in Methylcult were treated or not with 5 µg/mL
SLT-1 for 60 minutes, washed, and finally dispensed in microtiter wells
to measure colony formation. At day 20, the number of colonies were
counted for replicate wells at each cell concentration. Values
represent the average number of colonies (±SE) calculated from
counting colonies present in 8 to 12 replicate wells. HPC from this
patient were also shown to be negative for binding FITC-SLT-B (not
shown).
|
|
| |
DISCUSSION |
Shiga-like toxin 1, a ribosomal-inactivating protein produced by
pathogenic strains of E coli (eg, O157:H7), intoxicates cells expressing globotriaosylceramide (CD77, Gb3) on their
surface. It has recently been demonstrated that ex vivo treatment of
murine BM with SLT-1 can effectively purge SCID mice of a B-cell
lymphoma xenograft while sparing normal murine hematopoietic precursor cells, suggesting its potential use as an ex vivo purging
agent.7 This study demonstrates the expression of
Shiga-like toxin 1 receptors on 3 of the human cancers commonly treated
by autologous stem cell transplant (breast cancer, lymphoma, and
multiple myeloma) and proves the absence of SLT-1 receptor on human
CD34+ stem cells. Taken together, this evidence supports
the clinical use of SLT-1 as an ex vivo purging agent to deplete
malignant cells expressing receptors for SLT-1 from autologous stem
cell grafts.
The expression of SLT-1 receptors (SLT-R) was assessed on human breast
cancer cell lines and clinical biopsies of primary human breast cancer
tissues in light of the growing number of breast cancer patients
treated each year with stem cell transplants.16 Eighteen
breast cancer cell lines were tested. Thirteen of the 18 cell lines,
some derived from breast cancer metastases,42 expressed
SLT-R on their surface, whereas 80% of primary breast cancer biopsies
were SLT-1 receptor-positive, indicating the potentially broad
expression of SLT-1 receptors on tumor cells at both primary and
metastatic sites. These findings identify CD77/SLT-R as a marker on
clinical specimens of breast cancer and indicate that SLT-1 could be
used to purge these cells. Previous evidence for the expression of CD77
on breast tissue indicated some weak immunostaining of normal breast
ductal epithelium43-46 and the presence of CD77 in human
breast milk, possibly shed or secreted by ductal cells.47 Our results prove conclusively that CD77 is expressed on breast carcinomas. The previous finding of globo-H glycolipid on normal mammary gland epithelia and human mammary carcinomas demonstrate that
breast cells have the necessary biosynthetic enzymes to make CD77
(a precursor to globo-H) and more complex globo-series
glycolipids.48 Cell lines such as CAMA-1 and SKBR3 were
resistant to the toxin, whereas CD50 values observed for
other human breast cancer cell lines spanned several orders of
magnitude. Interestingly, these results also indicate that the
expression of SLT-1 receptors on cells is related to but not linearly
correlated with their sensitivity to the toxin.
Flow cytometry was performed on 134 tumor samples from patients with
hematological cancers stained with FITC-SLT-B. SLT-1 receptors are
predominantly expressed on follicle center cell lymphoma, diffuse large
B-cell lymphoma, and small lymphocytic lymphoma with or without chronic
lymphocytic leukemia. SLT-1 receptors are expressed on B-CLL/SL samples
in agreement with the expression of CD77 reported by
others.43,49-52 Previous work indicated the expression of
CD77 on cell lines established from multiple myeloma patients.53 We detected high levels of CD77 on BM plasma
cells from 5 multiple myeloma patients. Circulating clonotypic B cells, previously shown to be part of the myeloma clone,23-25,39
also bind SLT-1.
To determine whether SLT-1 might be an effective purging agent in
lymphoma and multiple myeloma, fresh patient cells were treated with
SLT-1 and purging was assessed using flow cytometry. We found that the
concentrations of SLT-1 required to mediate toxic effects on primary
malignant B cells (5 µg/mL) was considerably higher than that
required to kill breast cancer cell lines. For 2 lymphoma node biopsies
and B-CLL cells from 1 patient, substantial B-cell cytotoxicity was
detected 6 to 10 days after a pretreatment period with SLT-1. In the
case of multiple myeloma, pretreatment with SLT-1 mediated cytotoxicity
at days 6 to 10 for both circulating B cells and BM-localized plasma
cells. The prolonged delay in observing SLT-1-initiated cell death in
vitro implies that, in the case of ex vivo purging protocols, cellular
events leading to purging may occur after reinfusion. A more accurate
determination of the log kill of myeloma plasma cells after SLT-1
pretreatment was achieved by using a novel cellular limiting dilution
analysis for clonotypic transcripts.24 This RT-PCR assay
uses primers specific for the unique IgH rearrangement that
characterizes the myeloma clone in each patient. In untreated control
cultures of myeloma BM, essentially 100% of the cells were clonotypic.
However, in the SLT-1-treated cultures at 6 days of culture, the
limiting dilution analysis indicated that no clonotypic cells were
detectable, indicating a frequency of less than 1/3,000. Thus, SLT-1
pretreatment depleted myeloma plasma cells by greater than 3 log units.
Taken together with recent work showing that G-CSF-mobilized blood
from myeloma patients includes myeloma progenitors (Pilarski et al, manuscript submitted), these data suggest that SLT-1
purging of autologous stem cell transplants for multiple myeloma
patients may improve their survival.54
Hematopoietic stem cells in BM or peripheral blood represent a
subpopulation of CD34+ progenitor cells capable of
long-term repopulation of the immune and hematopoietic systems. The
absence of CD77 on CD34+ cells, early committed
progenitors, or circulating B and T cells suggests that they may resist
the effects of SLT-1. The expression of CD77 on hematopoietic cell
lineages has been primarily limited to a small number of activated T
cells and follicle center B cells and may be also a minor determinant
on monocytes and erythrocytes.55-58 Platelets may also be
CD77+.11,59 Colony-forming
unit-granulocyte-macrophage (CFU-GM) or CFU-C are
resistant to SLT-1, but a slight decrease in burst-forming unit-erythroid (BFU-E) counts7 suggests some sensitivity to the toxin. The survival of B cells from a posttransplant myeloma patient who had no detectable circulating B cells expressing the myeloma IgH clonotype indicates that normal human B cells escape SLT-1
toxicity. Using phenotypic analysis, we show here that normal HPC lack
the receptor for SLT-1. However, a key criterion for purging tumor
cells from stem cell grafts is that the purging agent must not damage
graft repopulating activity, making a functional assay for HPC mandatory.
To test directly the effects of SLT-1 on hematopoietic progenitor
cells, we pretreated MNC from G-CSF-mobilized blood with a
concentration of toxin shown to be toxic for primary malignant B cells
(5 µg/mL) and analyzed the survival of HPC, as measured by phenotypic
analysis. For unfractionated MNC from mobilized blood of cancer
patients, the numbers of CD34+45low HPC in
untreated and SLT-1-treated cultures were identical. For HPC-enriched
cells, pretreatment with SLT-1 resulted in even greater enrichment of
surviving HPC at day 10 of culture, indicating that HPC escape SLT-1
toxicity. However, ultimately, clinical use of SLT-1 requires a
demonstration that the functional activity of treated HPC is
unimpaired. To address this issue, enriched HPC from mobilized blood
were pretreated with SLT-1 and the number of CFU were enumerated.
Equivalent numbers of CFU were observed from either untreated or
SLT-1-pretreated HPC. A limiting dilution analysis of CFU indicated
that, for SLT-1-pretreated enriched HPC, the frequency of CFU was
1/12, identical to that in untreated control cultures. These findings
indicate that CFU also escape any SLT-1 toxicity. Thus, ex vivo purging
with SLT-1 appears to be clinically feasible. Transplantation of
toxin-treated and washed murine BM cells into SCID mice did not result
in toxicity to the host, suggesting that the levels of residual toxin
in the graft after washing steps are low.7
In conclusion, we have demonstrated that SLT-1 receptors are found on
the surface of breast cancer, B lymphoma, B-CLL, and myeloma B and
plasma cells. Malignant B cells in lymphoma, CLL, and myeloma were
effectively purged by a pretreatment with SLT-1. A greater than 3 log
depletion of clonogenic myeloma cells could be achieved with the toxin,
as monitored by RT-PCR in the case of 1 myeloma patient. Phenotypic
analysis and functional CFU assays conclusively show that hematopoietic
progenitors escape SLT-1 toxicity. The presence of SLT-1 receptors on
breast cancer, follicle center cell lymphoma, and multiple myeloma
cells, together with the demonstrated lack of SLT-1 toxicity towards
CD34+ stem cells, suggests a potential for SLT-1 as an ex
vivo purging agent in removing tumor cells from autologous stem cell grafts.
| |
ACKNOWLEDGMENT |
The authors thank Papar Laraya, Rose Hurren, Marijke Koekebakker,
Lori-Ann Webster, James Ho, Juliet Sheldon, and Denis Bouchard for
their technical help; Dr Hans Messner and Nazir Jamal for providing
peripheral blood stem cells; and Drs Brent Zanke and Heather Lochnan
for their helpful comments. In Edmonton, Eva Pruski provided
outstanding technical assistance, Dr Robert Coupland provided biopsies
of B-lymphoma nodes, and the Red Cross Apheresis Unit provided aliquots
of mobilized blood. We thank Agnieszka J. Szczepek for sequencing the
RT-PCR products shown in Fig 8. This work is dedicated to the memory of
Dr Ron Buick (1948-1996) for all his encouragement, advice, and support
over the years.
| |
FOOTNOTES |
Submitted September 30, 1998; accepted May 30, 1999.
Supported by a translational research grant to J.G. from the Leukemia
Society of America and by a grant from the Medical Research Council of
Canada to L.M.P. and A.R.B. E.C.L. was the recipient of a fellowship
from the Medical Research Council of Canada.
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 Jean Gariépy, PhD, Department of
Medical Biophysics, Room 7-117, Ontario Cancer Institute/Princess
Margaret Hospital, 610 University Ave, Toronto, ON, Canada, M5G 2M9.
| |
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Purging of Autologous Peripheral-Blood Stem Cells Using CD34 Selection Does Not Improve Overall or Progression-Free Survival After High-Dose Chemotherapy for Multiple Myeloma: Results of a Multicenter Randomized Controlled Trial
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A. Suzuki, H. Doi, F. Matsuzawa, S.-i. Aikawa, K. Takiguchi, H. Kawano, M. Hayashida, and S. Ohno
Bcl-2 antiapoptotic protein mediates verotoxin II-induced cell death: possible association between Bcl-2 and tissue failure by E. coli O157:H7
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TOP
ABSTRACT
INTRODUCTION