Cancer Research Annual Meeting 2010 EMT and Cancer Progression and Treatment
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Piloto, O.
Right arrow Articles by Small, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Piloto, O.
Right arrow Articles by Small, D.
[Cancer Research 66, 4843-4851, May 1, 2006]
© 2006 American Association for Cancer Research

Experimental Therapeutics, Molecular Targets, and Chemical Biology

IMC-EB10, an Anti-FLT3 Monoclonal Antibody, Prolongs Survival and Reduces Nonobese Diabetic/Severe Combined Immunodeficient Engraftment of Some Acute Lymphoblastic Leukemia Cell Lines and Primary Leukemic Samples

Obdulio Piloto1, Bao Nguyen1, David Huso2, Kyu-Tae Kim1, Yiwen Li3, Larry Witte3, Daniel J. Hicklin3, Patrick Brown1 and Donald Small1

1 Department of Pediatric Oncology and 2 Division of Comparative Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland and 3 ImClone Systems, Inc., New York, New York

Requests for reprints: Obdulio Piloto, Johns Hopkins University School of Medicine, 1650 Orleans Street, CRB 276, Baltimore, MD 21231. Phone: 410-955-1554; Fax: 410-955-8897; E-mail: opiloto1{at}jhmi.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The class III receptor tyrosine kinase FLT3 is expressed on the blasts of >90% of patients with B-lineage acute lymphoblastic leukemias (ALL). In addition, it is expressed at extremely high levels in ALL patients with mixed lineage leukemia rearrangements or hyperdiploidy and is sometimes mutated in these same patients. In this report, we investigate the effects of treating ALL cell lines and primary samples with human anti-FLT3 monoclonal antibodies (mAb) capable of preventing binding of FLT3 ligand. In vitro studies, examining the ability of two anti-FLT3 mAbs (IMC-EB10 and IMC-NC7) to affect FLT3 activation and downstream signaling in ALL cell lines and primary blasts, yielded variable results. FLT3 phosphorylation was consistently inhibited by IMC-NC7 treatment, but in some cell lines, IMC-EB10 actually stimulated FLT3 activation, possibly as a result of antibody-mediated receptor dimerization. Through antibody-dependent, cell-mediated cytotoxicity, such an antibody could still prove efficacious against leukemia cells in vivo. In fact, IMC-EB10 treatment significantly prolonged survival and/or reduced engraftment of several ALL cell lines and primary ALL samples in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. This occurred even when IMC-EB10 treatment resulted in FLT3 activation in vitro. Moreover, fluorescence-activated cell sorting and PCR analysis of IMC-EB10-treated NOD/SCID mice surviving 150 days post-leukemic cell injection revealed that FLT3 immunotherapy reduced leukemic engraftment below the level of detection in these assays (<0.001%). Furthermore, in vivo IMC-EB10 treatment did not select for resistant cells, because cells surviving IMC-EB10 treatment remain sensitive to IMC-EB10 cytotoxicity upon retransplantation. In vivo studies involving either partial depletion or activation of natural killer (NK) cells show that most of the cytotoxic effect of IMC-EB10 is mediated through NK cells. Therefore, such an antibody, either naked or conjugated to radioactive isotopes or cytotoxic agents, may prove useful in the therapy of infant ALL as well as childhood and adult ALL patients whose blasts typically express FLT3. (Cancer Res 2006; 66(9): 4843-51)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The class III receptor tyrosine kinase FLT3 is composed of five extracellular immunoglobulin-like domains that bind a ligand, a single transmembrane domain, and an intracellular kinase domain interrupted by a kinase insert (1–3). Ligand binding promotes receptor dimerization and activation of the kinase domain, leading to autotransphosphorylation of specific tyrosines (4). Activated FLT3 mediates signaling by phosphorylating target proteins and by serving as a docking site for SH2 domain containing proteins (5–8).

Normally, the FLT3 receptor is involved in differentiation, proliferation, and survival of multipotent hematopoietic stem cells as well as lymphoid and dendritic progenitor cells (9). Aberrant FLT3 expression/activation has been shown to play various roles in leukemogenesis, particularly in acute myelogenous leukemia (AML) and mixed lineage leukemia (MLL)–rearranged acute lymphoid leukemia (ALL) (reviewed in refs. 10–13). FLT3 may be activated by internal tandem duplication mutations in the juxtamembrane region or by point mutations in the kinase domain (14–16). Alternatively, wild-type (wt) FLT3 can be constitutively phosphorylated through overexpression of FLT3 along with ligand stimulation through an autocrine pathway (17–19). FLT3 has been shown to be expressed by >90% of B-lineage ALL blasts and is expressed at extremely high levels in ALL patients with MLL rearrangements or hyperdiploidy and is sometimes mutated in these same patients (20–22). These characteristics make FLT3 an attractive receptor for targeted therapy; thus, tyrosine kinase inhibitors (TKI) against FLT3 have been developed. Some of these TKIs selective for FLT3 are able to sufficiently decrease FLT3 activation to induce apoptosis in vitro and in vivo (23–28). Several FLT3 TKIs have been tested in phase I/II clinical trials in patients with AML. TKIs targeting FLT3 are selective but not specific and thereby inhibit various other kinases that lead to some toxicities observed in those trials. In addition, prolonged treatment with TKIs are likely to select for resistance through mutation of FLT3, as was observed in BCR-ABL with Gleevac treatment of chronic myelogenous leukemia (CML; ref. 29).

To overcome some of these limitations, we decided to investigate the targeting of FLT3 by monoclonal antibodies (mAb). A number of mAbs have been shown to successfully target cell surface receptors and interfere with vital signaling pathways (30). Besides inhibiting receptor activation, mAbs have the added ability of recruiting the host's immune system against the targeted cells and thereby killing cells that do not depend on receptor activation for survival. Anti-FLT3 mAbs IMC-EB10 and IMC-NC7 have been shown to inhibit wt and constitutively active mutant FLT3 in AML-derived cell lines and primary AML samples (31, 32). Furthermore, IMC-EB10 treatment of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice given AML blasts or cell lines resulted in reduced engraftment and/or prolonged survival without affecting engraftment of cord blood CD34+ cells (32). In this report, we examine the efficacy of anti-FLT3 immunotherapy in vitro and in vivo against ALL-derived cell lines and primary ALL samples and investigate the mechanism employed by IMC-EB10 to induce cytotoxicity in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents. Mouse monoclonal anti-phosphotyrosine antibody (4G10) and recombinant protein A-agarose were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Rabbit anti-phospho-signal transducers and activators of transcription 5 (anti-phospho-STAT5), anti-phospho-AKT, anti-AKT, anti-phospho-mitogen-activated protein kinase (anti-phospho-MAPK) p44/42, and anti-MAPK p44/42 antibodies were obtained from Cell Signaling Technologies, Inc. (Beverly, MA). Rabbit anti-human FLT3 and anti-STAT5 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Humanized anti-epidermal growth factor receptor (anti-EGFR) antibody IMC-C225 (Cetuximab; ImClone Systems, New York, NY) was used as a control. Asialo GM1 antisera was obtained from Wako Pure Chemical Industries (Osaka, Japan), and polyinosinic-polycytidylic acid [poly (I:C)] was obtained from Sigma (St. Louis, MO). Horseradish peroxidase–conjugated secondary antibody and enhanced chemiluminescence detection system were purchased from Amersham (Arlington Heights, IL).

IMC-EB10 and IMC-NC7 mAbs. IMC-EB10 and IMC-NC7 mAbs were engineered and binding analysis done as previously described (31, 32).

Cell lines. Human cell lines were cultured in RPMI 1640 (Life Technologies, Rockville, MD) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gimini Bio-Products, Woodland, CA) and penicillin/streptomycin (Life Technologies) at 37°C with 5% CO2.

Human samples. Human samples from pediatric ALL patients were obtained under an institutional review board–approved protocol at the Sidney Kimmel Cancer Center at Johns Hopkins Hospital. Leukemic blasts were separated by Ficoll-Hypaque (Amersham, Piscataway, NJ) density gradient centrifugation as previously described (33).

Western blot analysis. Western blots were done as previously described (32). Briefly, cells were treated for 1 hour with 10 µg/mL IMC-C225, IMC-EB10, or IMC-NC7 mAbs in fresh RPMI supplemented with 10% heat-inactivated FBS. Cells were then washed with cold PBS and lysed for 30 minutes at 4°C in NP40 lysis buffer. Lysates (500 µg) were incubated with rabbit anti-human FLT3 antibody overnight followed by incubation with protein A-agarose at 4°C for 2 hours. Protein A-agarose beads were washed with TBS-T and TBS, resuspended in SDS sample buffer, boiled for 7 minutes, and separated by 8% SDS-PAGE. Nonimmunoprecipitated total protein lysates were separated by 10% SDS-PAGE. Gels were blotted onto polyvinylidene fluoride microporous membrane (Millipore, Bedford, MA) and probed with the indicated antibody. Antibody binding was detected by incubation with a horseradish peroxidase–conjugated secondary antibody followed by chemiluminescence detection.

In vivo models of human leukemia: engraftment and survival. NOD/SCID mice were bred at Johns Hopkins University School of Medicine. Mice were sublethally irradiated (300 cGy) and injected with 0.5 x 106 cells from ALL-derived cell lines or 1 x 106 cells from ALL primary samples in 500 µL PBS by tail vein injection. Mice were given i.p. injections of 400 µg of IMC-C225 or IMC-EB10 at the indicated times. All animal procedures were conducted in conformity with institutional guidelines.

Flow cytometry. Cells were washed in chilled IFA buffer [0.01 mol/L HEPES, 0.15 mol/L NaCl, 1% NaN3, 4% FBS (pH 7.4)] and stained with human APC-conjugated CD45, FITC-conjugated CD19, PE-conjugated CD135, and/or murine PE-conjugated CD45 or DX5 (BD Biosciences, Inc., Palo Alto, CA) for 30 minutes at 4°C in the dark. Each sample was also stained with appropriate isotype controls. Stained cells were washed with cold IFA buffer and incubated with RBC lysis buffer (0.155 mol/L NH4Cl, 0.01 mol/L KHCO3, 0.1 mmol/L EDTA) for 3 minutes at room temperature and then washed again with cold IFA buffer. Cells were analyzed using a BD-FACSCalibur flow cytometer (BD Biosciences) and CellQuest software.

Quantitative real-time PCR. DNA was isolated from murine bone marrow and spleen cells using the QIAamp DNA Mini kit (Qiagen Sciences, Valencia, CA). Isolated DNA was quantified using the QuantiTect SYBR Green PCR Kit (Qiagen Sciences) and an iCycler iQ multicolor real-time PCR system (Bio-Rad, Richmond, CA). PCR cycles were 95°C for 15 seconds, 55°C for 30 seconds, and 72°C for 30 seconds. Cloned-actin cDNA was used as a standard for quantification using specific sense (5-TGCGTGACATTAAGGAGAAG-3) and antisense (5-GCTCGTAGCTCTTCTCCA-3) primers. Human-specific glyceraldehyde-3-phosphate dehydrogenase (h-GAPDH) primers (5-CAACGAATTTGGCTACAGCA-3 and 5-CCCCTCTTCAAGGGGTCTAC-3) were used to identify human DNA. Primers (5-ACCACAGTCCATGCCATCAC-3 and 5-TCCACCACCCTGTTGCTGTA-3) that amplify both murine and human GAPDH (m-GAPDH/h-GAPDH) DNA were used as loading controls. DNA samples from murine spleen/bone marrow cells added to human SEM-K2 cells at specific cell ratios were used as a standard curve for estimating the percentage of human cells. Gene copy numbers were determined from the threshold amplification cycle numbers using software supplied with the iCycler IQ Thermal Cycler. PCR products were visualized on a 2% agarose gel.

Histology. Tissue samples were fixed for >24 hours in 10% buffered formalin (Fisher Scientific, Fair Lawn, NJ), embedded in paraffin, and sectioned at 5 µm onto charged slides. Samples containing bone were decalcified with formic acid before sectioning. H&E staining was done on an automated stainer.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IMC-NC7 inhibits FLT3 activation along with downstream signaling pathways, whereas IMC-EB10 variably activates or inhibits these same pathways in ALL cell lines. FLT3 activation has been shown to play a role in leukemia, and inhibition of FLT3 activation in FLT3-dependent cells using TKIs results in apoptosis (8, 10–13, 23–27). The ability of IMC-EB10 and IMC-NC7 to affect FLT3 signaling was investigated using a number of ALL-derived cell lines expressing differing levels of cell surface FLT3 (Fig. 1A ). We first treated two ALL-derived cell lines (Karpas45 and NALM6), showing very low levels of FLT3 expression (Fig. 1A and B). Neither IMC-EB10 nor IMC-NC7 significantly affected the activation status of FLT3 or downstream signaling pathways when compared with IMC-C225 treatment (Fig. 1B, lanes 2 and 3 versus lane 1, lanes 5 and 6 versus lane 4). In contrast, IMC-NC7 treatment of ALL-derived cell lines expressing constitutively active FLT3-D835H (Hb1119) or constitutively active wt FLT3 (SEM-K2, REH, and RS411) resulted in significant suppression of FLT3 phosphorylation and activation of downstream pathways compared with treatment with control antibody, IMC-C225 (Fig. 1B, lane 9 versus lane 7, lane 12 versus lane 10, lane 15 versus lane 13, lane 18 versus lane 16). IMC-NC7-mediated inhibition of FLT3 phosphorylation is less dramatic in Hb1119 cells than in SEM-K2, REH, or RS411 cells possibly due to expression of FLT3 ligand–independent, mutant FLT3 on Hb1119 cells (Fig. 1B, lane 9 versus lanes 12, 15, and 18). IMC-EB10 treatment of Hb1119 and SEM-K2 cell lines also decreased the phosphorylation of FLT3 and downstream signaling proteins compared with IMC-C225 (Fig. 1B, lane 8 versus lane 7, lane 11 versus lane 10). However, when IMC-EB10 was incubated with REH and RS411 cell lines, FLT3 and downstream pathways were activated, possibly by favoring receptor dimerization upon antibody binding (Fig. 1B, lane 14 versus lane 13, lane 17 versus lane 16).


Figure 1
View larger version (32K):
[in this window]
[in a new window]
  		Figure 1. IMC-EB10 and IMC-NC7 can inhibit or activate FLT3 phosphorylation along with downstream STAT5, Akt, and MAPK signaling. A, ALL-derived cell lines were stained with CD135-PE or isotype control and analyzed by FACS. B, ALL-derived cell lines expressing wt FLT3 or FLT3-D835H (on Hb1119 cells) were treated with PBS (0), 10 µg/mL IMC-EB10 (E), or 10 µg/mL IMC-NC7 (N) for 1 hour. Immunoprecipitates and total protein extracts were resolved by 8% or 10% SDS-PAGE, respectively, and subjected to immunoblot analysis with the indicated phospho-specific antibodies. The same blots were then stripped and reprobed with protein-specific antibodies.

 
IMC-EB10 significantly reduces the engraftment of SEM-K2 cells in NOD/SCID mice but not of low FLT3-expressing Karpas45 and NALM6 cells. We previously found that IMC-NC7 has no in vivo efficacy in animal leukemia models due to a reduced ability to mediate antibody-dependent, cell-mediated cytotoxicity (ADCC; ref. 32). Therefore, only IMC-EB10 was further tested in vivo. A variety of treatment schedules of IMC-EB10 were used for NOD/SCID mice injected with SEM-K2 cells. Mice were injected i.p. with IMC-EB10 thrice per week starting 24 hours after cell injection, once starting 24 hours after cell injection, a total of thrice every other day starting 24 hours after cell injection or thrice per week starting 7 days after cell injection. A group of mice were treated with control antibody (IMC-C225) i.p. thrice per week starting 24 hours after cell injection. After 30 days after cell injection, mice were killed, and peripheral blood, spleen, and bone marrow cells were analyzed by flow cytometry. Each of the IMC-EB10 treatment schedules resulted in a significant reduction of human cells in the peripheral blood, spleen, and bone marrow compared with IMC-C225 treatment (Fig. 2A-C ). IMC-EB10 treatment typically reduced the presence of SEM-K2 cells to <1%. In contrast, IMC-EB10 treatment of NOD/SCID mice given REH or Hb1119 cells did not reduce leukemic engraftment (data not shown). IMC-EB10 also had no effect on reducing the engraftment of cell lines (Karpas45 and NALM6) expressing very low levels of FLT3 (data not shown).


Figure 2
View larger version (8K):
[in this window]
[in a new window]
  		Figure 2. IMC-EB10 significantly reduces engraftment of SEM-K2 cells in NOD/SCID mice. NOD/SCID mice given 0.5 x 106 SEM-K2 cells via tail vein injection were injected i.p. with 400 µg IMC-C225 thrice weekly starting 24 hours after cell injection as control treatment or with 400 µg IMC-EB10 thrice weekly starting 24 hours after cell injection, once starting 24 hours after cell injection, thrice every other day starting 24 hours after cell injection, or thrice weekly starting 7 days after cell injection. Thirty days after cell injection, mice were killed, and the presence of human cells was determined by flow cytometry using human-specific CD19-FITC and CD45-APC antibodies: (A) peripheral blood, (B) bone marrow, and (C) spleen.

 
IMC-EB10 prolongs survival of NOD/SCID mice given SEM-K2 cells. Mouse survival studies were done to determine if the reduced human engraftment shown in Fig. 2 would translate into improved survival. NOD/SCID mice given SEM-K2 cells were treated with IMC-C225 or IMC-EB10 using the same treatment regimens described previously. All IMC-EB10 treatment schedules resulted in significantly improved median survival compared with IMC-C225 treatment (Fig. 3A and B ).


Figure 3
View larger version (19K):
[in this window]
[in a new window]
  		Figure 3. IMC-EB10 prolongs survival of NOD/SCID mice given SEM-K2 cells. NOD/SCID mice given 0.5 x 106 SEM-K2 cells via tail vein injection were injected i.p. with 400 µg IMC-C225 thrice weekly starting 24 hours after cell injection as control treatment or 400 µg IMC-EB10 thrice weekly starting 24 hours after cell injection, once starting 24 hours after cell injection, thrice every other day starting 24 hours after cell injection, or thrice weekly starting 7 days after cell injection. A, mice were monitored daily for survival. B, mouse survival data from all IMC-EB10 treatment regimens combined and compared with IMC-C225 treatment. C, mice surviving 150 days after cell injection were killed and bone marrow and spleen cells analyzed by quantitative real-time PCR to determine the presence of human cells. A standard curve, where murine spleen/bone marrow cells were mixed with SEM-K2 cells at fixed ratios, was used to estimate the percentage of human cells.

 
To determine if IMC-EB10 treatment had eliminated all ALL cells in the mice surviving 150 days after cell injection (which comprise 19% of IMC-EB10-treated mice), those mice were killed and assessed for the presence of human cells using flow cytometry and/or quantitative real-time PCR. Fluorescence-activated cell sorting (FACS) analysis show that five of the six mice treated with IMC-EB10 that survived 150 days after cell injection had no detectable human cells in their spleen or bone marrow above background (data not shown). Quantitative real-time PCR analysis of these mice revealed that with the exception of one mouse treated with IMC-EB10 once starting 24 hours after cell injection, the percentage of human cells in the spleen or bone marrow of these mice is ≤0.001%, which was the limit of detection in the assay (Fig. 3C). DNA was not available from one mouse treated with IMC-EB10 thrice per week and surviving until day 150.

IMC-EB10 reduces the engraftment of ALL primary cells in NOD/SCID mice. To assess the ability of IMC-EB10 to reduce leukemia in vivo, we used NOD/SCID mice injected with ALL patient samples and treated them with PBS, IMC-C225, or IMC-EB10 thrice per week starting 24 hours after cell injection via i.p. injections. After 3 to 4 months, leukemic cells found in NOD/SCID mice derive from transplanted leukemic stem cells because only this small subpopulation of cells has the capability for sustained engraftment. After 3 to 4 months after cell injection, mice were killed, and the presence of human cells was determined by flow cytometry. IMC-EB10 treatments reduced the engraftment of two of four tested primary ALL samples in NOD/SCID mice; one of these samples is shown in Fig. 4A . Histologic analysis of the bone marrow from the IMC-EB10-treated mouse closest to the mean level of engraftment reveals normal appearing bone marrow with all lineages represented (Fig. 4B). In contrast, the bone marrow of mice treated with PBS or IMC-C225 are entirely replaced with sheets of leukemia cells. Western blot analysis of this ALL sample treated in vitro with IMC-EB10 shows activation of FLT3 and downstream Akt (Fig. 4C). Thus, activation of FLT3 by IMC-EB10 does not prevent the antibody from reducing leukemic engraftment in vivo (Fig. 4A and C).


Figure 4
View larger version (27K):
[in this window]
[in a new window]
  		Figure 4. IMC-EB10 significantly reduces engraftment of primary ALL blasts in NOD/SCID mice. NOD/SCID mice received 1 x 106 primary ALL blast via tail vein injection and were treated with 400 µg IMC-EB10 i.p. thrice weekly starting 24 hours after cell injection. Control mice received 200 µL PBS or 400 µg IMC-C225 i.p. thrice weekly starting 24 hours after cell injection. A, 14 weeks after cell injection, human engraftment was determined by flow cytometry using human-specific CD19-FITC and CD45-APC antibodies. B, bone marrow sections of mice were stained with H&E for morphology. C, ALL blasts were treated in vitro with 10 µg/mL IMC-C225 or IMC-EB10 for 1 hour. Immunoprecipitates and total protein extracts were resolved by 8% or 10% SDS-PAGE, respectively, and subjected to immunoblot analysis with the indicated phospho-specific antibodies. The same blots were then stripped and reprobed with protein specific antibodies.

 
IMC-EB10 treatment does not select for resistant cells in vivo. As shown in Fig. 2, IMC-EB10 treatment of NOD/SCID mice given SEM-K2 cells results in almost undetectable levels of human engraftment 30 days after cell injection. However, most of these IMC-EB10-treated mice eventually die of leukemia with symptoms (i.e., weight loss, white marrow, and large spleen) identical to those of IMC-C225-treated mice (Fig. 3; data not shown). To determine if IMC-EB10 treatment selects for resistant cells, a NOD/SCID mouse given SEM-K2 cells and treated thrice per week with IMC-EB10 that displayed symptoms of morbidity was killed 120 days after cell injection. Bone marrow from this mouse, consisting of 85% SEM-K2 cells as determined by FACS, was retransplanted into NOD/SCID mice. As a control, SEM-K2 cells grown in culture and not exposed to IMC-EB10 were also injected into NOD/SCID mice. Mice were injected once with IMC-C225 or IMC-EB10 24 hours after cell injection. Mice were killed 30 days after cell injection, and the presence of human cells was determined by flow cytometry. A single injection of IMC-EB10 was shown to reduce leukemic engraftment of the spleen or bone marrow to undetectable levels, even when the injected SEM-K2 cells are those that survived in an IMC-EB10-treated mouse (Fig. 5A and B ).


Figure 5
View larger version (8K):
[in this window]
[in a new window]
  		Figure 5. IMC-EB10 treatment of NOD/SCID mice given SEM-K2 cells do not select for resistant clones. NOD/SCID mice were injected via tail vein with 0.5 x 106 SEM-K2 cells grown in culture without IMC-EB10 or surviving IMC-EB10 treatment within NOD/SCID mice. Mice were injected once i.p. with 400 µg IMC-C225 or IMC-EB10 24 hours after cell injection. Thirty days after cell injection, mice were killed, and their (A) bone marrow and (B) spleen cells analyzed by flow cytometry for the presence of human cells using human-specific CD19-FITC and CD45-APC antibodies.

 
IMC-EB10 mediates cytotoxicity through natural killer cells in vivo. We previously showed that IMC-EB10 cytotoxicity is mediated in vitro through ADCC and requires natural killer (NK) cells. To investigate the role of NK cells in IMC-EB10-mediated cytotoxicity in vivo, we used Asialo GM1 antisera to reduce NK cell numbers in vivo (34–36). NOD/SCID mice were treated with an i.p. injection of 30 µL Asialo GM1 antisera + 70 µL PBS 3 days before and 4 and 11 days after SEM-K2 cell injection to suppress levels of NK cells. Pilot studies indicated that 3 days after a single injection of 30 µL Asialo GM1 antisera, NK cells in NOD/SCID mice were reduced by ~75% of their original level (data not shown). NOD/SCID mice injected with or without Asialo GM1 antisera were injected once i.p. with IMC-C225 or IMC-EB10 24 hours after SEM-K2 cells were injected. Thirty days after cell injection, mice were killed and assessed for the presence of human cells by flow cytometry. FACS analysis revealed that a reduction in NK cells numbers by Asialo GM1 antisera significantly impairs IMC-EB10-mediated cytotoxicity in vivo (Fig. 6A ).


Figure 6
View larger version (7K):
[in this window]
[in a new window]
  		Figure 6. IMC-EB10 mediates cytotoxicity in vivo through NK cells. A, NOD/SCID mice were injected with 0.5 x 106 SEM-K2 cells via tail vein and treated with 400 µg IMC-EB10 IP 24 hours after cell injection and/or with 30 µL of Asialo GM1 antisera given at days –3, +4, and +11 relative to when SEM-K2 cells were injected. Control mice were injected with 400 µg IMC-C225 i.p. 24 hours after cell injection. Mice were killed 30 days after cell injection and analyzed for human engraftment using flow cytometry and human-specific CD19-FITC and CD45-APC antibodies. B, NOD/SCID mice were injected with 0.5 x 106 RS411 cells via tail vein and treated with three doses of 400 µg IMC-C225 or IMC-EB10 i.p. every other day starting 24 hours after cell injection. Five days after cell injection, 125 µg poly (I:C) was injected i.p. Forty days after cell injection, mice were killed and analyzed for human engraftment using flow cytometry and human-specific CD19-FITC and CD45-APC antibodies.

 
Because Fig. 6A shows that NK cells mediate IMC-EB10 in vivo cytotoxicity, we explored the possibility of improving IMC-EB10's efficacy by activating NK cells in vivo; i.p. administration of poly (I:C) has been shown to activate NK cells in vivo, and pilot studies confirmed this observation (data not shown; refs. 37, 38). Pilot studies indicated that a single i.p. injection of 125 µg poly (I:C) given 5 days after leukemic cells are injected into NOD/SCID mice does not significantly reduce the engraftment of human cells (data not shown). Therefore, NOD/SCID mice given RS411 cells were injected i.p. with three doses of IMC-EB10 given every other day starting 24 hours after cell injection and/or 125 µg poly (I:C) given i.p. 5 days after cell injection. Control mice were treated with IMC-C225 24, 72 and 120 hours after cell injection. Mice were killed 40 days after cell injection, and human engraftment was examined by flow cytometry. IMC-EB10 treatment significantly reduced the engraftment of RS411 cells in NOD/SCID mice compared with IMC-C225 or poly (I:C) treatments (Fig. 6B). Furthermore, combining IMC-EB10 treatment with poly (I:C) resulted in a further decrease in human engraftment. Thus, the in vivo efficacy of IMC-EB10 can be improved by activating NK cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The high expression level of FLT3 on B-lineage ALL blasts and the roles that activated FLT3 plays in leukemogenesis give good credentials to FLT3 as a potential therapeutic target. A number of TKIs targeting FLT3 have been developed that possess both in vitro and in vivo efficacy, and several are being tested in clinical trials (23–27). Although FLT3 TKIs may prove useful in some clinical settings, nonetheless, they have limitations as therapeutic agents targeting FLT3-expressing leukemias: (a) All known FLT3 TKIs also have activity against a spectrum of other kinases, which can result in some toxicities at the concentrations required to induce cytotoxicity. (b) Chronic treatment with TKIs can lead to resistance as has been seen with Gleevac (29). (c) In the majority of cases of AML and ALL, FLT3 is not mutated, and it is not clear how important its signaling contributes to survival and proliferation in these cells. A mAb approach targeting FLT3 has several potential advantages. Compared with TKIs, mAbs have the added advantage of being specific, and thus less toxic, and of recruiting the host's immune system in inducing apoptosis and could also prove effective in the setting of FLT3 mutants selected for resistance to TKIs from chronic exposure.

We previously reported that IMC-EB10 and IMC-NC7 are two mAbs that bind FLT3 and block FLT3 ligand binding (31, 32). IMC-EB10 was shown to mediate ADCC in vitro and reduce engraftment of an AML primary sample in NOD/SCID mice without affecting the engraftment of normal cord blood CD34+ cells. In this report, we examined the in vitro and in vivo efficacy of using IMC-EB10 to treat ALL-derived cell lines and primary ALL samples and explored the mechanism employed by IMC-EB10 to induce cytotoxicity in vivo.

Leukemic cells consistently show up-regulated wt FLT3 expression/activation, although it is unclear how dependent they are on FLT3 signaling for survival (10–13). IMC-NC7 treatment consistently inhibited FLT3 phosphorylation, whereas IMC-EB10 treatment resulted in inhibition of FLT3 phosphorylation in some cell lines (Hb1119 and SEM-K2) but activation of FLT3 phosphorylation in others (REH and RS411). Both inhibition and activation of downstream phospho-STAT5, phospho-Akt, and phospho-MAPK correlated with FLT3 activation status in response to the antibodies. The bivalent nature of antibodies can result in the binding of two FLT3 monomers simultaneously, thus favoring receptor dimerization and activation. However, the ability of IMC-EB10 to induce FLT3 phosphorylation did not correlate with the level of surface FLT3 expression. The difference in the ability/inability of IMC-EB10 and IMC-NC7 to activate or inhibit FLT3 could result from differences in the epitopes recognized by each antibody.

Potentially, activation of FLT3 could provide a growth/survival advantage that could undermine attempts to induce cytotoxicity in leukemic cells. In any case, we could not find any evidence that either activation or inhibition of FLT3 signaling mediated by IMC-EB10 or IMC-NC7 treatment in vitro significantly affected ALL cell viability. One possible reason is that the antibodies may not reduce FLT3 phosphorylation beyond a critical threshold required to induce cytotoxicity. Even more likely, these ALL cell lines may not be completely dependent on FLT3 signaling for survival.

Although IMC-EB10 is not cytotoxic to ALL-derived cell lines in vitro, IMC-EB10 showed in vivo efficacy through ADCC. All IMC-EB10 treatment schedules significantly reduced the engraftment of SEM-K2 cells in NOD/SCID mice and prolonged their median survival compared with mice treated with a control antibody. The mice treated 7 days after leukemic injection had enhanced survival relative to control mice but shorter survival compared with mice whose treatment started 24 hours after injection. Reduced median survival due to delayed antibody treatment has also been observed with rituximab (39). This may be due to an increase in the ratio of leukemic cells to effector cells. Thus, combination treatment with chemotherapy first to reduce bulk disease may prove more effective.

Nineteen percent of the IMC-EB10-treated mice given SEM-K2 cells survived >150 days after cell injection. By quantitative real-time PCR and FACS analysis, those mice were found to have ≤0.001% human engraftment. Treatment with IMC-EB10 was also effective at reducing leukemic engraftment of two of four primary ALL samples at 3 to 4 months after leukemic cell injection, a time that reflects leukemic stem cell activity. This was true even when in vitro IMC-EB10 treatment resulted in activation of FLT3. This supports the hypothesis that IMC-EB10-mediated cytotoxicity is not dependent on inhibition of FLT3 signaling and is likely due to ADCC.

Although IMC-EB10 reduced the engraftment of several ALL-derived cell lines and primary samples, it was not efficacious against all cell lines and primary samples. IMC-EB10 treatment had no effect on NOD/SCID mice injected with two examples of low FLT3-expressing cells (Karpas45 and NALM6). This is not unexpected as a certain minimum level of surface FLT3 expression would be required to be effective, a phenomenon that has been observed with IMC-C225/EGFR and other antibodies (40). IMC-EB10 had no in vivo activity against the Hb1119 or REH cell lines nor against two of the four primary ALL samples that we tested, all of which express FLT3 at levels comparable with the cell lines and primary cells that were sensitive. The FLT3 activation status of these cells does not explain the inability of IMC-EB10 to kill these cells in vivo because IMC-EB10 was cytotoxic to an ALL primary sample and to RS411 cells in vivo, although in vitro IMC-EB10 treatment of these cells result in FLT3 activation. Expression of Fas ligand, MUC1, and/or tumor necrosis factor–related apoptosis-inducing ligand, which may have a role in blocking ADCC activity, has been associated with resistance to rituximab (41). However, FACS analysis of cell lines sensitive/resistant to IMC-EB10-mediated cytotoxicity did not show differential expression of these antigens. The more likely explanation would seem to be differences in their sensitivity to ADCC-induced apoptosis. The heterogeneous activity of IMC-EB10 has also been observed with other mAbs targeting CD20 or CD33 (42–45).

The development of resistance has been observed in patients treated with TKIs. Despite improved survival, most IMC-EB10-treated, SEM-K2-injected mice die, raising the possibility of selection of a resistant subpopulation of cells. However, retransplantation of leukemic cells surviving treatment showed they were still sensitive to IMC-EB10, indicating that treatment did not select for resistant clones.

One of the limitations of IMC-EB10 or any other mAb's efficacy in the NOD/SCID human leukemia model relates to the reduced NK cell activity of NOD/SCID mice (46). The multiple defects in innate immunity seen with NOD/SCID mice, thus, may underestimate the therapeutic potential of IMC-EB10. In addition, the in vivo efficacy of IMC-EB10 may be significantly greater in immune-competent hosts. We previously showed that NK cells are required for IMC-EB10-mediated in vitro ADCC against FLT3-expressing cells. In this report, we found that further decreasing the number of NK cells in vivo in NOD/SCID mice using Asialo GM1 antisera significantly interfered with the ability of IMC-EB10 to reduce the level of SEM-K2 cells. Moreover, activation of NK cells in vivo through administration of poly (I:C) improved the ability of IMC-EB10 to reduce the level of RS411 cells in NOD/SCID mice. Thus, these experiments further implicate NK cells as a key component in IMC-EB10-mediated cytotoxicity. In vivo activation of NK cells may well be a feasible method for improving the efficacy of IMC-EB10 against FLT3-expressing leukemias. In addition, IMC-EB10-mediated cytotoxicity will likely be less dependent on the host's immune system if IMC-EB10 is conjugated to a radionuclide or toxin as has been shown for a variety of antibodies (47).

In conclusion, the data show that IMC-EB10 reduces the engraftment and improves the survival of NOD/SCID mice injected with some ALL-derived cell lines and primary ALL samples. NK cell activity is a critical factor mediating IMC-EB10 cytotoxicity. Anti-FLT3 mAbs, like IMC-EB10, may prove efficacious against a wide range of FLT3 expressing malignancies, which include ALL, AML, and subsets of CML blast crisis, T-ALL, and chronic lymphocytic leukemia. These mAbs may also prove useful against TKI-resistant cells because their mode of action does not depend on inhibition of FLT3 signaling. Thus, mAbs targeting FLT3 are a specific and potentially effective therapeutic agent that should be further investigated and considered for clinical trials.


   Acknowledgments
 
Grant support: National Cancer Institute grants CA90668, CA70970, and CA100632 (D. Small); Leukemia and Lymphoma Society (D. Small); Burroughs Wellcome Trust (D. Small); and Grants RR00171 and CA62924 (D. Huso).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   Footnotes
 
Note: ImClone Systems, Inc. provided some of the funding for the studies reported here. In addition, several of the authors are employed by ImClone Systems, whose potential product was studied in this report.

Received 1/ 6/06. Accepted 2/16/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Rosnet O, Marchetto S, deLapeyriere O, Birnbaum D. Murine Flt3, a gene encoding a novel tyrosine kinase receptor of the PDGFR/CSF1R family. Oncogene 1991;6:1641–50.[Medline]
  2. Small D, Levenstein M, Kim E, et al. STK-1, the human homolog of Flk2/Flt-3, is selectively expressed in CD34+ human bone marrow cells and is involved in the proliferation of early progenitor/stem cells. Proc Natl Acad Sci U S A 1994;91:459–63.[Abstract/Free Full Text]
  3. Matthews W, Jordan CT, Wiegand GW, Pardoll D, Lemischka IR. A Receptor tyrosine kinase specific to hematopoietic stem and progenitor cell-enriched populations. Cell 1991;65:1143–52.[CrossRef][Medline]
  4. Lyman S, James L, Vanden Bos T, et al. Molecular cloning of a ligand for the flt3/flk-2 tyrosine kinase receptor: a proliferative factor for primitive hematopoietic cells. Cell 1993;75:1157–67.[CrossRef][Medline]
  5. Zhang S, Broxmeyer HE. Flt3 ligand induces tyrosine phosphorylation of gab1 and gab2 and their association with shp-2, grb2, and PI3 kinase. Biochem Biophys Res Commun 2000;277:195–9.[CrossRef][Medline]
  6. Zhang S, Fukuda S, Lee Y, et al. Essential role of signal transducer and activator of transcription (Stat)5a but not Stat5b for Flt3-dependent signaling. J Exp Med 2000;192:719–28.[Abstract/Free Full Text]
  7. Lavagna- Sevenier C, Marchetto S, Birnbaum D, Rosnet O. The CBL-related protein CBLB participates in FLT3 and interleukin-7 receptor signal transduction in pro-B cells. J Biol Chem 1998;273:14962–7.[Abstract/Free Full Text]
  8. Tse KF, Mukherjee G, Small D. Constitutive activation of FLT3 stimulates multiple intracellular signal transducers and results in transformation. Leukemia 2000;14:1766–76.[CrossRef][Medline]
  9. Lyman SD, Jacobsen SE. c-kit ligand and Flt3 ligand: stem/progenitor cell factors with overlapping yet distinct activities. Blood 1998;91:1101–34.[Free Full Text]
  10. Levis M, Small D. FLT3: ITDoes matter in leukemia. Leukemia 2003;17:1738–52.[CrossRef][Medline]
  11. Brown P, Small D. FLT3 inhibitors: a paradigm for the development of targeted therapeutics for paediatric cancer. Eur J Cancer 2004;40:707–21, discussion 722–4.
  12. Gilliland DG, Griffin JD. The roles of FLT3 in hematopoiesis and leukemia. Blood 2002;100:1532–42.[Abstract/Free Full Text]
  13. Stirewalt DL, Radich JP. The role of FLT3 in haematopoietic malignancies. Nat Rev Cancer 2003;3:650–65.[CrossRef][Medline]
  14. Nakao M, Yokota S, Iwai T, et al. Internal tandem duplication of the flt3 gene found in acute myeloid leukemia. Leukemia 1996;10:1911–8.[Medline]
  15. Abu-Duhier FM, Goodeve AC, Wilson GA, Care RS, Peake IR, Reilly JT. Identification of novel FLT-3 Asp835 mutations in adult acute myeloid leukaemia. Br J Haematol 2001;113:983–8.[CrossRef][Medline]
  16. Yamamoto Y, Kiyoi H, Nakano Y, et al. Activating mutation of D835 within the activation loop of FLT3 in human hematologic malignancies. Blood 2001;97:2434–9.[Abstract/Free Full Text]
  17. Zheng R, Levis M, Piloto O, et al. FLT3 ligand causes autocrine signaling in acute myeloid leukemia cells. Blood 2004;103:267–74.[Abstract/Free Full Text]
  18. Hawley TS, Fong AZ, Griesser H, Lyman SD, Hawley RG. Leukemic predisposition of mice transplanted with gene-modified hematopoietic precursors expressing flt3 ligand. Blood 1998;92:2003–11.[Abstract/Free Full Text]
  19. Drexler HG. Expression of FLT3 receptor and response to FLT3 ligand by leukemic cells. Leukemia 1996;10:588–99.[Medline]
  20. Carow CE, Levenstein M, Kaufmann SH, et al. Expression of the hematopoietic growth factor receptor FLT3 (STK-1/Flk2) in human leukemias. Blood 1996;87:1089–96.[Abstract/Free Full Text]
  21. Birg F, Courcoul M, Rosnet O, et al. Expression of the FMS/KIT-like gene FLT3 in human acute leukemias of the myeloid and lymphoid lineages. Blood 1992;80:2584–93.[Abstract/Free Full Text]
  22. Armstrong SA, Mabon ME, Silverman LB, et al. FLT3 mutations in childhood acute lymphoblastic leukemia. Blood 2004;103:3544–6.[Abstract/Free Full Text]
  23. Tse KF, Novelli E, Civin CI, Bohmer FD, Small D. Inhibition of FLT3-mediated transformation by use of a tyrosine kinase inhibitor. Leukemia 2001;15:1001–10.[CrossRef][Medline]
  24. Levis M, Allebach J, Tse KF, et al. A FLT3-targeted tyrosine kinase inhibitor is cytotoxic to leukemia cells in vitro and in vivo. Blood 2002;99:3885–91.[Abstract/Free Full Text]
  25. Yee KW, O'Farrell AM, Smolich BD, et al. SU5416 and SU5614 inhibit kinase activity of wild-type and mutant FLT3 receptor tyrosine kinase. Blood 2002;100:2941–9.[Abstract/Free Full Text]
  26. Weisberg E, Boulton C, Kelly LM, et al. Inhibition of mutant FLT3 receptors in leukemia cells by the small molecule tyrosine kinase inhibitor PKC412. Cancer Cell 2002;1:433–43.[CrossRef][Medline]
  27. O'Farrell AM, Abrams TJ, Yuen HA, et al. SU11248 is a novel FLT3 tyrosine kinase inhibitor with potent activity in vitro and in vivo. Blood 2003;101:3597–605.[Abstract/Free Full Text]
  28. Clark JJ, Cools J, Curley DP, et al. Variable sensitivity of FLT3 activation loop mutations to the small molecule tyrosine kinase inhibitor MLN518. Blood 2004;104:2867–72.[Abstract/Free Full Text]
  29. Gorre ME, Mohammed M, Ellwood K, et al. Clinical resistance to STI-571 cancer therapy caused by BCR-ABL gene mutation or amplification. Science 2001;293:876–80.[Abstract/Free Full Text]
  30. Harris M. Monoclonal antibodies as therapeutic agents for cancer. Lancet Oncol 2004;5:292–302.[CrossRef][Medline]
  31. Li Y, Li H, Wang MN, et al. Suppression of leukemia expressing wild-type or ITD-mutant FLT3 receptor by a fully human anti-FLT3 neutralizing antibody. Blood 2004;104:1137–44.[Abstract/Free Full Text]
  32. Piloto O, Levis M, Huso D, et al. Inhibitory anti-FLT3 antibodies are capable of mediating antibody-dependent cell-mediated cytotoxicity and reducing engraftment of acute myelogenous leukemia blasts in nonobese diabetic/severe combined immunodeficient mice. Cancer Res 2005;65:1514–22.[Abstract/Free Full Text]
  33. Levis M, Tse KF, Smith BD, Garrett E, Small D. A FLT3 tyrosine kinase inhibitor is selectively cytotoxic to acute myeloid leukemia blasts harboring FLT3 internal tandem duplication mutations. Blood 2001;98:885–7.[Abstract/Free Full Text]
  34. Young WW, Jr., Hakomori SI, Durdik JM, Henney CS. Identification of ganglio-N-tetraosylceramide as a new cell surface marker for murine natural killer (NK) cells. J Immunol 1980;124:199–201.[Abstract]
  35. Habu S, Fukui H, Shimamura K, et al. In vivo effects of anti-asialo GM1. I. Reduction of NK activity and enhancement of transplanted tumor growth in nude mice. J Immunol 1981;127:34–8.[Abstract]
  36. Kawase I, Urdal DL, Brooks CG, Henney CS. Selective depletion of NK cell activity in vivo and its effect on the growth of NK-sensitive and NK-resistant tumor cell variants. Int J Cancer 1982;29:567–74.[Medline]
  37. Djeu JY, Heinbaugh JA, Holden HT, Herberman RB. Augmentation of mouse natural killer cell activity by interferon and interferon inducers. J Immunol 1979;122:175–81.[Abstract/Free Full Text]
  38. Djeu JY, Heinbaugh JA, Vieira WD, Holden HT, Herberman RB. The effect of immunopharmacological agents on mouse natural cell-mediated cytotoxicity and on its augmentation by poly I:C. Immunopharmacology 1979;1:231–44.[CrossRef][Medline]
  39. Di Gaetano N, Cittera E, Nota R, et al. Complement activation determines the therapeutic activity of rituximab in vivo. J Immunol 2003;171:1581–7.[Abstract/Free Full Text]
  40. Patel D, Lahiji A, Melchior M, et al. Centuximab induces antibody-dependent cellular cytotoxicity in vitro which is dependent on EGFR levels. Keystone Symposia, Antibody-Based Therapeutics for Cancer. 2005; Abstract #129.
  41. Treon SP, Mitsiades C, Mitsiades N, et al. Tumor cell expression of CD59 is associated with resistance to CD20 serotherapy in patients with B-cell malignancies. J Immunother 2001;24:263–71.[CrossRef][Medline]
  42. Matsui H, Takeshita A, Naito K, et al. Reduced effect of gemtuzumab ozogamicin (CMA-676) on P-glycoprotein and/or CD34-positive leukemia cells and its restoration by multidrug resistance modifiers. Leukemia 2002;16:813–9.[CrossRef][Medline]
  43. Amico D, Barbui AM, Erba E, Rambaldi A, Introna M, Golay J. Differential response of human acute myeloid leukemia cells to gemtuzumab ozogamicin in vitro: role of Chk1 and Chk2 phosphorylation and caspase 3. Blood 2003;101:4589–97.[Abstract/Free Full Text]
  44. Golay J, Zaffaroni L, Vaccari T, et al. Biologic response of B lymphoma cells to anti-CD20 monoclonal antibody rituximab in vitro: CD55 and CD59 regulate complement-mediated cell lysis. Blood 2000;95:3900–8.[Abstract/Free Full Text]
  45. Smith MR. Rituximab (monoclonal anti-CD20 antibody): mechanisms of action and resistance. Oncogene 2003;22:7359–68.[CrossRef][Medline]
  46. Shultz LD, Schweitzer PA, Christianson SW, et al. Multiple defects in innate and adaptive immunologic function in NOD/LtSz-scid mice. J Immunol 1995;154:180–91.[Abstract]
  47. von Mehren M, Adams GP, Weiner LM. Monoclonal antibody therapy for cancer. Annu Rev Med 2003;54:343–69.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 BloodHome page
O. Piloto, M. Wright, P. Brown, K.-T. Kim, M. Levis, and D. Small
Prolonged exposure to FLT3 inhibitors leads to resistance via activation of parallel signaling pathways
Blood, February 15, 2007; 109(4): 1643 - 1652.
[Abstract] [Full Text] [PDF]

Home page
 ASH Education BookHome page
D. Small
FLT3 Mutations: Biology and Treatment
Hematology, January 1, 2006; 2006(1): 178 - 184.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Piloto, O.
Right arrow Articles by Small, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Piloto, O.
Right arrow Articles by Small, D.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online