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Experimental Therapeutics |
Department of Molecular Pharmacology [L. M. W., S. M. G., R. A. B., C. L. M., L. C. H., P. J. H., P. M. P., M. K. D.], Animal Resource Center [C. M. S.], and Department of Tumor Cell Biology [R. A. A.], St. Jude Children’s Research Hospital, Memphis, Tennessee 38105
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1% metastatic tumor burden. All eight samples were purged to the level of detection by reverse transcription-PCR (samples from seven patients) or clonogenic potential (sample from one patient), whichever assay was used. The described adenovirus/rabbit carboxylesterase/CPT-11 (irinotecan, 7-ethyl-10[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin) virus-directed enzyme prodrug method may be useful for patients whose tumor burdens exceed 1% at the time of stem cell harvest. Assessment of purging efficacy with additional samples from NB patients is ongoing. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Current purging methods for NB use monoclonal antibodies directed against cell surface markers to isolate either tumor cells (12) or hematopoietic stem cells (8 , 13) . These systems have been shown to reduce the tumor burden of the graft by up to 2–4 logs (8 , 12) . However, these methods appear to be effective only if the tumor burden at the time of stem cell harvest is less than 1%, and even then, residual tumor cells have been detected after purging (3 , 8, 9, 10 , 12, 13, 14) . Our goal was to develop a purging protocol that would be effective in reducing the tumor cell number to a level undetectable by RT-PCR even when the tumor burden exceeded 1%.
The VDEPT method described below uses a replication-deficient Ad to deliver the cDNA encoding rCE (AdRSVrCE) selectively to NB cells. rCE efficiently converts the prodrug CPT-11 (irinotecan) to its active metabolite, SN-38 (7-ethyl-10-hydroxycamptothecin). Human tumor cells that express the rabbit CE are up to 500-fold more sensitive to CPT-11 than cells that express only endogenous human CEs (Ref. 15 ; data not shown), thus potentially providing a favorable therapeutic index. This VDEPT approach has been effective in purging mixtures of 10–25% cultured human NB-1691 NB cells/PBMNCs (16) , with a decrease of at least 6 logs of tumor cells. We now describe the purging efficacy and lack of toxicity of the method using an in vivo mouse model of disseminated NB and marrow reconstitution of irradiated NOD/SCID mice and in a pilot study with bone marrow aspirates from eight NB patients.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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CPT-11 was a gift from Dr. J. P. McGovren (Pharmacia Upjohn Co., Kalamazoo, MI). A CPT-11 stock solution of 10 mM was made in 100% methanol and stored at -20°C for no more than 3 weeks. Dilutions of the stock solution were made with water immediately before use.
Human PBMNCs and Human CD34+ Cells.
PBMNCs were separated from peripheral blood collected from healthy volunteers, using Ficoll-Hypaque (Histopaque-1077; Sigma Diagnostics, St. Louis, MO) according to the manufacturer’s instructions. Granulocyte colony-stimulating factor-mobilized peripheral CD34+ cells were purchased from Poietics of Clonetics (Walkersville, MD). These cell preparations, which contain 
95% human CD34+ cells, were stored in liquid nitrogen until the day of use, when they were thawed quickly and washed with 0.9% NaCl before use.
Generation and Characterization of AdRSVrCE.
Construction and characterization of the E1a-/E3-deleted, replication-deficient Ad containing the RSV promoter and the cDNA encoding a rCE have been described previously (15, 16, 17, 18)
.
The amount of virus used in each experiment is reported as the MOI, which is defined as the number of pfu/total number of cells. The percentage of active virus particles (pfu) compared with total virus particles was 1%.
Mouse Model of Disseminated NB.
The initial description of this model and its recapitulation of the pattern of dissemination of NB in pediatric patients have been published (19)
. In the initial study, the injection of 5 x 106 NB-1691 cells into the tail vein of SCID mice resulted in the death of 100% of mice from disseminated disease in a predictable manner (a median of 42 days). In the current study, various numbers of NB-1691 cells (100 cells to 1 x 106 cells) were suspended in sterile saline and injected i.v. into SCID mice. Mice were observed daily, and the day of death was monitored. Animals in obvious distress were euthanized, and the day of sacrifice was recorded as the day of death. The decision to euthanize was made by an animal caregiver having no knowledge of the experimental protocol. Mice were housed and treated in accordance with a protocol approved by the SJCRH Committee for Animal Care and Use.
Assessment of Toxicity to Human Hematopoietic Progenitor Cells.
A standard in vitro assay was used to assess the ability of human hematopoietic progenitor cells to form colonies in methylcellulose supplemented with growth factors, as published previously (16)
.
In Vitro Assessment of Cytotoxicity to NB-1691 Cells.
Clonogenic assays were performed by standard methods, also as described previously (16)
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Assessment of Toxicity to NOD/SCID Marrow Repopulating Cells.
NOD/LtSz-Prkdcscid/J mice were purchased from Jackson Laboratories (Bar Harbor, ME) or were obtained from Dr. Patrick Kelly (SJCRH). The ability of human CD34+ cells to repopulate the marrow of NOD/SCID mice was determined using a technique modified from published methods (20, 21, 22, 23)
. All treatments were approved by the SJCRH Committee for Animal Care and Use.
Mice (8–12 weeks old) were irradiated with a total of 350 cGy delivered from a 137Cs source at 100 cGy/min, and human CD34+ cells or mixtures of purged NB-1691/CD34+ cells were injected by tail vein 1–3 h after irradiation. In agreement with prior studies, 90–100% of male and female mice receiving radiation survived the above-mentioned dose of radiation (20) . Animals were housed in Barrier III facilities and maintained on standard, nonsterile chow and water. All animals received 50 µl of reconstituted anti-asialo GM1 (Wako Chemicals, Richmond, VA) on days 1 (the day of irradiation), 4, and 11. NB cells, human hematopoietic cells, and mixtures of these cell types were suspended in PBS at concentrations such that 150 µl of cell suspension contained the number of cells to be injected.
SCID mice were used to assess purging efficacy (the method described above), and NOD/SCID mice were used to assess toxicity to marrow repopulating cells because NB-1691 cells do not form tumors in NOD/SCID mice.
Toxicity Testing after Irradiation and Marrow Engraftment.
Toxicity testing was done 1, 4, and 8 weeks after injection of human cells. Peripheral blood was collected from each mouse via the retro-orbital sinus, using a narrow bore glass Pasteur pipette. Blood for a complete blood count was collected in EDTA. Complete blood counts were done using a Hemavet 3700 Multispecies Hematology Analyzer (CDC Technologies, Oxford, CT). This analyzer provides a five-part differential (segmented neutrophils, lymphocytes, monocytes, basophils, and eosinophils), as well as other blood indices including total WBC, RBC Hgb, hematocrit, mean corpuscular volume, mean corpuscular Hgb, mean corpuscular Hgb concentration, and platelets. Reticulocyte smears were prepared in the St. Jude Animal Resource Center Diagnostic Laboratory and sent to Ani-Lytic, Inc. (Gaithersburg, MD) for interpretation. Reticulocyte smears were prepared by mixing equal volumes of EDTA blood and New Methylene Blue N. The mixture was allowed to stand for 30 min at room temperature and then smeared onto a glass slide.
Blood for serum chemistry analysis was collected without anticoagulant and allowed to clot. The clot was removed from the serum, and serum chemistry analysis was performed using a VetScan Chemistry Analyzer (Abaxis, Inc., Sunnyvale, CA), which provides a diagnostic panel that includes albumin, alkaline phosphatase, ALT, amylase, TBil, blood urea nitrogen, calcium (Ca2+), total cholesterol, creatinine, glucose, potassium (K+) total protein, and globulin. Other chemistry tests such as those for AST, 
-glutamyl transferase, sodium, phosphorus, chloride, protein electrophoresis, and triglycerides were prepared and sent to Ani-Lytics, Inc. for analysis.
At the end of each experiment (8 week time point), each mouse was anesthetized using an admixture of ketamine (100 mg/ml) and xylazine (20 mg/ml) administered i.p. at a dose of 0.2 ml/mouse. Each mouse was exsanguinated, and the blood was collected according to the procedures described above. After exsanguination, liver, lung, spleen, gonads, and bone marrow were collected from each mouse. All tissues were submitted to the St. Jude Animal Resource Center Histology Laboratory for routine H&E staining. A partial listing of results obtained from the above-mentioned tests is included in this article.
Immunohistochemical Staining of Human CD45+ Cells in Mouse Marrow.
Antihuman CD45+ conjugated to PE was purchased from BD PharMingen (San Diego, CA). PE-conjugated isotype-matched mouse immunoglobulin was used as a negative control. Immunofluorescence staining was done by the method reported previously (24)
, except that after harvesting marrow from both femurs of each mouse and washing of cells with PBS, immunostaining was done on unfixed cells. All reagents were used and procedures were done at 4°C. Samples were protected from light during staining and analysis.
Flow Cytometric Analysis.
Samples (5 x 105 cells) from unmanipulated mice, mice receiving control CD34+ cells, or mice receiving purged samples of 10% NB-1691/90% CD34+ cells were resuspended in 0.5 ml of saline and counterstained with PI. The percentage of human CD45+ cells was quantitated using a fluorescence-activated cell-sorting Calibur flow cytometer (Becton Dickinson, San Jose, CA) by collecting PE fluorescence at 585 ± 21 nm and PI fluorescence at wavelengths greater than 670 nm. Viable cells were selected by electronic gating for single cells with low PI fluorescence. The percentage of viable cells with positive levels of PE fluorescence (human CD45+) was determined by comparison with background levels of PE fluorescence in control samples containing cells from normal mouse marrow.
Purging and Assessment of Purging Efficacy and Toxicity in Preclinical Models.
Human hematopoietic cells or mixtures of NB/hematopoietic cells (1–2.5 x 107, unless otherwise indicated) were placed in tissue culture flasks having a surface area of 75 cm2 and containing 20 ml of Iscove’s modified Dulbecco’s medium supplemented with 10% fetal bovine serum and growth factors as reported previously (16)
. Cells were exposed to the indicated MOI of AdRSVrCE for 24 h, at which time virus was removed. Cells were then washed twice, and virus-free medium was added to the flasks. Forty-eight h after the removal of virus, cells were incubated with CPT-11 for 4 h, at which time drug was removed, and cells were frozen, injected into mice, or plated for progenitor cell assays and clonogenic assays or continued in culture as appropriate. In NOD/SCID marrow reconstitution assays to assess the toxicity of the purging method, control CD34+ cells were incubated in vitro for 76 h before injection into the mice but not exposed to Ad or drug, in parallel with purged cell preparations. Using a hemocytometer, trypan blue-excluding cells were counted immediately before injection into the mice, and 3 x 106 viable CD34+ cells were injected into each animal in both the purged and unpurged groups. (For samples containing mixtures of NB cells and CD34+ cells, the actual number of CD34+ cells was assumed to be 90% of the total cell number because apoptosis of the NB cells was not apparent until 5–10 days after exposure to CPT-11, and NB cells were indistinguishable from CD34+ cells at the time of injection into the mice.)
Collection of Patient Bone Marrow Aspirate Samples.
Bone marrow (5–10 ml) was collected in an EDTA tube by aspiration of the iliac crest of patients with known marrow metastases, who were receiving therapy for high-risk NB. Polymorphonuclear cells were isolated by Ficoll separation (16)
. Nucleated cells were then processed according to the described purging protocol. Bone marrow biopsies obtained at the same time as the aspirates were reviewed by the attending pathologist, who estimated the percentage of tumor burden by conventional light microscopy. Whereas microscopic evaluation of tumor burden is not quantitative, and methods such as real-time RT-PCR for tumor cell markers may eventually replace the current standard methods, microscopic estimates of tumor burden are included (Fig. 5)
as the current standard to which other methods are compared. Informed consent was obtained from all patients according to institutional guidelines.
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Statistical Analysis.
Data in Table 1
were analyzed using a one-way ANOVA with a Bonferroni post test. P < 0.05 was considered statistically significant.
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RESULTS |
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25–50%. AdRSVrCE alone (100 MOI) decreased the clonogenic potential of NB cell lines by 10–50%. These data documented that AdRSVrCE or CPT-11 alone was insufficient to eradicate the clonogenic potential NB cells. We also demonstrated previously by two in vitro assays, clonogenic potential and RT-PCR, that the AdRSVrCE/CPT-11 VDEPT method effectively reduced the number of NB cells in a 10% NB-1691/90% PBMNC mixture to undetectable levels (15 , 16) . The next step in assessing the utility of this method for eventual clinical application was to evaluate the efficacy and potential toxicities of the approach in in vivo mouse models.
Experiments to assess purging efficacy were done by mixing 10% NB-1691 cells with 90% human PBMNCs and injecting either purged or unpurged mixtures i.v. into the tail vein of SCID mice. Because i.v. injection of 5 x 106 NB-1691 cells produces disseminated disease in 100% of SCID mice (19)
, we anticipated that all of the mice injected with unpurged cell preparations would develop disseminated disease. However, the sensitivity and reliability of this model in detecting the small numbers of viable NB cells that might remain after purging were unknown. Therefore, we first determined the number of NB-1691 cells required to produce disseminated disease in this SCID mouse model. Fig. 1a
shows survival curves for five groups of SCID mice injected with the indicated number of NB-1691 tumor cells. The data demonstrate that infusion of 1000 NB cells produced disseminated disease and death, in a dose-dependent manner. Mice receiving only 100 NB-1691 cells showed no evidence of disease for >1 year. Mice receiving 105 or 106 cells developed advanced disease requiring euthanasia 73–96 days after injection. The results show that this in vivo assay is capable of detecting 1000 viable NB-1691 cells. The data also suggest that even in immune-compromised SCID mice, there is a level of NB cells that is tolerated and does not result in systemic disease.
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shows survival curves for SCID mice that received i.v. injection of either purged or unpurged samples. All 14 mice receiving unpurged samples developed disseminated NB that required euthanasia 73–96 days after injection. Conversely, all 14 mice receiving purged samples survived for more than a year after injection with no evidence of disease (P < 0.001).
Reassessment of Viral MOI on Purging Efficacy
Data in Fig. 1
indicate that exposure of mixtures of 10% NB-1691/90%PBMNCs exposed to a MOI of 50 of AdRSVrCE + 5 µM CPT-11 reduced the level of NB cells to less than 100 cells. These results corroborate previous results obtained using in vitro assays.
Although the above results were encouraging, we considered it likely that eliminating primary NB cells would be more difficult than purging cultured human NB cells. Accordingly, because previous results indicated that CPT-11 concentrations greater than 5 µM were toxic to human PBMNCs and human CD34+ progenitor cells (16) but that viral MOIs of less than 500 did not appear toxic to these hematopoietic cell populations, we investigated the utility of increasing the virus:cell ratio used in the purging protocol.
Fig. 2
demonstrates the effect of CPT-11 alone or in combination with an adenoviral MOI of 50 and 100 on the clonogenic potential of NB-1691 cells. A dose-dependent effect is seen both for CPT-11 concentration and also for viral concentration (MOI). Furthermore, a viral MOI of 100 increases the sensitivity of NB-1691 cells to CPT-11 by a factor of 5. We concluded that if, in fact, purging with a MOI of 100 could be shown to be nontoxic to progenitor and NOD/SCID repopulating cells, then using a MOI of 100 would be preferable in future translational studies. Toxicity testing in NOD/SCID mice was therefore done with groups of mice receiving CD34+/NB-1691 cells that had been exposed to adenoviral MOIs of 50 or 100, in combination with 5 µM CPT-11.
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shows the percentage of human CD45+/total nucleated cells in mouse marrow from mice receiving purged NB/CD34+ cell mixtures versus unmanipulated CD34+ cells. In the group of mice receiving 3 x 106 unmanipulated CD34+ cells (control mice), one of nine mice failed to engraft (indicated by the filled diamond). In the eight control animals that did engraft, the percentage of human CD45+ cells in the marrow ranged from 0.60% to 5.28%. For mice receiving mixtures of NB/CD34+ cells that had been exposed to a viral MOI of 50 and a CPT-11 concentration of 5 µM, eight of eight mice engrafted, with percentages of human cells in the marrow of 1.28–10.75%. Similarly, in the group of mice receiving cells purged with an adenoviral MOI of 100 plus 5 µM CPT-11, all 10 mice reconstituted their marrow with percentages of human cells ranging from 0.36% to 16.92%. There was no statistical difference in the percentage of human cells in the marrows of the three groups of mice. We conclude that the purging with adenoviral MOIs of 50 and 100 produced no detectable toxicity to human CD34+ NOD/SCID reconstituting cells.
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reports the mean values ± SD for the week 4 time point. No significant differences were seen in values obtained from samples of mice receiving unmanipulated CD34+ cells compared with purged cell mixtures (Table 1)
. Furthermore, multiple statistical analyses comparing values obtained at the three different time points for each group of mice or at a single time point for all three groups of mice failed to show any differences between mice that received unmanipulated CD34+ cells and irradiated, "transplanted" mice that received purged 10% NB/90% CD34+ cell mixtures (data not shown). We did observe several individual mice in each group with AST levels higher than the normal range, and these values are reflected by the relatively large SD for AST levels reported in Table 1
. However, each of these individual mice had normal ALT and TBil levels, verifying that liver function in these mice was within normal range. We conclude that liver and kidney function in all of the mice was within normal limits.
Assessment of Toxicity and Efficacy of Purging on Patient Bone Marrow Samples by RT-PCR and Clonogenic Assay
Documentation of the Sensitivity of RT-PCR to Detect NB-1691 Cells.
To assess the ability of RT-PCR to detect low numbers of NB cells, we made 10-fold dilutions ranging from 1 NB-1691 cell in 103 PBMNCs to 1 NB cell in 107 PBMNCs and extracted RNA from a total of 5 x 107 cells of each mixture. RT-PCR products for TH and N-MYC obtained with RNA extracted from dilutions of 1:103 to 1:106 for TH and 1:106 and 1:107 for N-MYC are shown in Fig. 4
. Also shown is a negative control in which no RNA was used in the reverse transcription reaction and positive controls in which primers for ß-actin were used to verify the integrity of the RNA used in the reaction. Whereas the sensitivity of RT-PCR depends on the level of expression of a given transcript in a specific cell line or tumor, the data show that when NB-1691 cells are used as the "standard," the message for TH is detectable at a level of 1:105 cells, and the message for N-MYC is detectable at a level of 1:106 or 1:107 cells. Results shown are from one of five replicate experiments. Results among the five experiments were reproducible, with the exception of some variability as to whether N-MYC transcript was/was not detected at a dilution of 1:107 cells, suggesting that this concentration is at the borderline of detection.
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Fig. 5
shows primary data from four of the patient samples. RT-PCR using RNA from bone marrow samples obtained from three of the patients (Fig. 5a)
detected N-MYC transcripts in the unpurged aliquot, but in not in the purged aliquots. Two of these three tumors also expressed TH as seen by the RT-PCR product in the unpurged aliquots, but no TH signal was present in the purged samples. No signal was detected in the control reaction to which no RNA was added (data not shown). The attending pathologist reviewed bone marrow biopsies obtained on the same day as the aspirates and estimated the tumor burden by conventional light microscopy for the three patients to be 15%, 2%, and 1%, as indicated.
Fig. 5b
shows results from a "clonogenic assay" with a fourth sample obtained from a patient who had relapsed after treatment with stem cell transplant. The bone marrow biopsy showed nearly complete replacement of the marrow space by metastatic tumor cells. Therefore, beginning with a total cell number of 2 x 107, if purging were successful there would have been an insufficient number of cells remaining to extract the 2 µg of RNA required for RT-PCR. Instead, purged and unpurged aliquots of this sample were plated and maintained in culture for 8 weeks. Unpurged control samples grew readily in culture; no cells were detected in the flask containing purged cells (Fig. 5b)
. Cells in the unpurged flask expressed N-MYC and TH (data not shown).
A summary of purging results from all eight patients is shown in Table 2
. Seven of the eight clinical samples were completely purged using the AdRSVrCE virus to the level of RT-PCR detection. The purge failure (patient 8) was likely due to a low level of rCE expression (data not shown). We have subsequently used a slightly modified Ad to successfully purge a sample obtained from patient 8 (see "Discussion").
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DISCUSSION |
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The rationale for the specific application of rCE/CPT-11 for purging NB cells was based on several observations. First, at the MOI used, the Ad specifically transduces NB cells compared with hematopoietic cells (16)
. Second, overexpression of rCE sensitizes cells that express endogenous levels of human CEs to CPT-11 by up to 500-fold (15)
.4
Third, CPT-11 has demonstrated significant, dose-dependent antitumor activity against NB xenografts (27)
, and clinical responses have been seen in patients with high-risk NB in a Phase I trial (28)
. These studies suggest that the high intracellular levels of SN-38 achievable with rCE/CPT-11 VDEPT may produce effective NB cell kill, and results with clinical specimens (Fig. 5
and Table 2
) are encouraging. However, because one of eight samples failed to purge with AdRSVrCE, we have begun to evaluate additional viral constructs, and we have identified promoters that produce higher levels of rCE in both NB cell lines and primary tumor cells than the RSV promoter. Interestingly, a sample from patient 8, the single purge failure seen thus far, was successfully purged using an AdCMVrCE construct (data not shown). Further evaluation of additional constructs is ongoing.
Autologous stem cell transplantation is routinely used to treat high-risk NB and appears important in improving disease-free survival. Controlled studies to assess the benefit of purging in NB have not yet been completed; it is not known whether purging is essential in the treatment of this disease, but several observations suggest that purging provides some clinical benefit. Adult lymphoma patients who receive stem cell grafts free of residual disease show improved survival compared with patients who receive contaminated grafts (29, 30, 31) . Also, up to 50% of stem cell grafts collected from NB patients in apparent remission are contaminated by tumor cells that can be tumorigenic (32) and contribute to relapse (11) . Thus, for patients with bone marrow involvement, purging is likely an important component of successful therapy, and it has become part of many cooperative group treatment protocols (1 , 3 , 14 , 33) .
In addition to reducing the likelihood of tumor cell reinfusion, more effective purging methods may also offer other advantages. Improved purging efficacy may make possible the option of earlier stem cell harvesting, resulting in more efficient collection of stem cells (34) and more rapid engraftment (35) . Earlier harvest may protect stem cells from certain deleterious effects of chemotherapy such as secondary leukemias, which occur in up to 7% of children receiving contemporary regimens for high-risk NB (36) .
The described rCE/CPT-11 VDEPT method can be performed with either bone marrow grafts or peripheral blood stem cells and appears to be feasible as well as relatively effective and nontoxic. Also, in contrast to currently available purging methods, the described VDEPT approach is efficacious with tumor burdens estimated to be greater than 1%. Whereas we do not envision that stem cell harvest would be done when patients have 25% NB cells in their marrow, a method such as rCE/CPT-11 VDEPT may have utility in patients whose marrow contains 1–5% tumor cells (because these patients are not eligible for standard purging protocols) or when early harvest would likely yield more viable stem cells.
Laboratory manipulations involve only maintaining the cells in culture for a total of 76 h and exposing the cells to virus and CPT-11 during this time. We did note that the apparent viability of NOD/SCID repopulating cells that have been maintained in culture for 76 h, even with recommended concentrations of specific growth factors, may be lower than that of CD34+ cells that have been stored at -80°C until the day of use. Accordingly, the NOD/SCID engraftment experiments (Fig. 3)
were done by injecting 3 x 106 viable CD34+ cells/mouse (plus or minus NB cells and purging). This number of cells was chosen because it is the lowest number of peripheral CD34+ cells that can reproducibly repopulate the marrow of NOD/SCID mice that receive 350 cGy of radiation (20)
. Therefore, if the purging procedure were toxic to the subset of cells responsible for marrow reconstitution in the mice, engraftment failures in the mice receiving purged cells compared with unmanipulated cells should have been detectable. No such engraftment failures were seen. One hundred percent of mice receiving purged cells engrafted, compared with 89% of the mice receiving unpurged CD34+ cells. However, it should be noted that in preliminary experiments (data not shown) in which we injected 3 x 106 CD34+ cells that had never been maintained in culture ex vivo into each of six mice, the percentage of CD45+ human leukocytes in the marrow of those mice ranged from 7.41% to 67.4%. It is controversial whether the precise percentage of CD45+ cells detected in mouse marrow is a more meaningful parameter than the presence versus absence of human CD45+ cells (20
, 21)
because few mice receiving these low levels of peripheral blood CD34+ cells have been studied. We conclude that whereas the purging protocol itself is not toxic to NOD/SCID repopulating cells, and 100% of transplanted mice engrafted with human hematopoietic cells, there may be some decrease in the percentage of human cells in the NOD/SCID marrow 8 weeks after cell infusion if CD34+ cells are maintained ex vivo for 3 days before injection into the mice. The relevance of this observation as it might apply to autologous stem cell rescue is unknown.
In conclusion, we describe an approach to stem cell purging for NB that may decrease tumor cell contamination of hematopoietic cell grafts to levels below those detectable by RT-PCR, even when the initial level of tumor cell contamination exceeds 1%. Such purging efficacy may not only reduce the likelihood of tumor cell reinfusion but may also allow administration of high-dose consolidation therapy to more patients in a more timely manner. Although additional innovative therapies may ultimately be required to cure high-risk patients, this purging method may help optimize existing therapies. We are evaluating additional patient samples to assess the efficacy of the purging protocol for a variety of NB patients. If we continue to observe the efficacy seen with samples in the pilot study, a clinical trial will be proposed for patients with levels of bone marrow disease greater than 1% for whom conventional purging would be inappropriate.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by Grants CA23099, CA21765, and CA76202 from NCI and by American Lebanese Syrian Associated Charities. 
2 To whom requests for reprints should be addressed, at Department of Molecular Pharmacology, St. Jude Children’s Research Hospital, 332 North Lauderdale, Memphis, TN 38105. Fax: (901) 521-1668; E-mail: mary.danks{at}stjude.org
3 The abbreviations used are: NB, neuroblastoma; Ad, adenovirus; ALT, alkaline aminotransferase; AST, aspartate aminotransferase; CE, carboxylesterase; MOI, multiplicity of infection (expressed as pfu/number of cells); NOD/SCID, non-obese diabetic severe combined immunodeficient; PE, phycoerythrin; PI, propidium iodide; PBMNC, peripheral blood mononuclear cell; pfu, plaque-forming unit(s); rCE, rabbit liver carboxylesterase; SJCRH, St. Jude Children’s Research Hospital; TBil, total bilirubin; VDEPT, virus-directed enzyme prodrug therapy; RT-PCR, reverse transcription-PCR; RSV, Rous sarcoma virus; Hgb, hemoglobin; TH, tyrosine hydroxylase.
4 M. K. Danks and P. M. Potter, unpublished observations.
Received 12/10/01. Accepted 6/27/02.
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REFERENCES |
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 C. M. Thirukkumaran, J. M. Luider, D. A. Stewart, T. Cheng, S. M. Lupichuk, M. J. Nodwell, J. A. Russell, I. A. Auer, and D. G. Morris Reovirus oncolysis as a novel purging strategy for autologous stem cell transplantation Blood, July 1, 2003; 102(1): 377 - 387. [Abstract] [Full Text] [PDF]
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