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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 1 (July 1), 1999:
pp. 52-61
Engraftment of MDR1 and NeoR Gene-Transduced Hematopoietic
Cells After Breast Cancer Chemotherapy
By
Jeffrey A. Moscow,
Hui Huang,
Charles Carter,
Kenneth Hines,
JoAnne Zujewski,
Georgie Cusack,
Cathy Chow,
David Venzon,
Brian Sorrentino,
Yawen Chiang,
Barry Goldspiel,
Susan Leitman,
Elizabeth J. Read,
Andrea Abati,
Michael M. Gottesman,
Ira Pastan,
Stephanie Sellers,
Cynthia Dunbar, and
Kenneth H. Cowan
From the National Cancer Institute, National Heart, Lung and Blood
Institute, and the Clinical Center, National Institutes of Health,
Bethesda, MD; St Jude's Children Research Center, Memphis, TN; and
Genetic Therapy, Inc, a Novartis company, Gaithersburg, MD.
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ABSTRACT |
To determine whether the multidrug resistance gene MDR1
could act as a selectable marker in human subjects, we studied
engraftment of peripheral blood progenitor cells (PBPCs) transduced
with either MDR1 or the bacterial NeoR gene in six breast
cancer patients. This study differed from previous MDR1 gene
therapy studies in that patients received only PBPCs incubated in
retroviral supernatants (no nonmanipulated PBPCs were infused),
transduction of PBPCs was supported with autologous bone
marrow stroma without additional cytokines, and a control gene (NeoR)
was used for comparison with MDR1. Transduced PBPCs were
infused after high-dose alkylating agent therapy and before
chemotherapy with MDR-substrate drugs. We found that hematopoietic
reconstitution can occur using only PBPCs incubated ex vivo, that the
MDR1 gene product may play a role in engraftment, and that
chemotherapy may selectively expand MDR1 gene-transduced
hematopoietic cells relative to NeoR transduced cells in some patients.
This is a US government work. There are no restrictions on its use.
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INTRODUCTION |
THE ABILITY TO DEVELOP cellular
resistance to anticancer drugs is a property of tumor cells but not of
normal cells, including hematopoietic progenitor cells. Although tumor
cell populations progressively recruit and use mechanisms of drug
resistance, the ability of hematopoietic cells to withstand the toxic
effects of chemotherapy seems to diminish. Thus, the clinical response to chemotherapy in the treatment of recurrent cancer often results in
the familiar pattern of dose reduction and disease progression.
Gene-therapy techniques offer the potential to transfer the drug
resistance mechanisms used by tumors into hematopoietic progenitor cells.1-3 In vitro studies have shown that transgenic
expression of the MDR1 gene, which encodes the drug-efflux pump
P-glycoprotein, can confer a characteristic pattern of pleiotropic drug
resistance to previously drug-sensitive cells.4,5
Utilization of the MDR1 gene for hematopoietic cell protection
is attractive in the context of breast cancer therapy because the
MDR1 gene can confer resistance to several of the active
chemotherapeutic agents used in the treatment of this disease,
including paclitaxel and doxorubicin, both of which have considerable
hematopoietic toxicity.
The ability of this gene to protect hematopoietic progenitor cells from
chemotherapy-induced myelotoxicity was first shown in transgenic mice
in which the human MDR1 cDNA was constitutively expressed in
hematopoietic cells and the animals were shown to be protected from the
myelosuppressive effects of chemotherapeutic drugs.6
Several murine models have since shown that direct retroviral-mediated
gene transfer of MDR1 could be used to introduce and express
this gene in hematopoietic cells.7-10 Results from these
studies showed that hematopoietic cells transduced with the
MDR1 gene have preferential survival after treatment of the animals with MDR drugs. Therefore, because the MDR1 gene can
protect blood-forming cells from toxicity, it may also serve as an in vivo selectable marker that could be used to selectively expand the
numbers of hematopoietic cells containing this transgene.
Several groups have conducted clinical MDR1 gene therapy trials
to examine whether the MDR1 gene could act as a selectable marker in the clinical setting.11-13 In each of these
studies, cancer patients received a conditioning regimen of
myeloablative chemotherapy and subsequent autologous transplantation
with MDR1-transduced hematopoietic stem cells (HSCs)
supplemented with genetically unmanipulated HSCs. Two of these trials
did not present information regarding the effects of MDR1 gene
marking on subsequent chemotherapy-induced myelotoxicity. In the third
study there was some suggestion of possible protection from
chemotherapy-induced myelotoxicity despite low levels of MDR1
gene marking in vivo; however, too few patients were studied to draw
any conclusions.13 In all three MDR1 gene-therapy trials, the level of gene marking detected at the time of
reconstitution has been low (<5%), thus limiting any potential
benefit from the myelosuppressive effects of MDR
drugs.11-13
In the current study, we examined the feasibility of reconstituting
patients with hematopoietic cells exposed to retroviral transduction
conditions without simultaneous reinfusion of nonmanipulated hematopoietic progenitor cells. The ex vivo culture conditions necessary for viral transduction may lead to engraftment
defects,14 and thus give the nonmanipulated cells a
significant advantage in competition for engraftment. The conditioning
regimen in this study, single-agent thiotepa (350 mg/m2),
was chosen to be less intensive than previously used high-dose chemotherapy regimens, such as ifosfamide-carboplatin-etoposide (ICE), to obviate the need for simultaneous reinfusion of
unmanipulated peripheral blood progenitor cells (PBPCs). Therefore,
this study differed from previous MDR1 gene-transfer studies in
that hematopoietic rescue depended solely on PBPCs that had been
incubated in retroviral supernatants.
The ability of the MDR1 gene to protect hematopoietic cells
from the myelotoxicity of anticancer drugs was determined by
reconstituting patients with gene-modified CD34+ cells, one
half of which were transduced with a retroviral vector containing the
MDR1 multidrug resistance gene (G1MD), while the other half
were transduced with a retroviral vector containing the neomycin
resistance (NeoR) gene (G1Na.40). The transductions were supported with
autologous irradiated stroma, unlike previous clinical MDR1
gene-therapy studies which have relied on cytokines to support viral
transduction. After recovery, patients received four cycles of
paclitaxel followed by four cycles of either doxorubicin or
vinblastine, all of which are known substrates for the MDR1 drug efflux
pump. The comparison of the levels of MDR1 to NeoR transgenes
in peripheral blood cells was used to determine whether PBPCs
containing the MDR1 gene were selectively protected from myelotoxic effects of MDR1 substrate chemotherapy.
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PATIENTS AND METHODS |
Patient selection.
The treatment protocol MB361 was approved by the National Institutes of
Health (NIH) Institutional Biosafety Committee, the NIH Recombinant DNA
Advisory Committee, and the National Cancer Institute (NCI)
Institutional Review Board. Patients with metastatic breast cancer were
eligible for the study. Patients with metastatic breast cancer who
declined to participate in the gene-therapy procedures and other
patients with high-risk breast cancer were offered enrollment on
another study that used the same chemotherapy schema.
Treatment plan.
Before chemotherapy, bone marrow aspirates were obtained for diagnostic
purposes and to establish autologous stromal cell cultures using
procedures previously described.15 The protocol chemotherapy schema is shown in Fig 1A.
Patients were first treated with four 2-week cycles of MLF, which
consisted of sequential methotrexate (250 mg/m2 intravenous
[IV] at hour 0), 5-fluorouracil (500 mg/m2 IV at hours 4 and 25), and leucovorin (250 mg/m2 at hour 24). At least 14 days after the last MLF cycle, when the absolute neutrophil count
(ANC) exceeded 1,200/µL and platelets exceeded 90,000/µL, patients were then treated with two 3-week cycles
of cyclophosphamide (4 g/m2 over 4 hours) with mesna
2-mercaptoethanesulfonate uroprotection. Patients received filgrastim
10 µg/kg/d beginning on day 2 of the first cyclophosphamide cycle and
underwent apheresis when the white blood cell (WBC) count increased to
greater than 5,000 cells/µL after reaching the nadir level.
Peripheral blood progenitor cells were harvested during recovery from
cyclophosphamide using a Fenwal CS3000 (Baxter Corp, Deerfield, IL) or
a Cobe Spectra (COBE, Lakewood, CO) apheresis device. All patients
underwent two apheresis procedures each, with a mean of 15.2 L of blood processed per procedure. Adequate numbers of PBPCs were collected in
all gene-therapy patients after the first cyclophosphamide cycle. The
collected mononuclear cells were CD34+ enriched using a
Ceprate stem cell concentrator column (Cellpro, Bothell, WA) according
to the manufacturer's instructions.16 An aliquot (5 × 106) of CD34-selected cells was examined for breast
cancer cell contamination by immunohistochemistry using a panel of
anti-keratin antibodies. All of the CD34+ samples were
negative for tumor cell contamination, with the limit of detection of 1 tumor cell in 105 CD34+ cells. At least 1.5 × 106 CD34-selected cells per kilogram were stored
without further manipulation for hematopoietic rescue in the event of
engraftment failure. These cells were placed in freezing medium
containing 50% human AB serum, 40% Plasmalyte-A (Baxter), 10%
dimethyl sulfoxide (DMSO; Tera Pharmaceuticals, Salt Lake City, UT), 10 µg/mL Darnase (Genentech, Thousand Oaks, CA), and 15 U/mL heparin
(Fujisawa, Deerfield, IL). The cells were cryopreserved in a controlled
rate freezer and stored in liquid nitrogen.
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| Fig 1.
(A) MB361 chemotherapy schema. Bone marrow aspirate for
stroma culture was obtained before the first cycle of chemotherapy.
Vinblastine was substituted for doxorubicin after patients exceeded a
lifetime cumulative doxorubicin dose of 550 mg/m2. (B) A
diagram of the PCR assay showing the position of the primers used to
detect the MDR1 transgene and the relative positions of their
annealing sequences on the human genome. The numbering system shows
that of the G1MD vector. The MDR1 cDNA starts at nt 1479 and
ends at nt 5339. The MDR1 ATG start codon is at position
1491.
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After recovery from the second cyclophosphamide cycle, patients were
treated with thiotepa (350 mg/m2 on day 1) followed by
reinfusion on day 4 of the cryopreserved PBPCs that had been incubated
in viral supernatant. Patients subsequently received daily injections
of filgrastim (5 µg/kg). After hematologic recovery, patients
received four 3-week cycles of paclitaxel (175 mg/m2 IV
over 24 hours) followed by four 3-week cycles of doxorubicin (60 mg/m2). Vinblastine (1.5 mg/m2/d for 5 days)
was administered in place of doxorubicin when the total lifetime
doxorubicin dose exceeded 550 mg/m2.
Transduction of hematopoietic cells.
For retroviral transductions, CD34+ cells were collected by
centrifugation at 290g for 10 minutes and at a concentration of 1 to 3 × 105 cells/mL in two equal portions of
transduction medium (TM). TM consisted of three parts Dulbecco's
Modified Eagles Medium (GIBCO, Grand Island, NY) supplemented with 10%
fetal bovine serum (Biowhittaker, Walkersville, MD) and either 1 part
supernatant from amphotrophic retroviral vector producer cell line G1MD
clone 5 (0.5 to 0.6 × 106 vector/mL) or 1 part
supernatant from amphotrophic retroviral G1Na (NeoR) vector producer
cell line (3.5 to 5.1 × 106 vector/mL) (Genetic
Therapy Inc, Gaithersburg, MD). TM was also supplemented with 4 µg/mL
protamine sulfate (Fujisawa, Deerfield, MI). Autologous bone marrow
stromal cells (0.5 to 2.0 × 106 cells) from each
patient were plated in 162-cm2 flasks (Costar, Cambridge,
MA) in DMEM containing 10% fetal bovine serum and incubated overnight
at 37°C and then irradiated (1,000 cGy). CD34+ cells (1 to 3 × 105 /mL) in TM containing supernatants from
retroviral producer cell lines were placed in flasks containing
irradiated autologous stromal cells and incubated at 37°C in 5%
CO2 for 18 to 24 hours. At 24 and 48 hours the nonadherent
cells were collected by centrifugation at 745g for 10 minutes,
resuspended in fresh TM (containing fresh G1MD or G1Na supernatant),
and incubated at 37°C in 5% CO2. Adherent cells were
harvested at 72 hours by incubation with Trypsin-EDTA (GIBCO-BRL,
Gaithersburg, MD), combined with nonadherent cells and
collected by centrifugation at 745g for 10 minutes. Aliquots were taken for polymerase chain reaction (PCR) analysis of G1MD and
G1Na vector DNA and for testing for bacterial and fungal contamination. The remaining cells were resuspended in freezing medium and stored in
liquid nitrogen.
Detection of transgenes in hematopoietic cells.
A sample of peripheral blood was collected before each cycle and was
used for isolation of mononuclear and polymorphonuclear cells. A double
gradient was formed by layering an equal volume of HISTOPAQUE-1077 over
HISTOPAQUE-1119 (Sigma, St Louis, MO). Whole blood was carefully
layered onto the upper HISTOPAQUE-1077 medium, followed by
centrifugation at 700g for 30 minutes. Mononuclear cells were
found at the plasma/1077 interface whereas cells of the granulocytic
series were found at the 1077/1119 interface. The two layers were
harvested, washed with HEPES-Buffered Standard Saline (Biofluids,
Rockville, MD), and collected by centrifugation for 10 minutes at
700g. Aliquots of cell preparations were counted with a
hematocytometer and stained with Hema stain (Biochemical Sciences, Inc,
Swedesboro, NJ) to document the purity of the collection. The remaining
cells were concentrated again by centrifugation and resuspended in a
cell lysis solution (Gentra Systems Inc, Research Triangle Park, NC).
All samples were stored at  80°C until DNA extraction.
DNA was isolated from the granulocytes and monocytes using the Puregene
DNA isolation kit (Gentra Systems Inc). After the frozen samples were
thawed and incubated with RNase A, protein was precipitated from the
lysate and pelleted by centrifugation. The supernatant was collected,
added to 3 vol of isopropyl alcohol, and the DNA pellet was then formed
by centrifugation at 15,000g for 1 minute. The DNA pellet was
washed once with 70% alcohol, resuspended in DNA hydration solution,
and the concentration determined by ultraviolet spectrophotometry.
Care was taken to avoid potential contamination of patient specimens
with PCR products. DNA extraction and PCR assays were performed in
separate rooms. Vector DNA was detected with a PCR assay using the
following MDR1 primers which span nucleotides (nt) 3737 to 4436 (exons 18 to 23): 5'-TGA AAC AAA ACG ACA GAA TAG TAA C-3',
5'-AAT ACT AAC AGA ACA TCC TCA AAG C-3'). The amplified fragment is 699 bp (see Fig 1B). These primers span more than 10 kb on
the MDR1 genomic gene17 and were chosen to reduce
the possibility of amplification of endogenous genomic MDR1
sequences. Previous studies have shown that, due to cryptic splice
sites within the MDR1 cDNA, G1MD producer cell lines generate
both full-length and spliced transcripts resulting in full-length and
truncated provirus in target cells.18 The shortened
provirus produces a truncated MDR1 mRNA that results in a large
deletion of coding sequences. The upstream G1MD primer used for viral
detection in these studies is located just upstream of the cryptic
splice acceptor site. Therefore, the PCR assay recognizes only
full-length proviral sequences. The reaction mixtures were incubated at
94°C for 3 minutes, followed by 40 cycles at 94°C for 30 seconds, 58°C for 20 seconds, 72°C for 50 seconds, and a final
extension of 10 minutes at 72°C.
Semiquantitative estimates of the relative percent of transgene copy
number were made by comparing the relative signal intensity of the G1MD
PCR reaction from patient-derived DNA with the relative signal
intensity obtained from a control G1MD dilution series. The dilution
series was made from DNA extracted from a cloned MCF-7 breast cancer
cell line containing a single copy of the MDR1 transgene per
cell that was serially diluted in DNA extracted from nontransduced
CD34+ cells. These serial dilutions of control DNA were
amplified by PCR at the same time as patient samples. PCR products (10 µL) were separated by electrophoresis on a 2% 3:1 agarose gel,
stained with SYBR Green (FMC, Rockland, ME), photographed using an
electronic camera, and the band intensity was determined using NIH
Image software. The densitometry of the dilution series was measured and served as a reference for the relative amount of the MDR1 transgene contained in hematopoietic cells obtained from patients.
A nested MDR1 PCR assay was developed using primers located
within the first amplified PCR fragment and which span nt 3808 to 4171 and exons 18 to 21 (5'-CAT TTT TCC TTC AGG GTT TCA C-3', 5'-GTT CTT TCT TAT CTT TCA GTG CTT G-3'). This primer pair
produces a 363-bp product (see Fig 1B). The PCR conditions for the
nested MDR1 reaction were 94°C for 3 minutes, then 25 cycles at 94°C for 30 seconds, 58°C for 20 seconds, 72°C
for 30 seconds, and a final extension of 10 minutes at 72°C. The
PCR reaction for actin was performed as a control at the same time
using primers 5'-CAT TGT GAT GGA CTC CGG AGA CGG-3' and
5'-CAT CTC CTG CTC GAA GTC TAG AGC-3' and used the same
conditions as the initial MDR1 PCR reaction. The limit of
detection of the nested PCR assay was .01% G1MD vector copy per cell.
PCR for the NeoR gene was performed using the
NeoR outer primers (5'-GGC CAG ACT GTT ACC ACT
CC-3', 5'-CAG CCG ATT GTC TGT TGT GC-3') and nested
primers (5'-CGG ATC GCT CAC AAC CAG TC-3', 5'-AGC CGA
ATA GCC TCT CCA CC-3') and gave a band size of 502 bp.19 The reaction conditions were 95°C for 2 minutes,
20 cycles of 95°C for 1 minute, 60°C for 1.5 minutes, 72°C
for 2 minutes for the first PCR and 26 cycles of 95°C for 1 minute,
60°C for 1.5 minutes, 72°C for 2 minutes for the nested PCR,
followed by a final extension of 8 minutes at 72°C. In some
reactions, [32P]dCTP was added to the nested PCR
reaction. PCR products (10 µL) were separated by electrophoresis on a
2% 3:1 agarose gel, stained with SYBR Green (FMC) and photographed
using a CCD camera. Band intensity was determined using NIH Image
software. Radiolabeled PCR fragments were detected by autoradiography.
The limit of detection of the PCR assay was .01% G1Na vector copy per cell.
Detection of helper virus.
Posttransplantation peripheral blood mononuclear cellular DNA was
screened by PCR for recombinant helper virus genome as previously described.19 Conditions for amplification were 95°C for
2 minutes, followed by 25 cycles at 95°C for 1 minute and 72°C
for 1.5 minutes, and final extension at 72°C for 10 minutes.
Retroviral supernatants were also tested for replication-competent
helper virus using coculture amplification on mus dunni cell
line.20 All samples were negative for helper virus.
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RESULTS |
The protocol chemotherapy schema is shown in Fig 1A. Of the first nine
patients to enroll in the study, six patients underwent PBPC
harvest, ex vivo retroviral transduction with the MDR1
and NeoR vectors, reinfusion after thiotepa chemotherapy, and
completion of eight subsequent cycles of chemotherapy with the MDR
drugs paclitaxel, doxorubicin, or vinblastine. Two patients did not complete the study because their bone marrow stromal cells harvested before the beginning of chemotherapy did not grow in vitro, and one
patient withdrew consent. The characteristics of the patients who
received MDR1 and NeoR gene-transduced CD34+ cells
are shown in Table 1.
The PBPCs used for retroviral transductions were harvested during the
first cycle of cyclophosphamide treatment. Apheresis was initiated when
the WBC count first exceeded 5,000 cells/µL, and all six patients
underwent two apheresis procedures each. As shown in
Table 2, the mean blood CD34 cell count
increased from 28.9 cells/µL to 112.6 cells/µL from the first to
the second day of apheresis. The corresponding increase in the WBC
count was from 6,850 cells/µL on day 1 to 18,500 cells/µL on day 2 of apheresis. The stem cell collections were thus performed during the
period of exponential increase in the CD34 count in the peripheral blood.
For each patient an aliquot of 1.5 × 106
CD34-selected cells/kg was incubated separately with each vector (G1MD
and G1Na). Because the purity of the CD34 selected product was
approximately 80%, the average number of CD34 cells incubated in each
vector was approximately 1.2 × 106 CD34+
cells/kg. After 3 days in culture in the presence of viral supernatant and autologous irradiated stroma, the number of surviving CD34 cells
decreased to approximately 65% of the original number. Therefore, as
shown in Table 3, the mean total number of
CD34+ cells available for hematopoietic rescue after
thiotepa chemotherapy was only 1.5 × 106
CD34+ cells/kg (G1MD, 0.78 × 106
CD34+ cells/kg; G1Na, 0.74 × 106
CD34+ cells/ kg). In contrast to other MDR1
gene-therapy protocols,10-13 in this trial we did not
reinfuse any PBPCs that had not been cultured in viral supernatant.
In this study retroviral transductions were supported solely with
irradiated, autologous stroma; we did not add any additional cytokines
to the culture. To determine the relative transduction efficiency of
cells after incubation with retroviral supernatants for 72 hours, DNA
obtained from cells at the end of the transduction period was analyzed
by PCR for the presence of vector DNA and compared to a serial dilution
of DNA from a control cell line containing a single copy of G1MD or
G1NA vector per cell. As shown in Table 3, the mean transduction
efficiency after incubation with G1MD vector was estimated to be
0.26%, while the mean transduction efficiency of cells incubated with
G1Na vector was 30.3%. This difference in transduction efficiency
between the two vectors was due in part to the 6- to 10-fold difference
in titer of viral supernatants for G1MD (0.5 to 0.6 × 106 vector/mL) and G1Na (3.5 to 5.1 × 106
vector/mL), and in part to a cryptic splice site in the G1MD vector
that renders half of the G1MD proviruses undetectable by our PCR assay
due to a large deletion of the MDR1 coding region (see Patients
and Methods). The difference in transduction efficiency resulted in a
100-fold difference in the dose of CD34+ cells containing
the two vectors; the mean total estimated dose containing full-length
G1MD-transduced CD34+ cells was 0.19 × 104 cells/kg (range, 0.17 to 0.22 × 104
cells/kg), while the mean number of reinfused NeoR-containing CD34+ cells was 19.8 × 104 cells/kg
(range, 8.1 to 30.5 × 104/kg cells/kg).
Despite the low number of CD34+ cells reinfused which
carried the MDR1 transgene, we were able to detect the presence
of the MDR1 transgene after hematopoietic recovery in all 6 patients (Fig 2). In patient 1, no G1MD
vector DNA signal was detectable in granulocytes immediately after
hematopoietic recovery from high-dose thiotepa chemotherapy (the limit
of detection being 0.01% relative gene copy number), nor was vector
DNA detected after treatment with four cycles of paclitaxel (pre-cycle
1 through pre-cycle 5). However, G1MD vector DNA was apparent in
granulocytes after two cycles of doxorubicin (pre-cycle 6 and pre-cycle
7) and after treatment with two cycles of vinblastine (pre-8 and post-8). MDR1 and NeoR transgene levels in polymorphonuclear
cells of the next five patients are also shown in Fig 2. In the second patient, the MDR1 transgene was not detected immediately after hematologic recovery after infusion of G1MD vector-marked cells, but
it was detected in granulocytes after the third cycle of paclitaxel and
following treatment with four cycles of doxorubicin (pre-cycle 4 through post-cycle 8). In patient 3, no G1MD vector was detectable in
granulocytes at hematopoietic reconstitution (pre-cycle 1), but it was
detected after the second cycle of paclitaxel. In this patient there
was no detectable G1MD vector DNA in granulocytes after the fourth
cycle of paclitaxel chemotherapy. For patients 4, 5, and 6, the G1MD
transgene was initially present in granulocytes (Fig 2) after
hematological recovery from thiotepa, but it was lost during the course
of therapy with paclitaxel, and remained undetectable after therapy
with either doxorubicin or vinblastine. A similar pattern in
MDR1 transgene detection was seen in the examination of
monocytes (Fig 3).
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| Fig 2.
PCR assay showing the presence of MDR1 and NeoR
transgene in granulocytes for all patients enrolled on the study. In
each set of MDR1 panels, the top gel shows the nested
MDR1 primers, and the bottom panels show the control for the
amount of DNA placed in PCR reaction using primers for the actin gene.
The NeoR gels show the results for the nested NeoR primers.
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| Fig 3.
PCR assay showing the presence of the MDR1
transgene in monocytes obtained from the patients enrolled in the
study. In each set of MDR1 panels, the top gel shows the nested
MDR1 primers, and the bottom panel shows the control for the
amount of DNA placed in PCR reaction using primers for the actin
gene.
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By reinfusing an equal number of PBPCs transduced with the control gene
NeoR in the study design, we were able to discern the specific effect
of transgenic expression of the MDR1 gene on both engraftment
and on subsequent response of transduced cells to chemotherapy. Despite
the much higher transduction efficiency of the G1Na vector, the NeoR
transgene was only faintly detectable (=.01% NeoR gene copy) in
granulocytes of three patients (Fig 2). In patients 1 and 5, G1Na
vector DNA signal was seen only in peripheral granulocytes obtained at
hematopoietic reconstitution from high-dose thiotepa (pre-cycle 1), but
it was undetectable thereafter. In patient 4, a low level of G1Na
marking was observed intermittently after treatment with paclitaxel and
doxorubicin (pre-cycles 4, 6, and 8, and post-cycle 8), suggesting that
the level of marking was constant and near the limit of detection of
0.01%.
Semiquantitative PCR analysis was used to determine the level of G1MD-
and G1Na-transduced granulocytes in each patient at the time of
hematopoietic reconstitution and after each cycle of MDR chemotherapy
by comparing the PCR signal strength of the transgene with serial
dilutions of DNA from a control cell line containing a single vector
copy per cell. As shown in Fig 4A, of the
three patients that had undetectable levels (<.01%) of G1MD vector
in granulocytes at the time of hematopoietic reconstitution, all three
patients had increased G1MD marking at some time after chemotherapy to
levels >1.0% (=1 G1MD vector per 100 cells). One of the other three
patients with low levels of gene marking at the time of reconstitution
became negative after the first cycle of chemotherapy, while the other
two patients who were initially G1MD positive remained at a low level
of G1MD marking during the first cycle of chemotherapy and then
decreased to an undetectable level. In contrast, as shown in Fig 4B,
only one patient converted from undetectable marking with G1Na at
hematopoietic reconstitution to a positive level of marking on
chemotherapy, but the highest level achieved was only 0.01%.
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| Fig 4.
Semiquantitative PCR analysis G1MD (A) and G1Na (B)
marking in granulocytes at hematopoietic recovery and during MDR
chemotherapy. Semiquantitative PCR analysis for G1MD and G1Na was
performed on DNA from granulocytes obtained at hematopoietic
reconstitution and during each cycle of chemotherapy. The maximal level
of marking for each vector at hematopoietic recovery and at any time
during chemotherapy is depicted for each patient. The patient samples
are referred to by patient number.
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This is the first study of hematopoietic gene therapy in which the
PBPCs that had been cultured in viral supernatant were not supplemented
with unmanipulated PBPCs before infusion in patients. Therefore,
hematological recovery of these patients from high dose thiotepa is of
interest. Since we contemporaneously enrolled other breast cancer
patients (high-risk stages II, III, and IV) on a study (MB381) which
used the same chemotherapy schema as was used in the gene therapy study
(MB361), we compared the hematologic recovery of the gene-therapy
patients with patients receiving the same chemotherapy, but whose PBPC
collections were not incubated in viral supernatant after CD34
selection. As shown in Table 4, patients
enrolled on MB361 received a lower dose of PBPCs owing to the loss of
CD34 cells during the retroviral transduction process, with the gene
therapy patients receiving a median dose of 1.4 × 106
CD34+ cell/kg compared with a median CD34+ dose
of 2.4 × 106 cells/kg for the non-gene-therapy
patients. Nevertheless, there was little difference in the median time
to recovery of a granulocyte count >500 cells/µL (11.0 ± 0.9 v 10.0 ± 0.4 days) or to a granulocyte count of
>1,200/µL (12.5 ± 0.8 v 11.0 ± 0.8 days),
and only a 2.5-day difference (14.5 ± 2.7 v 12.0 ± 1.5 days) in time to platelet count >20,000/µL (without requiring
platelet transfusions). One gene-therapy patient and two
non-gene-therapy patients required reinfusion of additional,
unmanipulated stored PBPCs to aid hematological recovery (granulocytes
>1,200/µL and platelets >90,000/µL) to continue the
chemotherapy regimen.
All six gene-therapy patients were treated for metastatic breast
cancer. Two of the six patients had measurable disease at the time of
enrollment, and both of these patients achieved a partial response with
the chemotherapy regimen (see Table 1). All of the other four patients
had evaluable disease only and showed improvement with chemotherapy.
However, these patients were unevaluable for overall response. All six
patients completed the chemotherapy regimen, which took approximately
11 months from the time of enrollment on the study.
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DISCUSSION |
In this report, we have explored the concept of using gene therapy to
enhance the effectiveness of anticancer chemotherapy by imparting drug
resistance on normal tissues.1-3 This strategy holds out
the promise not only of making bone marrow cells more resistant to
chemotherapy, but also of exploiting drug-resistance genes to help
deliver and expand cells cotransduced with other therapeutic genes that
could be used in the treatment of both cancer and nonmalignant disorders.
Previous clinical studies of MDR1 gene transfer into
hematopoietic stem cells have shown that patients can be reconstituted with MDR1 gene-marked hematopoietic cells. However, the levels of gene marking in the previous studies were low.11-13 Two
factors common to each of these trials may have contributed to this
outcome. First, to promote proviral integration of the retroviral
transgene, ex vivo culture conditions included incubation of
CD34+ cells with the cytokines interleukin-3 (IL-3), IL-6,
and stem cell factor; these cytokines stimulate quiescent, pluripotent hematopoietic cells to enter the cell cycle and become susceptible to
retroviral integration into the host cell genome. Incubation of PBPCs
under these conditions can result in expansion of the number of cells
in culture after the incubation period, increased retroviral
transduction efficiency, and increased number of colony-forming units
(CFUs) containing the transgene. However, animal studies have shown
that incubation of hematopoietic stem cells with these cytokines
results in an engraftment defect.21,22 Furthermore, the
fraction of CFUs containing an MDR1 transgene after incubation in cytokines, which was thought to be a surrogate marker for
transduction of pluripotent stem cells, has not correlated with the
long-term engraftment of hematopoietic cells containing the
transgene.12 In addition, the stimulation of cell division
by these cytokines may have irreversibly placed the primitive stem
cells in the CD34-selected collection on a path toward differentiation
and ultimately apoptosis.
Second, in the interest of safety, previous MDR1 gene-therapy
studies reinfused stored, unmanipulated PBPCs along with the PBPCs
incubated in viral supernatant and cytokines. Uncultured and
unmanipulated PBPCs may have a significant competitive advantage over
cultured PBPCs for engraftment resulting in decreased gene marking
efficiencies in vivo. After reinfusion of equal numbers of cultured and
nonmanipulated hematopoietic progenitors, animal studies indicate that
only one tenth of the cells that repopulate the marrow come from cells
that were incubated in tissue-culture conditions.14
In the current MDR1 gene-transfer clinical study, the trial
design was modified in an attempt to optimize transduction and engraftment of gene-marked pluripotent stem cells. First, the retroviral transduction was supported with autologous, irradiated stroma rather than using the cytokines commonly used for ex vivo transduction IL-3, IL-6, and stem cell factor. Second, we reinfused only PBPCs incubated in retroviral supernatant; we did not supplement the PBPC rescue with stored, unmanipulated PBPCs. Third, the
conditioning regimen in this study was single-agent thiotepa (350 mg/m2). Although this dose of thiotepa produces significant
myelosuppression, it may be less toxic to the bone marrow
microenvironment than standard conditioning regimens. It is possible
that any of these changes may have facilitated MDR1 gene
transfer into a stem cell compartment that could successfully engraft
and expand with subsequent breast cancer chemotherapy.
All six patients entered on this trial had evidence of G1MD marking in
granulocytes during treatment with MDR chemotherapy (paclitaxel,
doxorubicin, and vinblastine). In the first three patients, no G1MD
vector DNA was detected in granulocytes or monocytes at the time of
hematopoietic reconstitution after treatment with thiotepa. However,
G1MD marking became apparent in both granulocytes and monocytes during
treatment with MDR chemotherapy in all three patients. In contrast,
G1MD vector DNA was detected immediately after hematopoietic
reconstitution in patients 4, 5, and 6, but it was lost in both
granulocytes and monocytes early on after treatment in these patients
as well as in patient 3.
These results suggest that in the first two patients, G1MD transduction
occurred in pluripotent stem cells with long-term engrafting potential.
In contrast, G1MD transduction in the other patients most likely
occurred in progenitor cells that were ultimately destined for
elimination by programmed cell death. Although G1MD-transduced cells
may be protected from MDR chemotherapy, the survival of MDR1-transduced hematopoietic progenitor cells in patients is also dependent on the state of differentiation of the hematopoietic cell in which the gene is transduced ex vivo. An alternative
explanation is that the first two patients received paclitaxel at 250 mg/m2 with granulocyte colony-stimulating factor (G-CSF)
support for all four cycles, while the remaining four patients received
paclitaxel at 175 mg/m2 with G-CSF only if needed
(ANC <500/µL for >3 days). This raises the
possibility that the paclitaxel dose may not have been high enough in
the last group of patients to select for the MDR1 transgene, or
that the G-CSF administration resulted in prestimulation of primitive
cells and contributed to the selection of the MDR1 transgene, as has been shown experimentally to be the case with stem cell factor.23
In the current trial we used the NeoR gene (G1Na vector) as a control
to compare two equal populations of CD34+ cells, which
differed only in the gene that was inserted into the host genome.
Although the transduction efficiency of the NeoR vectors under our
culture conditions was almost two orders of magnitude higher than the
transduction efficiency of the MDR1 vector, we were surprised
that we were able to detect the MDR1 transgene in peripheral
blood granulocytes more frequently and at higher levels than the NeoR
gene. All six patients marked for MDR1, whereas only three of
six patients showed NeoR marking. Semiquantitative PCR analysis
indicated that the highest level of NeoR gene marking, obtained in
patient 4, was only 0.01% of total peripheral blood granulocytes.
These results are comparable with previous studies by us using
stroma-supported transduction of NeoR15 (and C.D.,
unpublished observations, 1998): in a total of six
patients, only three showed intermittent NeoR marking and none showed
sustained engraftment of NeoR-marked cells. In comparison, the level of
MDR1 marking observed in all six patients ranged from 0.01% to
1% of the total peripheral blood granulocytes (Fig 4). These results
suggest that the transgenic MDR1 expression may have had a
beneficial effect on hematopoietic cell reconstituting ability, even
when it did not lead to long-term repopulation and expansion with MDR
drug therapy.
These results are consistent with a recent competitive repopulation
experiments in mice by Bunting et al.24 In these studies, murine bone marrow cells transduced with a Harvey-based retroviral vector containing the human MDR1 gene (HaMDR1) during cytokine stimulation showed a substantial increase in multipotent repopulating cells compared to control marrow cells transduced with a retroviral vector containing a mutant dihydrofolate reductase gene.24
The mechanism whereby MDR1 transduction might enhance long-term
engraftment of cells is unclear. Recent studies have indicated that
MDR1 overexpression can protect cells from undergoing
apoptosis.25,26 Thus, MDR1 transduction of
hematopoietic progenitor cells might reduce their susceptibility to
programmed cell death. Alternatively, it is possible that MDR1
overexpression in primitive hematopoietic cells could reduce their
propensity to differentiate toward committed progenitor cells in vitro
and in vivo.24
Bunting et al24 also found that hematopoietic cells
transduced with the Harvey-based HaMDR1 vector and then cultured for an
additional 12 days in the presence of cytokines produced a myeloproliferative disorder in recipient mice which was not observed in
mice reinfused with cells immediately after transduction. The report of
myeloproliferative disorders following reinfusion of cells containing
the MDR1 transgene raises important safety concerns. However,
it is not clear how the prolonged incubation in cytokines and the
Harvey-based vector interacted with MDR1 to produce this effect. The current clinical trial used a Moloney-based retroviral vector for both the MDR1 and NeoR vectors and did not subject cells to prolonged incubation after transduction. No
preclinical6-8,27 or clinical11-13 studies have
reported myeloproliferation in recipients of MDR1 transduced
cells, nor has the NeoR Maloney-based vector been associated with
hematopoietic disorders in recipient patients.19,28
This trial of MDR1 gene therapy occurred in the context of a
pilot trial of prolonged sequential high-dose chemotherapy for breast
cancer. Rather than use autologous stem cell rescue as consolidation,29 we employed a sequential, continuous
chemotherapy strategy that would optimize the utilization of the
MDR1 transgene if improvement in transduction efficiency could
make MDR1-mediated hematopoietic protection clinically
beneficial. The sequential nature of the chemotherapy schema was
suggested by the findings of Bonadonna et al,30 who
demonstrated the superiority of sequential doxorubicin followed by
sequential cyclophosphamide, methotrexate, and 5-fluorouracil, as
opposed to alternating these two regimens, as adjuvant therapy in
breast cancer patients with four or more positive lymph nodes. The
treatment plan began with an antifolate-based induction regimen of
methotrexate, fluorouracil and leucovorin based on a regimen used in
colon cancer.31 We then harvested and reinfused PBPCs in
the context of high- dose alkylating agent chemotherapy. After
engraftment, therapy was consolidated with the MDR1 drugs paclitaxel
and doxorubicin. This treatment design permitted the engraftment of
MDR1 hematopoietic stem cells in patients before the initial
administration of chemotherapeutic agents that are substrates for the
drug-resistance transgene.
This study shows that patients can be reconstituted with PBPCs
incubated in retroviral supernatants after treatment with high-dose thiotepa and that, under these conditions, no unmanipulated cells need
to be included in the stem cell rescue. It is possible that long-term
engraftment resulted primarily from residual hematopoietic cells and
not from the cultured, but not genetically altered, reinfused stem
cells. Thus, it is not known whether long-term engraftment of
gene-marked cells was enhanced or diminished by the use of a
myelotoxic, rather than a myeloablative, preparative regimen.
The design of this study allowed us to examine the effect of
MDR1 both on engraftment as well as on in vivo selection with MDR drugs. In all six patients, the higher level of engraftment of
MDR1-containing cells relative to NeoR, despite a lower transduction efficiency, suggests that MDR1 overexpression may have had a
beneficial effect on the engraftment potential of hematopoietic cells.
However, clear assessment of the potential of the MDR1
transgene for in vivo selection was limited both by the fact that only
two of six patients showed evidence of in vivo expansion, and by the
inability to perform functional studies on the MDR1-containing
engrafted cells due to the low transduction efficiency.
Therefore, although the ability of retroviral vectors containing
MDR1 to confer drug resistance in hematopoietic lineages in
animal models is not in doubt,7-10 the ability of this
transgene to confer clinical drug resistance in human hematopoietic
tissues has yet to be conclusively shown. The low efficiency of
hematopoietic reconstitution of patients with gene-transduced cells
capable of long-term engraftment remains the major limitation to
widespread application of MDR1 gene therapy for therapeutic
benefit. Additional studies with greater numbers of patients are needed
to identify strategies to increase the efficiency of gene transduction
in hematopoietic stem cells with long-term engraftment and self-renewal potential.
| |
FOOTNOTES |
Submitted January 6, 1999; accepted March 8, 1999.
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 Kenneth H. Cowan, MD, PhD, UNMC/Eppley
Cancer Center, Eppley Institute for Research in Cancer, 600 S 42nd St,
Omaha, NE 68198-6085.
| |
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TOP
ABSTRACT
INTRODUCTION