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NEOPLASIA
From the Hematology Branch, National Heart, Lung, and
Blood Institute, and Laboratory of Pathology, Division of Clinical
Sciences, National Cancer Institute, National Institutes of Health,
Bethesda, MD.
Chromosomal translocations involving the platelet-derived growth
factor Constitutively activated tyrosine kinases are
increasingly being recognized as fusion oncoproteins in hematologic
malignancies, the best known example being bcr-abl in chronic
myelogenous leukemia (CML) associated with the Philadelphia
chromosome.1 More recently fusion proteins have been
described involving the platelet-derived growth factor The tyrosine kinases in the abl, PDGF Here we describe cloning of a new PDGF Patient
Cytogenetics
Metaphase fluorescence in situ hybridization (FISH) was also performed on marrow. Fresh slides were made and G-banded, and images were captured as described above. The slides were then destained and dehydrated in an ethanol series (70%, 85%, 100%), immersed in 70% formamide/2 × standard sodium citrate (SSC) for 5 minutes, and hybridized with either LSI CSF1R SpectrumOrange/D5S721, D5S23 SpectrumGreen, or LSI p53 SpectrumOrange probes (VYSIS, Downers Grove, IL). After 24 hours of incubation at 37°C, the slides were washed in 50% formamide/2 × SSC at 45°C. The signals were visualized using a 4',6-diamidino-2-phenylindole-2HCl (DAPI) counterstain and a Zeiss fluorescence microscope with a Chroma filter set that includes DAPI, fluorescein isothiocyanate (FITC), and rhodamine filters. Digital images were recaptured on Cytovision. Southern blotting For Southern blotting on patient material, 1.5 × 107 mononuclear bone marrow cells were isolated by Ficoll sedimentation from fresh bone marrow. Peripheral blood mononuclear cells from a healthy volunteer were used as a control. After overnight incubation in proteinase K, genomic DNA was phenol-chloroform extracted using standard methods.22 DNA (10 µg) from the patient and control were restriction enzyme digested overnight at 37°C with either BamHI (Promega, Madison, WI), EcoRI (Promega, WI), HindIII (Boehringer Mannheim, Mannheim, Germany), or PvuII (Boehringer Mannheim). After electrophoretic separation on a 1% agarose gel and overnight transfer to HYbond N nylon membrane (Amersham, Arlington Heights, IL), the membrane was probed with a 1.1-kb (HindIII-XhoI fragment) genomic PDGF R
probe23 (generously provided by Dr Gary Gilliland [Boston, MA]), using standard Southern blot technique.22
The probe was 32P-labeled using Prime-It RmT random primer
labeling kit (Stratagene, La Jolla, CA). The membrane was then exposed
to a Storm 860 PhosporImager (Molecular Dynamics, Sunnyvale, CA). For
Southern blotting on mouse splenic cells, spleens were gently crushed
through a nylon mesh and DNA was extracted as described above. DNA was
digested with BamHI before electrophoresis and blotting. A
300-bp polymerase chain reaction (PCR)-generated probe against
enhanced green fluorescence protein (eGFP) was used to probe
the membrane.
Molecular cloning of breakpoint The t(5;17) breakpoint was molecularly characterized using a 5'/3' RACE (rapid amplification of complementary DNA [cDNA] ends) kit (Roche Molecular Biochemicals, Indianapolis, IN) as described initially by Frohman.24 Briefly, total messenger RNA (mRNA) from patient peripheral blood mononuclear cells (Ficoll separated) and normal control bone marrow stroma was extracted using RNA-STAT (Tel-Test, Friendswood, TX) according to the manufacturer's protocol. Then mRNA from patient peripheral blood mononuclear cells was extracted using QuickPrep Micro mRNA purification kit (Amersham Pharmacia Biotech, Piscataway, NJ). Total RNA and mRNA were reverse transcribed using AMV reverse transcriptase and reverse PDGFR primer
2369PR (5'-TAGATGGGTCCTCCTTTGGTG-3').5 After cDNA
purification (High Pure PCR purification kit, Roche Molecular
Biochemicals), a 5' poly-A tail was appended using terminal
transferase. The tailed cDNA was amplified using Expand High Fidelity
polymerase (Roche Molecular Biochemicals) using the following PCR
conditions: 94°C for 2 minutes, 94°C for 15 seconds, annealing at
58°C for 30 seconds, elongation for 40 seconds, cycle elongation of
20 second for each cycle after 10 cycles (for a total of 35 cycles) using the forward Qo-T16V RACE primer
(5'-GACCACGCGTATCGATGTCGACT(16)V-3') and a reverse PDGFR
primer PDGFR-Po (5'-GTAACGTGGCTTCTTCTGCCA-3').5
The diluted first-round PCR product (1:20) was reamplified in a nested PCR reaction with the same conditions as the first round using a
forward Qi RACE primer (5'-GACCACGCGTATCGATGTCGAC-3') and a reverse
PDGFR primer PDGFR-Pi
(5'-TGAGGATGAGAAGGGAGATGATGG-3').5 The nested PCR products
from both patient and healthy control were TA-cloned into pCR2.1-TOPO
vector (TOPO TA Cloning Kit, Invitrogen, Carlsbad, CA) and vector
inserts from selected colonies were sequenced using an automated
sequencer. The DNA sequences were analyzed by the BLAST algorithm
(http://www.ncbi.nlm.nih.gov/BLAST/).
Reverse transcription-PCR of fusion breakpoints or native
Rabaptin-5 and PDGF R 3' and 5' PDGFR/Rabaptin-5 3') were
designed. Patient, negative control total RNA (1 µg), and negative
control water were reverse transcribed using MuLV Reverse Transcriptase
and Random Hexamers primers (Perkin-Elmer, Norwalk, CT). The cDNA was
amplified using Taq DNA polymerase (Perkin-Elmer) in a
nested PCR using the following cycle conditions for both outer and
inner cycle: 95°C for 2 minutes followed by 35 cycles of 95°C for 1 minute, 60°C for 1.5 minutes, 72°C for 2 minutes, and final
extension of 72°C for 8 minutes. Primers for amplifying the
5'Rabaptin-5/PDGFR 3' breakpoint were: RP2151F
(5'-AAGCACAGCCTGCATGTGTC-3'), RP2574R (5'-GGTCCACGTAGATGTACTCA-3')
(outer) and RP2269F (5'-CAGCAGACCACGTAGAAGAA-3'), RP2433R
(5'-CTGAGATCACCACCACCTTA-3') (inner). The 5' PDGFR/Rabaptin-5 3'
primers were: PR1684F (5'-AGCCGAACATCATCTGGTCT-3'), PR2106R (5'-GCTGTTCAACGGTAGCCTTA-3') (outer) and PR1785F
(5'-GAGACTAACGTGACGTACTG-3'), PR2034R (5'-GACTCTCAAGC- TGTTGAGAC-3')
(inner). The same PCR conditions were used to amplify the native genes,
PDGFR or rabaptin-5. Primers PR1684F, RP2574R
(outer) and PR1785F, RP2433R (inner) were used for PDGFR.
Primers RP2151F, PR2106R (outer) and RP2269F, PR2034R (inner) were used
for rabaptin-5.
Construction of a Rabaptin-5/PDGF R expression
plasmid, Rabaptin-5 cDNA sequences were excised from a plasmid
(provided by Dr Marino Zerial, Heidelberg, Germany) and PDGFR cDNA
sequences from a tel/PDGFR retroviral vector in the MSCV
retroviral backbone containing both the tel/PDGFR and eGFP cDNA with
an IRES25 (provided by Dr Gary Gilliland, Boston, MA). The
tel-PDGFR sequence was flanked by EcoRI
restrictions sites. To generate a unique 5' restriction site, the MSCV
plasmid was partially digested with EcoRI, generating a
singly cut plasmid, and a oligonucleotide linker containing a
SwaI site and EcoRI sticky ends was ligated into
the plasmid. The cDNA generated from patient bone marrow cells was
amplified using reverse transcription (RT)-PCR to generate a 603-bp
fragment spanning the Rabaptin-5/PDGFR breakpoint. The breakpoint
region included unique SacII (in the PDGFR
portion of the gene) and SapI (in the rabaptin-5
portion of the gene) restriction enzyme sites. Furthermore, another
SacII site was generated at the 5' end of the PCR product
using a tailed PCR primer. The amplified breakpoint product was
SacII digested and ligated into the SacII site of
tel/PFGDR. After ligation, the breakpoint sequence and orientation
were confirmed by sequencing. To generate the full-length
Rabaptin-5/PDGFR construct, the full-length Rabaptin-5 plasmid was
digested using SpeI (upstream of the 5' end and Kozak
consensus sequence) and BamHI (downstream of the SapI site). The resulting 2.5-kb fragment was blunt-ended
using Pfu DNA polymerase (PCR Polishing Kit, Stratagene) and
subsequently digested with SapI. This fragment containing a
5' blunt end and 3' SapI site was ligated into the MSCV
plasmid containing the tel-PDGFR with the Rabaptin-5/PDGFR
breakpoint after digestion with SapI and SwaI
(blunt end cutting enzyme). Correct ligation of all ligation sites was
confirmed by sequencing. We also generated a mutant
rabaptin-5/PDGFR containing a Lys>Arg mutation in amino acid 635 of the PDGFR. This mutant renders the tyrosine
kinase domain of the PDGFR inactive.9 The
Lys635Arg tel/PDGFR plasmid (provided by Gary Gilliland, Boston, MA)
was SacII/BamH1 digested and the resulting 884-bp
fragment ligated into the same restriction sites of the
Rabaptin-5/PDGFR MSCV plasmid.
Transformation of Ba/F3 cells Bosc-23 cells were transfected with the MSCV plasmids using calcium phosphate precipitation.26 Retroviral supernatant was collected 48 hours after transfection, filtered (Millex HV, 45 µm) and 1 × 106 Ba/F3 cells incubated per 1 mL supernatant with 10 µg/mL polybrene. Twenty-four hours later cells were placed in fresh RPMI media, with 10% fetal calf serum (FCS) and interleukin-3 (IL-3) (1 ng/mL). Then, 48 hours after retroviral infection the cells were flow sorted and GFP+ cells were collected and expanded in RPMI media with 10% FCS and 10% WEHI-conditioned media, as a source of IL-3.Murine bone marrow transplant Six days before harvesting BALB/c bone marrow cells, 5-fluorouracil (Fluka, Milwaukee, WI), 150 mg/kg, was administered by intraperitoneal injection. Bone marrow was harvested by flushing femurs and tibia and 9 × 106 bone marrow cells were plated on RetroNectin-coated plates (100 mm; Takara, Japan) in 10 mL Dulbecco modified Eagle medium (DMEM) containing 10% FCS, murine IL-3 (10 ng/mL), human-IL-6 (50 ng/mL), human-Flt-3 (100 ng/mL), and rat stem cell factor (SCF; 100 ng/mL) for 48 hours. At 48 and 72 hours, 5 mL media was replaced with retroviral supernatant from transiently transfected Bosc-23 cells (as described above) supplemented with the same cytokines as during the 48-hour prestimulation. At 96 hours the cells were collected and 1 × 106 cells were injected into the tail vein of lethally irradiated BALB/c mice (radiation dose: 450 cGy twice, 6 hours apart). Two weeks after transplantation blood counts were monitored biweekly by retro-orbital phlebotomy. Premorbid diseased animals were killed by CO2 asphyxiation.Western blotting Ba/F3 cells were washed in phosphate-buffered saline (PBS) and suspended in lysis buffer containing complete protease inhibitor cocktail (Boehringer Mannheim). After addition of sodium dodecyl sulfate (SDS) sample buffer and boiling, the samples were run on 8% SDS-polyacrylamide gels, transferred to a nitrocellulose membrane, and immunoblotted using a rabbit anti-PDGFR antibody (P-20, C-terminal;
1:2000; Santa Cruz Biotechnology, Santa Cruz, CA), followed by a
secondary antirabbit horseradish peroxidase (HRP)-conjugated antibody
(1:20 000). Bound antibodies were detected with SuperSignal (Pierce,
Rockford IL) enhanced luminol and oxidizing reagent as specified by the manufacturer.
A novel translocation in a patient with CMML involving the
PDGF R loci on 5q (Figure 1B), and also
telomeric to p53 on 17p (Figure 1C).
Southern blot analysis on patient DNA from bone marrow cells using a
genomic PDGF
Cloning of the t(5;17)(q33;p13) breakpoints To identify the fusion partner of the translocated PDGFR, 5'RACE-PCR (Figure
3A) was performed. Briefly, patient and
normal marrow stroma control mRNA were reverse transcribed and a
5'poly-A tail was added. cDNA was amplified, using nested PCR with
forward RACE primers and reverse PDGFR-specific primers. The normal
stroma mRNA resulted in a strong band (Figure 3B) and subsequent
cloning and sequencing confirmed amplification of the native
PDGFR. Use of 5'RACE on patient-derived total mRNA and
polyA mRNA failed to reveal an amplified band, but instead showed an
amplification smear (Figure 3B). The PCR amplification product from
both the poly-A and total mRNA samples was TA-cloned and 24 isolated
colonies were subsequently purified and sequenced. One of 24 colonies
contained a 454- nucleotide insert with a partial PDGFR
sequence fused to a novel gene partner. None of the other 23 colonies
contained a partial PDGFR sequence. BLAST search on the
454-nucleotide insert revealed the 3' PDGFR sequence to
be fused to rabaptin-5. Previous physical mapping has
localized rabaptin-5 to band 17p13 near, but telomeric to
p53. Our FISH analysis showing p53 is centromeric to the breakpoint on 17p (Figure 1C) supports the finding that PDGFR is fused to rabaptin-5. Analysis of the
breakpoint sequence revealed an in-frame fusion (Figure 3C). RT-PCR
using primers spanning the breakpoint on patient and normal bone marrow
stroma mRNA revealed patient-specific expression of the 5'
Rabaptin-5/PDGFR 3' (Figure 4),
confirming the 5'RACE-PCR results. The reciprocal fusion construct,
that is, 5' PDGFR/Rabaptin-5 3', was not detected but both native
PDGFR and Rabaptin-5 mRNA were expressed in patient and normal bone
marrow stroma (Figure 4).
Rabaptin-5/PDGF R fusion protein occurs at amino acid residue 739, fusing 85% of the native Rabaptin-5 to the transmembrane and
intracellular portion of the PDGFR. The novel fusion protein has
1318 amino acids and a predicted molecular weight of 150 kd. It
includes 3 and one-half of the 4 coiled-coil domains, the Rab4
and tuberous sclerosis binding sites, and the caspase-3 cleavage sites,
but lacks the Rab5 binding site (Figure 5).
Rabaptin-5/PDGF R, we
generated a bicistronic MSCV-based retroviral plasmid containing Rabaptin-5/PDGFR, as well as eGFP. We also generated a
kinase-inactive Rabaptin-5/PDGFR mutant, carrying a single amino
acid substitution in the active tyrosine kinase domain of the PDGFR
portion of fusion protein. These constructs were compared to the
previously described fusion oncogene, tel/PDGFR, and an
empty vector carrying only eGFP. The IL-3-dependent murine
hematopoietic cell line Ba/F3 was infected with these retroviral
vectors. As shown (Figure 6A), cells
infected with the 4 different vectors all had a similar growth curve in
the presence of IL-3. When IL-3 was not present, the cells infected
with the MSCV empty vector (eGFP alone) and the kinase-inactive mutant
of Rabaptin-5/PDGFR died. This growth pattern in the absence or
presence of IL-3 was identical to what was seen in Ba/F3 cells without
retroviral infection (data not shown). In contrast, cells infected with
either Rabaptin-5/PDGFR or tel/PDGFR were IL-3 independent
(Figure 6A). The Rabaptin-5/PDGFR and tel/PDGFR transduced cells
had a similar growth rate without IL-3. The expression of the fusion
genes was confirmed by Western blotting (Figure 6B). The IL-3
independence of these cells suggests a transforming property of the
novel PDGFR fusion oncogene. The lack of effects of the
kinase-inactive mutant confirms the importance of the kinase domain,
suggesting that the novel fusion protein transforms cell lines through
constitutive activation of the tyrosine kinase domain, similar to other
tyrosine kinase fusion oncogenes.
To confirm the transforming property of rabaptin-5/PDGF
Fusion oncogenes generated as a consequence of reciprocal
chromosomal translocations are commonly seen in hematologic
malignancies. The consequence is invariably disruption of key
regulatory pathways involved in cell growth or survival. Fusion
oncoproteins in myeloproliferative disorders commonly involve
deregulated protein tyrosine kinases such as abl, PDGF Clathrin-mediated endocytosis is a process by which cells internalize selected components of the plasma membrane, thus clearing receptor-bound hormones and growth factors, internalizing channels, and transporters and recycling synaptic vesicles.18 The net result of clathrin-mediated endocytosis with regard to mitogenic signaling is attenuation, through the down-regulation of surface signaling receptors.27 Rab proteins are small GTPases that regulate vesicular transport in endocytosis and exocytosis pathways, where they temporally and spatially coordinate the vesicular transport process. To date more than 40 distinct Rab proteins have been identified; each is believed to be associated with a particular organelle or pathway. Twelve Rab proteins have been localized to the endocytic pathway, including the Rabaptin-5 interacting proteins, Rab4 and Rab5. They are both present in early endosomes, Rab4 in early and recycling endosomes and Rab5 in clathrin-coated vesicles and early endosomes.28 The Rab proteins are tightly regulated by accessory proteins that modulate Rab protein activity by controlling membrane association, nucleotide binding, and hydrolysis.29 Rabaptin-5 is a critical accessory protein for Rab5 and Rab4. Rabaptin-5 exists as a homodimer (mediated by coiled-coil domains) in the cell cytosol,15 and is recruited to early endosomes by Rab5 in a GTP-dependent manner.14 There, Rabaptin-5 stabilizes Rab5 in a GTP-bound active form by down-regulating GTP hydrolysis.30 Once in the early endosomes, Rabaptin-5, through its interaction with Rab5, is an essential and rate-limiting component of the endocytic process required for homotypic fusion between early endosomes and heterotypic fusion between clathrin-coated vesicles and early endosomes.14,31 There is increasing evidence that disruption of the endocytosis process might have a role in malignant transformation.27,32 Mutations in internalization domains of both epidermal growth factor receptors and granulocyte colony-stimulating factor receptors (found in patients with Kostman syndrome developing acute myelogenous leukemia) have been shown to transform cell lines.33,34 Further evidence for a role of disruption in endocytotic pathways in hematologic malignancies is the presence of other endocytosis-related proteins in chromosomal translocations. AF-1p (Eps15)35 and EEN (SH3p8)36 are both fused as C-terminal partners to MLL, a gene commonly involved in a variety of hematologic malignancies. The CALM (AP180) gene has been found as an N-terminal fusion partner to the AF10 gene both in the human cell line U93737 and in patients with hematologic malignancies.38 Finally, the tumor suppressor tuberin (tuberous sclerosis complex-2) is a Rab5 GTPase activating (GAP) protein, and interacts directly with Rabaptin-5.17 The tumor suppressor activity of tuberin is encoded by functional domains in the C-terminus that contains the GAP activity.39 The previously described PDGF In summary, we have cloned a novel PDGF
We are grateful to Gary Gilliland and Ema Anastasiadou (Boston, MA)
for providing tel-PDGF
Submitted December 26, 2000; accepted June 11, 2001.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Magnus K. Magnusson, Hematology Branch, National Heart, Lung, and Blood Institute, Bldg 10, Rm 7C103, 9000 Rockville Pike, Bethesda, MD 20892; e-mail: magnussm{at}nhlbi.nih.gov.
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B. Delaval, S. Letard, H. Lelievre, V. Chevrier, L. Daviet, P. Dubreuil, and D. Birnbaum Oncogenic Tyrosine Kinase of Malignant Hemopathy Targets the Centrosome Cancer Res., August 15, 2005; 65(16): 7231 - 7240. [Abstract] [Full Text] [PDF]
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A. Tefferi and D. G. Gilliland The JAK2V617F Tyrosine Kinase Mutation in Myeloproliferative Disorders: Status Report and Immediate Implications for Disease Classification and Diagnosis Mayo Clin. Proc., July 1, 2005; 80(7): 947 - 958. [Abstract] [PDF]
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A. Pardanani and A. Tefferi Imatinib targets other than bcr/abl and their clinical relevance in myeloid disorders Blood, October 1, 2004; 104(7): 1931 - 1939. [Abstract] [Full Text] [PDF]
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J. Chen, I. R. Williams, J. L. Kutok, N. Duclos, E. Anastasiadou, S. C. Masters, H. Fu, and D. G. Gilliland Positive and negative regulatory roles of the WW-like domain in TEL-PDGF{beta}R transformation Blood, July 15, 2004; 104(2): 535 - 542. [Abstract] [Full Text] [PDF]
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C. Morerio, M. Acquila, C. Rosanda, A. Rapella, C. Dufour, F. Locatelli, E. Maserati, F. Pasquali, and C. Panarello HCMOGT-1 Is a Novel Fusion Partner to PDGFRB in Juvenile Myelomonocytic Leukemia with t(5;17)(q33;p11.2) Cancer Res., April 15, 2004; 64(8): 2649 - 2651. [Abstract] [Full Text] [PDF]
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J. L. Vizmanos, F. J. Novo, J. P. Roman, E. J. Baxter, I. Lahortiga, M. J. Larrayoz, M. D. Odero, P. Giraldo, M. J. Calasanz, and N. C. P. Cross NIN, a Gene Encoding a CEP110-Like Centrosomal Protein, Is Fused to PDGFRB in a Patient with a t(5;14)(q33;q24) and an Imatinib-Responsive Myeloproliferative Disorder1 Cancer Res., April 15, 2004; 64(8): 2673 - 2676. [Abstract] [Full Text] [PDF]
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K. Wilkinson, E. R. P. Velloso, L. F. Lopes, C. Lee, J. C. Aster, M. A. Shipp, and R. C. T. Aguiar Cloning of the t(1;5)(q23;q33) in a myeloproliferative disorder associated with eosinophilia: involvement of PDGFRB and response to imatinib Blood, December 1, 2003; 102(12): 4187 - 4190. [Abstract] [Full Text] [PDF]
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J. Sohal, V. T. Phan, P. V. Chan, E. M. Davis, B. Patel, L. M. Kelly, T. J. Abrams, A. M. O'Farrell, D. G. Gilliland, M. M. Le Beau, et al. A model of APL with FLT3 mutation is responsive to retinoic acid and a receptor tyrosine kinase inhibitor, SU11657 Blood, April 15, 2003; 101(8): 3188 - 3197. [Abstract] [Full Text] [PDF]
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 J. F. Apperley, M. Gardembas, J. V. Melo, R. Russell-Jones, B. J. Bain, E. J. Baxter, A. Chase, J. M. Chessells, M. Colombat, C. E. Dearden, et al. Response to Imatinib Mesylate in Patients with Chronic Myeloproliferative Diseases with Rearrangements of the Platelet-Derived Growth Factor Receptor Beta N. Engl. J. Med., August 15, 2002; 347(7): 481 - 487. [Abstract] [Full Text] [PDF]
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M. K. Magnusson, K. E. Meade, R. Nakamura, J. Barrett, and C. E. Dunbar Activity of STI571 in chronic myelomonocytic leukemia with a platelet-derived growth factor beta receptor fusion oncogene Blood, July 18, 2002; 100(3): 1088 - 1091. [Abstract] [Full Text] [PDF]
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| Copyright © 2001 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
receptor in chronic myelomonocytic leukemia
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Abstract
Introduction
(PML-RAR
