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Immunomagnetic Purging of Ewing’s Sarcoma From Blood and Bone Marrow: Quantitation by Real-Time Polymerase Chain Reaction

From the Pediatric Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, and Department of Pediatrics, University of Maryland School of Medicine, Baltimore, MD; Department of Pediatrics, Walter Reed Army Medical Center, Washington, DC; and Department of Pediatrics, Memorial Sloan-Kettering Cancer Center, New York, NY.

Address reprint requests to Margret E. Merino, MD, Pediatric Oncology Branch, National Cancer Institute, National Institute of Health, 9000 Rockville Pk, Bethesda, MD 20892-1928; email: merinom{at}mail.nih.gov

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
PURPOSE: A propensity for hematogenous spread with resulting contamination of autologous cell products complicates cellular therapies for Ewing’s sarcoma. We used a new approach to purge artificially contaminated cellular specimens of Ewing’s sarcoma and show the capacity for real-time polymerase chain reaction (PCR) to quantify the contamination level of Ewing’s sarcoma in such specimens.

PATIENTS AND METHODS: Binding of monoclonal antibody (MoAb) 8H9 to Ewing’s sarcoma cell lines and normal hematopoietic cells was studied using flow cytometry. Using real-time PCR–based amplification of t(11;22), levels of Ewing’s contamination of experimental and clinical cellular products were monitored. Purging was accomplished using immunomagnetic-based depletion. Monitoring of the function of residual hematopoietic progenitors and T cells was performed using functional assays.

RESULTS: MoAb 8H9 shows binding to Ewing’s sarcoma but spares normal hematopoietic tissues. Nested real-time PCR is capable of detecting contaminating Ewing’s sarcoma cells with a sensitivity of one cell in 10⁶ normal cells. After 8H9-based purging, a 2- to 3-log reduction in contaminating Ewing’s sarcoma was shown by real-time PCR, with purging to PCR negativity at levels of contamination of 1:10⁶. Levels of contamination in clinical samples ranged from 1:10⁵ to 10⁶. Therefore, 8H9-based purging of clinical samples is predicted to reduce tumor cell contamination to a level below the limit of detection of PCR.

CONCLUSION: These results demonstrate a new approach for purging contaminated cellular products of Ewing’s sarcoma and demonstrate the capacity of real-time PCR to provide accurate quantitative estimates of circulating tumor burden in this disease.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
CURRENT CONCEPTS hold that Ewing’s sarcoma is a systemic disease from the time of onset, demonstrated by the observation that more than 90% of patients with clinically localized disease recur distantly if treated with local measures alone.¹ Indeed, the generally accepted factor responsible for the recent improvement in survival rates observed in patients with clinically localized disease is the control of hematogenously disseminated micrometastasis via neoadjuvant multiagent chemotherapy.² Recently, molecular monitoring has been used to detect circulating Ewing’s sarcoma cells by identification of the tumor-specific t(11;22).^3,4 Using this approach, hematogenous dissemination has been confirmed in a substantial number of patients with Ewing’s sarcoma. West et al³ found a 25% incidence of positivity for the Ewing’s sarcoma-specific t(11;22) in the peripheral blood or bone marrow from patients with clinically localized disease, and higher rates have been observed in other series and in patients with overt metastatic disease.^5-7 In some reports, evidence for positivity in peripheral blood persisted after initiation of chemotherapy, suggesting that ongoing dissemination may occur intermittently throughout treatment protocols.

In an attempt to improve survival in high-risk patients with Ewing’s sarcoma, several groups have studied the use of high-dose chemotherapy with bone marrow or peripheral-blood progenitor-cell transplantation.^8-20 Survival rates of up to 40% in poor-risk patients have been reported after high-dose combined therapy and autologous progenitor cells in contrast with historical survival rates of 0% to 20% with chemotherapy/radiation therapy alone.^8,9 One factor complicating the use of autologous progenitor cell products in therapies for Ewing’s sarcoma is the propensity for hematogenous dissemination with resultant tumor contamination of autologous progenitor cell products. In one report, despite CD34-based positive selection for progenitor cells, autologous peripheral-blood progenitor preparations were shown to contain EWS/FLI1 translocation–positive cells in 54% of samples evaluated.⁶ Although the true clinical impact of contaminating tumor cells in autologous products remains unclear, genetically marked tumor cells residing in autologous bone marrow have been shown to be present at disease relapse in patients with neuroblastoma and AML.^21,22 Similar concerns regarding the potential for autologous cell preparations to contribute to disease recurrence arise in the context of immune-based therapy trials that are currently being undertaken and involve the transfer of autologous T cells collected before the initiation of therapy.²³

To date there has been no method reported for purging autologous hematopoietic cells of Ewing’s sarcoma. In this article, we evaluated a monoclonal antibody–based purging technique that allowed us to reduce the tumor burden in contaminated bone marrow and peripheral-blood specimens by 2 to 3 logs. Further, we show that this purging technique results in a polymerase chain reaction (PCR)–negative product when performed at levels of tumor contamination found in patient samples.

	PATIENTS AND METHODS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Monoclonal Antibody Production
Hybridoma 8H9 was derived from the fusion of Balb/c splenocytes with SP2/0 myeloma as previously described.^24,25 Lymphocytes derived from these mice were fused with SP 2/0 mouse myeloma cell lines. Clones were selected for specific binding on enzyme-linked immunosorbent assay (ELISA). The 8H9 hybridoma secreting an immunoglobulin (Ig)G1 monoclonal antibody was selected for further characterization after subcloning. 8H9 was produced by ascites induction in Balb/c mice and purified sequentially by Protein G affinity chromatography and SP-Sepharose chromatography in 0.1 M of citrate-phosphate buffer (pH 4.2). Purity of the antibody was verified by sodium dodecyl sulfate Polyacrylomide Gel Electrophoresis and immunoreactivity by ELISA and immunofluorescence analysis.

Cell Preparations
Peripheral-blood mononuclear cells (PBMCs). PBMCs used in tumor-spiking experiments were obtained by Ficoll-based (Pharmacia Biotech, Piscataway, NJ) density gradient separation of the fresh buffy coat fraction of normal healthy donor blood units obtained at the Department of Transfusion Medicine, Clinical Center, National Cancer Institute (NCI), according to approved protocols. For analysis of T-cell reactivity to anti-CD3 monoclonal antibody after purging, PBMCs were T-cell enriched using a negative selection column (R & D Biosystems, Minneapolis, MN), which resulted in a T-cell purity of approximately 80%.

Nonmobilized apheresis samples analyzed for contamination were obtained as part of NCI Pediatric Oncology Branch 97-0052 after informed consent. Leukapheresis procedures were done using the CS3000 Plus (Fenwal Division, Baxter, Deerfield, IL), which processed 5 to 15 L of blood volume. As part of this immunotherapy protocol, countercurrent centrifugal elutriation was performed. This procedure separates cells on the basis of size and density and thus results in the separation of peripheral-blood mononuclear fractions derived from apheresis into lymphocyte-rich and monocyte-rich fractions. The apheresis product was elutriated using a Beckman J-6M centrifuge equipped with a JE 5.0 rotor (Beckman Instruments, Palo Alto, CA) in HBSS without magnesium, calcium, and phenol red (BioWhittaker, Walkersville, MD) at a centrifuge speed of 3,000 rpm (1,725 x g).²⁶ Cell fractions (450 to 550 mL each) were collected at flow rates of 120, 140, and 190 mL/min during centrifugation and at 190 mL/min with the rotor off. The first two fractions (120 mL/min and 140 mL/min) are typically enriched for lymphocytes, and the last two fractions (190 mL/min and rotor off) are enriched for monocytes. All fractions were cryopreserved in 10% DMSO (Cryoserv; Research Industries, Salt Lake City, UT), RPMI with penicillin, streptomycin, and L-glutamine and 25% fetal calf serum (FCS).

Progenitor cells. CD34⁺ cells used for purging experiments were selected using the Miltenyi Variomax direct isolation system (Miltenyi, Auburn, CA) from cryopreserved granulocyte colony-stimulating factor (G-CSF)–mobilized peripheral hematopoietic progenitor cells from a patient with Ewing’s sarcoma. The hematopoietic progenitor cells were obtained for therapeutic use according to approved protocols and after obtaining informed consent, and were used for research purposes after the patient’s death. These cells were not positive by reverse transcriptase (RT) PCR for Ewing’s sarcoma and were, therefore, artificially contaminated with TC-71 at the concentrations shown for the purging experiments. Unmodified bone marrow used for purging experiments and enriched CD34⁺ populations used in the colony-forming unit (CFU) assays were obtained from fresh human marrow collected from normal volunteers according to approved protocols and after obtaining informed consent (Poietics Laboratories, Gaithersburg, MD). The mononuclear fraction was obtained by Ficoll-based density gradient separation and subsequently enriched for CD34⁺ cells by the Miltenyi Variomax direct CD34 selection system.

Tumor cell lines. Ewing’s sarcoma cell lines used for screening included TC-71, 5838, RD-ES, CHP100, and A4573, which have been previously reported,²⁷ and NCI-EWS-94(JR) and NCI-EWS-95(SB), which are cell lines derived from patients treated at the NCI, which have also been previously reported.²⁷ EWS-925 was a cell line derived from a patient with isolated intrarenal recurrence of Ewing’s sarcoma treated with resection at the University of Maryland.

Flow Cytometry Analysis
Flow cytometric analysis was performed using the Becton Dickinson FacsCalibur machine (Becton, Dickenson and Co, San Jose, CA). Fluorescence data were collected using a three-decade log amplification on 10,000 viable gated cells as determined by forward and side light scatter intensity. Monoclonal antibodies used for immunofluorescence included MoAb 8H9, murine IgG1 isotype, goat antimouse IgG1-FITC, CD3-PE (S4.1), CD34-PE (581) Caltag (Burlingame, CA), CD99-FITC (TU12) (Pharmingen, San Diego, CA). For immunofluorescence analysis, cells were incubated with antibody at a concentration of 1 µg/10⁶ cells for 20 minutes at 4°C, then washed with phosphate-buffered saline with 0.2% human serum albumin and 0.1% sodium azide. For 8H9 and isotype staining, this was incubated with goat antimouse fluorescein isothiocyanate conjugate for 10 minutes at 4°C and washed before analysis.

Immunomagnetic Purging
Cell products were spiked with tumor cells from the Ewing’s sarcoma cell lines TC-71, EWS-NCI-94, and EWS-NCI-96 at the levels of contamination indicated for individual experiments shown. For purging of CD34⁺ peripheral-blood progenitor cells, a total of 10 x 10⁶ cells were spiked. One x 10⁶ cells were analyzed for prepurged and postpurged PCR. For PBMC and unmodified bone marrow specimens, 30 to 80 x 10⁶ cells were spiked with TC71 and 10 to 50 x 10⁶ cells were analyzed for pre- and postpurged PCR. For purging, cells were incubated at 4°C with monoclonal antibody 8H9 at a concentration of 1 µg/10⁶ total cells for 20 minutes and washed with buffer (phosphate-buffered saline, 0.5% BSA, 2 mmol/L EDTA). Cells were then incubated with rat antimouse IgG1 magnetic beads at a ratio of 1:1 for 20 minutes at 4°C. Purging was accomplished using the Miltenyi Variomax system wherein the sample is run over the Miltenyi AS depletion column with a flow resistor of 24G. Cells from the depleted fraction were then washed with 3 mL of buffer. The positively selected fraction of cells was removed by releasing the column from the magnet and washing with 3 mL of buffer and analyzed by PCR. In cases where viability of the positively selected fraction was evaluated, the positive fraction was pelleted and resuspended in RPMI with 10% FCS, L-glutamine (4 µM), penicillin (100 units/mL), and streptomycin (100 µg/mL) and placed in an incubator at 37°C with 5% CO₂ for 5 days.

Conventional PCR
For analysis of contamination of patient apheresis fractions, RNA was extracted from 20 to 50 x 10⁶ cells using TRIzol Reagent (Life Technologies, Rockville, MD) or guanidinium isothiocyanate/CsCl method.²⁸ After cDNA was generated from 250 ng of RNA using a random hexamer, PCR was performed with the Perkin Elmer (Boston, MA) GeneAmp PCR system 2400 using ESPB1 and ESBP2 primers and the following conditions: 40 cycles at 95°C for 30 seconds for denaturation, at 60°C for 30 seconds for annealing, and at 72°C for 30 seconds for elongation and then 72°C for 7 minutes. To assess the integrity and quantity of RNA, a PCR reaction with GAPDH primers was performed for each patient sample. Ten microliters of each PCR product were run on 1.3% Tris-borate EDTA agarose gel and transferred to a nylon membrane. A [phosphorus-32]gamma-adenosine triphosphate 20-mer oligonucleotide probe was generated using T4 polynucleotide kinase. The membrane was hybridized using ExpressHyb Hybridization Solution (Clontech, Palo Alto, CA) according to the manufacturer’s instructions. The membrane was then exposed to Kodak Xomat film (Kodak, Rochester, NY) for 24 to 144 hours.

Real-Time Quantitative PCR
Real-time quantitative PCR was performed using the Lightcycler Instrument (Roche Molecular Biochemicals, Indianapolis, IN). RNA was extracted from 10 to 50 x 10⁶ cells from all samples except for the CD34⁺ population, in which 1 x 10⁶ cells were used. The Trizol phenol/chloroform extraction or RNA-Easy columns (Qiagen, Valencia, CA) were used for RNA extraction. The First Strand Synthesis kit (Roche) was used to generate cDNA from 1 µg of RNA from each sample. PCR was then run on 5 µL of cDNA on the Lightcycler instrument with primers ESBP1 and ESBP2 for 40 cycles. The real-time PCR conditions were as follows: initial denaturation at 95°C for 60 seconds, then 40 cycles of denaturation at 95°C for 1 second, annealing at 58°C for 10 seconds, and elongation at 72°C for 13 seconds. Fluorescence was measured at the end of each annealing phase fluorescence, and F2/F1 was plotted versus cycle number. In cases where nested PCR was performed, an initial 20 cycles of PCR were carried out with the primer pair ESBPI and ESBP2 using conventional PCR at 95°C for 30 seconds for denaturation, 60°C for 30 seconds for annealing, 72°C for 30 seconds for elongation, and 72°C for 7 minutes. This was followed by 40 additional cycles using quantitative PCR with the above described settings using 2 µL of the product of the first reaction and the primer pair EWS 696-FLI1 1041. By conventional PCR, primer pair ESBP1-ESBP2, and EWS 696-FLI1 1041 generate fragments of 310 and 205 base pairs, respectively. Both sets of primers are outside the breakpoint of the EWS/FLI 1 translocation. In the initial evaluation of the real-time PCR, both nested and nonnested Lightcycler PCR products were confirmed by size using 1% TAE agarose gel with ethidium bromide (data not shown). Hybridization probes spanning the EWS/FLI 1 breakpoint were used to detect target template in the Lightcycler reaction. To provide a positive control and to quantitate total amplified RNA, G6PD was amplified from 5 µL of cDNA and analyzed using the sequence-specific hybridization probes G6PDHP1 and G6PDHP2. All real-time PCR reactions were 20-µL reactions with primer concentrations of 0.8 µM, hybridization probe concentrations of 0.2 µM, and MgCl₂ concentrations of 5 µM. Two µL of DNA Master Hybridization Probes mix (Roche Molecular Biochemicals, Mannheim, Germany) was used. On all hybridization probes, the 5' probe (HP1) was labeled at the 3'end with fluorescein, and the 3' probe (HP2) was labeled at the 5' end with Lightcycler Red 640 and phosphorylated at the 3' end (Genset Oligos, Paris, France). The cycle crossing number was ascertained at the point at which all samples had entered the log linear phase. The cycle crossing number was used to determine log-cell concentration according to a standard curve. The standard curve was generated by amplifying 5 µL of cDNA derived from 1 µg of RNA from 10 to 50 x 10⁶ normal PBMCs spiked with TC-71 tumor cells at decreasing concentrations from 1:10 to 1:10⁷.

Sequences
[Phosphorus-32] Gamma Probe 5'TACTCTCAGCAGAACA- CCTATG

Primers

ESBP1 5' CGA CTA GTT ATG ATC AGA GCA 3'

ESBP2 5' CCG TTG CTC TGT ATT CTT ACT GA 3'

EWS 696 5' AGC AGC TAT GGA CAG CAG 3'

FLI 1 1041 5' TTG AGG CCA GAA TTC ATG TT 3'

G6PD1 5' CCG GAT CGA CCA CTA CCT GGG CAA G 3'

G6PD2 5' GTT CCC CAC GTA CTG GCC CAG GAC CA 3'

Lightcycler Hybridization Probes

1. EWSHP1 5' TAT AGC CAA CAG AGC AGC AGC TAC –F 3'
   
2. EWSHP2 5' LC RED 640 –GGC AGC AGA ACC CTT CTT –P 3'
   
3. G6PDHP1 5' GTT CCA GAT GGG GCC GAA GAT CCT GTT G –F 3'
   
4. G6PDHP2 5' LC RED 640 –CAA ATC TCA GCA CCA TGA GGT TCT GCA C –P 3'
   

OKT3-Mediated Proliferation of Purged T-Cell Specimens
One x 10⁸ CD3-enriched cells were spiked with Ewing’s sarcoma at a level of 1:10³. Cells from prepurged and postpurged samples were added in triplicate to a 96-well plate at a concentration of 2 x 10⁵ cells/well containing decreasing concentrations of plate bound anti-CD3 antibody OKT3 (Ortho Biotech Inc, Raritan, NJ) from 100 µg/mL to 3 µg/mL. Cells were incubated with 200 µL of RPMI with 10% FCS, L-glutamine, penicillin, and streptomycin for a 48 hours and then pulsed with 1 µCi of tritium ([³H]) thymidine per well. Cells were collected after 18 hours of pulsing, and [³H] incorporation was enumerated using the TopCount NXT (Packard, Meriden, CT). Subtracting background activity with media alone generated net counts.

CFU Assays
CD34⁺ cells were enriched from prepurged and postpurged samples from fresh human bone marrow using the Miltenyi direct CD34⁺ progenitor isolation kit. Thirty-five x 10⁶ bone marrow mononuclear cells from each sample were run over a positive selection (size MS) column yielding a CD34⁺-enriched population with estimated purities of more than 70%.²⁹ One thousand cells were plated in triplicate in methylcellulose media (MethoCultGF+H4435, Stem Cell Technologies, Vancouver, BC) containing recombinant human (rh) stem-cell factor, rh granulocyte-macrophage colony-stimulating factor, rh interleukin-3, rh interleukin-6, rh G-CSF, and rh erythropoietin. CFUs were counted after 14 days of culture. Colony-forming assays pre- and post-8H9 purging were also performed using cryopreserved CD34⁺-selected peripheral-blood progenitor cells obtained from a patient with Ewing’s sarcoma using the technique stated above. In addition, myeloid progenitor activity from CD34⁺-selected cells obtained from normal donor marrow (Poietics, Rockville, MD), was evaluated using a CELISA assay (BioWhittaker Inc, Rockville, MD),³⁰ pre- and postpurging with 8H9.

	RESULTS

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Monoclonal Antibody 8H9 Binding
To identify a potential reagent that could be used to target contaminating Ewing’s sarcoma cells, monoclonal antibodies induced via immunization with neuroblastoma were screened for cross-reactivity with Ewing’s sarcoma. Monoclonal antibody 8H9 was observed to bind to nine of nine Ewing’s sarcoma cell lines evaluated (Fig 1). For comparison, anti-CD99 was used as an antibody, which is known to show substantial reactivity for Ewing’s sarcoma cells.^31,32 The level of reactivity was variable, with some cell lines showing diminished levels of reactivity compared with CD99, whereas two lines (EWS-NCI-95 and RD-ES) showed increased reactivity compared with CD99. Importantly, lymphoid and hematopoietic populations showed no reactivity with 8H9, as shown in Fig 2A (CD3-gated PBMC) and Fig 2B (CD34-gated bone marrow cells), whereas CD99 showed significant binding to T-cell populations. Thus 8H9 showed significant binding to all Ewing’s cell lines tested, with no evidence of reactivity with normal hematopoietic cell populations. These results suggested that 8H9 could prove useful as an agent for immunomagnetic-based purging.

View larger version (37K): [in this window] [in a new window]   Fig 1. Reactivity of 8H9 with Ewing’s sarcoma cell lines. Flow cytometric analysis of 8H9-binding to 9 Ewing’s sarcoma cell lines, designated in the upper right corner of the histogram. FL1 fluorescence of isotype (dashed), CD99 (gray), and 8H9 (black).

View larger version (21K): [in this window] [in a new window]   Fig 2. Lack of reactivity of 8H9 with T cells or bone marrow progenitor cells. CD3+ cells from peripheral blood (top panel) and CD34+ cells from bone marrow (bottom panel) are analyzed for isotype (red), CD99 (blue), and 8H9 (green).

View larger version (24K): [in this window] [in a new window]   Fig 3. Real-time PCR analysis of t(11;22) in PBMCs spiked with Ewing’s sarcoma. (A) Nonnested PCR of contaminated PBMCs. The relationship between crossing cycle and log cell number is shown. (B) Nested PCR of contaminated PBMCs. (C) Relationship between crossing cycle and log-cell number from 3B samples.

View larger version (21K): [in this window] [in a new window]   Fig 4. Quantitative PCR analysis of purging. (A) Nonnested PCR of pre- and postpurged bone marrow. (B) Nested PCR of pre- and postpurged CD34-selected cells contaminated at 1:103. (C and D) Nested PCR of pre- and postpurged PBMCs from a normal donor contaminated at 1:100 and 1:106, respectively.

View larger version (23K): [in this window] [in a new window]   Fig 5. Quantitative PCR analysis of apheresis fractions collected from patients with high-risk Ewing’s sarcoma. Patient A shows contamination of all fractions at 1:105 to 1:106. Patient B shows contamination in the lymphocyte (120 mL/min) fraction at approximately 1:106. Patient C shows contamination in several fractions at approximately 1:106.

View larger version (60K): [in this window] [in a new window]   Fig 6. Progenitor colony-forming assays. Mean CFUs from CD34-selected cells from bone marrow from a normal healthy donor are plotted for prepurged and postpurged samples. Samples were analyzed in triplicate.

View larger version (14K): [in this window] [in a new window]   Fig 7. OKT3 mediated T-cell proliferation. Normal donor T cells were evaluated for proliferation to anti-CD3 as measured by [3H] thymidine uptake. Counts per million (y axis) are plotted v OKT3 concentration (x axis) for prepurged () and postpurged (•) samples.

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

An ideal purging method should target only tumor cells and show no binding to normal cell populations. The identification of such a tumor-specific antigen has historically posed a challenge in Ewing’s sarcoma. Although CD99 typically shows high expression on Ewing’s sarcoma cells, it is also expressed on T cells (Fig 2A) and CD34⁺ hematopoietic progenitor cells,³⁷ making it unsuitable for purging hematologic products. Monoclonal antibody 8H9 was initially developed because of its reactivity with neuroblastoma but was subsequently reported to react with 19 of 19 fresh Ewing’s sarcoma/primitive neuroectodermal tumors. Importantly, these previous results confirm that 8H9 reactivity is not limited to established cell lines.²⁵ Our results (Fig 1) showed reactivity with all Ewing’s cell lines evaluated. Because this antibody shows no reactivity with T cells and CD34⁺ cells, it is ideally suited for purging. Indeed, we demonstrated a 2 to 3-log reduction of Ewing’s sarcoma in all experiments after 8H9-incubation and one run over the negative selection column. In the clinical setting of autologous hematopoietic progenitor-cell transplantation, the combination of positive selection for CD34⁺ cells, which results in an approximate 2-log passive depletion of tumor,^7,38,39 and 8H9-purging of tumor cells would be expected to result in up to 5 logs of depletion, which is predicted to be well below the limit of detection using currently available techniques. Further, even in the setting of autologous T-cell transplantation, as potentially used in the context of immune reconstitutive therapies,²³ the use of 8H9-based purging with its 2 to 3-log reduction will substantially diminish the tumor burden contained in autologous cellular products.

This is the first report of using MoAb 8H9 to purge Ewing’s sarcoma. Interestingly, 8H9 also shows reactivity with many sarcomas, including rhabdomyosarcoma and osteosarcoma (data not shown). This introduces the possibility of a sarcoma-specific antibody with potential applications in immune-directed therapy. In addition, identification and characterization of the tumor-specific epitope, which binds to 8H9, could offer important insight into the biology of these tumors. Such studies are currently underway. Further, we observed that Ewing’s sarcoma cells positively selected using 8H9 retain their growth potential and are able to be maintained in cell cultures. This property has the potential to aid in the generation and study of tumor cell lines derived from patients with pediatric sarcomas, which is currently difficult because of limitations of tumor size and surgical accessibility of primary tumors. We are currently investigating whether Ewing’s sarcoma cells derived from apheresis or bone marrow samples in patients with metastatic disease that are positively selected and grown in culture could provide a source of tumor samples for further biologic study.

RT-PCR is a sensitive tool for monitoring minimal residual disease (MRD).⁴⁰ It remains unclear, however, whether evidence of small amounts of residual tumor by molecular analysis is predictive for relapse in solid tumors, and data in the literature are conflicting. de Alava et al⁵ evaluated MRD in Ewing’s sarcoma patients and showed a correlation between PCR positivity and disease relapse. In that report, however, some patients remained PCR-positive without disease relapse. Using real-time PCR, it is now possible to quantitate starting templates and compare starting template amounts between samples obtained at different time points. Real-time quantitative PCR has been used as a tool to monitor MRD in leukemia patients^41,42 and may be useful in the evaluation of disease response⁴³ and in predicting relapse in patients by the detection of increasing levels of tumor-specific transcript. This is the first article on the use of real-time quantitative PCR to detect and quantify the Ewing’s sarcoma-specific t(11;22). It is possible that quantitative PCR could allow for further identification of patients with a high risk of relapse by the detection of increasing amounts of Ewing’s transcripts over time.

Because contamination of peripheral blood by solid tumors is likely to be relatively low (in the range of 1:10⁵ to 1:10⁶ in this series), the sensitivity of the analysis must be high to allow for the detection of low levels of circulating tumor in patients with solid tumors. The level of sensitivity of our technique detected one Ewing’s sarcoma cell in 10⁶ normal cells with nested PCR from 10 x 10⁶ cells. Thus this assay was capable of amplifying product from 10 contaminating cells. It is possible that the level of sensitivity would be even greater if RNA was extracted from larger cell numbers. The positive immunomagnetic-selection procedure used for purging could also provide an approach for tumor enrichment for monitoring MRD, diagnostic confirmation, or staging evaluation in the setting of metastatic disease. Indeed, cells eluted from the column were positive by PCR analysis, which would be predicted to increase the sensitivity of PCR detection of contaminating Ewing’s sarcoma in patient samples. One caveat that should be noted is that the quantitative technique relies on the assumption that the level of expression of t(11;22) is consistent among cell lines and patient samples. This may not be the case, however, and may lead to under- or overestimation of the absolute level of tumor burden when comparing patient samples with a standard curve. Such limitations would not preclude evaluation of changes in the level of PCR positivity of an individual patient over time, wherein substantial changes in the level of expression of t(11;22) may be less likely.

In this article, we have demonstrated a purging technique that reduces tumor burden in artificially contaminated products by at least 2 to 3 logs. This approach resulted in a PCR-negative result in specimens contaminated at a level similar to that observed in patients. The demonstration that CFU assays on progenitor cells as well as CD3-induced T-cell proliferation are normal after purging demonstrates no detrimental effects on normal progenitor-cell and T-cell function, making this a potentially feasible addition to autologous protocols. We conclude that immunomagnetic purging via negative selection using MoAb 8H9 warrants evaluation in clinical trials to reduce tumor burden administered to patients. The application of such a technique may aid in evaluating the role of tumor contamination to disease relapse.

	ACKNOWLEDGMENTS

 
We acknowledge the generous contributions of the Charles and Dana Nearburg Foundation. We thank the Department of Transfusion Medicine of the National Institutes of Health (NIH) for assistance in obtaining the patient apheresis specimens; Patricia Dinndorf, MD, at Children’s National Medical Center for her assistance in obtaining patient progenitor cells; Gretchen Schwartz, MD, Medicine Branch, NCI, NIH, for her assistance in the CFU assays; and Allan Wayne, MD, Pediatric Oncology Branch, NCI, NIH, for his careful review of the manuscript.

	REFERENCES

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
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Submitted November 27, 2000; accepted May 8, 2001.

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