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`
`Non-transferrin-bound iron in myelodysplastic syndromes: A marker of
`ineffective erythropoiesis?
`
`Article  in  The Hematology Journal · February 2000
`DOI: 10.1038/sj/thj/6200028 · Source: PubMed
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`The Hematology Journal (2000) 1, 153 158
`© 2000 The European Haematology Association All rights reserved 1466 4680/00 $15.00 a
`www.nature.com/thj
`
`Non-transferrin-bound iron in myelodysplastic syndromes: a marker of
`ineffective erythropoiesis?
`
`Agostino Cortelezzi*:'*, Chiara Cattaneo'*, Silvia Cristiani', Lorena Duca’, Barbara Sarina’,
`Giorgio L Deliliers*, Gemino Fiorelli? and Maria Domenica Cappellini?
`
`‘Servizio Autonomo di Ematologia Diagnostica, Ospedale Maggiore, IRCCS, Milano, Italy; ?Centro Anemie Congenite,
`Dipartimento di Medicina Interna, Ospedale Maggiore, IRCCS, Milano, Italy; *Centro Trapianti di Midollo, Ospedale Maggiore,
`IRCCS, Milano, Italy
`
`Introduction:
`Iron overload is usually observed in patients (even untransfused) with
`myelodysplastic syndromes (MDS), and contributes towards the generation of low molecular
`weight iron complexes or non-transferrin-bound iron (NTBI), which in turn favors oxidative
`DNA damage and consequent apoptosis.
`Materials and methods: Levels of NTBI and lipid peroxidation were evaluated by means of
`free serum malondyaldehyde (MDA)in untransfused MDSpatients and wetried to correlate
`them with ineffective erythropoiesis, apoptosis and the pattern of in vitro growth.
`Results: NTBI levels were found to be significantly higher in low-risk than in high-risk MDS
`patients, as well as in patients with a lower myeloid/erythroid ratio. MDA was found to be
`uniformly higher in the MDSpatients as a whole than in normal controls. The bone marrow
`progenitor cells in the MDSpatients with high NTBI levels showed a higher degree of
`apoptosis, but this difference was notstatistically significant. Patients with a leukemic growth
`pattern had lower NTBI levels than those with a non-leukemic pattern.
`Conclusion: These data suggest
`ineffective
`that NTBI
`is
`related to the degree of
`erythropoiesis and that it contributes towards inducing apoptosis in MDS bone marrow
`precursors. The presence of leukemic growth is associated with low NTBI levels, probably
`due to increased iron consumption by blastcells.
`The Hematology Journal (2000) 1, 153-158
`
`Keywords:
`
`non-transferrin-bound iron; myelodysplastic syndromes; dyserythropoiesis; apoptosis
`
`Introduction
`
`Iron overload, due to the presence of ineffective
`erythropoiesis and to the increased absorption follow-
`ing the anemic state,
`is usually observed in patients
`affected by myelodysplastic syndromes (MDS), even
`untransfused.' Jensen et al.? have demonstrated that
`iron chelation by desferrioxamine in transfusion-
`dependent MDS patients can improve hemoglobin
`levels by reducing transfusion need,
`thus indirectly
`suggesting
`that
`iron overload may worsen the
`ineffective erythropoiesis and the level of anemia.
`Excess body iron contributes towards the generation
`of low molecular weight
`iron complexes or non-
`
`*Correspondence: A Cortelezzi, Servizio Autonomo di Ematologia
`Diagnostica, Ospedale Maggiore Policlinico, Via F. Sforza 35, 20 122
`Milano,Italy;
`Tel: +39 (0) 0255 033429/3345; Fax: +39 (0) 02550 033380;
`E mail: cortelez@polic.cilea.it
`*A Cortelezzi and C Cattaneo contributed equally to this work
`Received 29 September 1999; accepted 22 February 2000
`
`transferrin-bound iron (NTBI), which in turn favors
`the formation of potentially toxic oxygen derivatives.>*
`NTBI
`is more readily taken up by cells
`than
`transferrin-bound iron is,» and contributes towards
`increasing the labile pool of cytoplasmic iron (LIP).°
`The degree of cell damage induced by NTBI can be
`deduced from the level of lipid peroxidation, which is
`knownto be a consequence ofan increase in LIP
`Increased bone barrow (BM) cell apoptosis is a
`common finding in MDS, particularly in patients at
`low risk of leukemic evolution.’ '° Although many
`factors are likely to be involved in this process, such as
`an increased ratio of pro- vs anti-apoptotic proteins
`(bel2 family),"' the real etiopathogenesis of MDSis far
`from being completely understood. It has recently been
`reported that the high serum levels of tumor necrosis
`factor-a (TNF-a) observed in MDSpatients may lead
`to apoptosis by inducing the generation of free
`radicals, which can induce oxidative DNA damage.” "*
`In particular, Peddie et al.'? have demonstrated that
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`NTBI levels in myelodysplastic syndromes
`A Cortelezzi et al
`
`154
`
`MDS CD34* cells have an increased level of oxidized
`pyrimidine nucleotides.
`the levels of
`On the basis of these observations,
`NTBI and malondyaldehyde (MDA), an index oflipid
`peroxidation, were evaluated in serum taken from
`untransfused MDSpatients, with the aim of revealing
`their possible role in ineffective erythropoiesis and
`apoptosis.
`
`Materials and methods
`
`Patients
`
`Thirty-three untransfused patients with primary MDS
`(36 to 88 years old, median age 70) and ten normal
`controls were
`enrolled after having given
`their
`informed consent. The patients wereclassified accord-
`ing to the FABcriteria as having refractory anemia
`(RA, 12 cases), RA with ring sideroblasts (RARS,nine
`cases), RA with excess of blasts (RAEB, 10 cases), and
`RAEBin transformation (RAEB-t, two cases). Patients
`with RA or RARSwere classified as low risk (LR),
`and those with RAEB or RAEB-t as high risk (HR).
`The characteristics of the patients are summarized in
`Table 1.
`
`Table 1 Patient characteristics
`
`Diagnosis
`RA
`
`RARS
`
`RAEB
`
`RAEB t
`
`Patient
`1
`2
`3
`4
`5
`6
`7
`8
`9
`10
`ll
`12
`13
`14
`15
`16
`17
`18
`19
`20
`21
`22
`B
`24
`25
`26
`27
`28
`29
`30
`31
`32
`33
`
`Sex
`M
`F
`M
`F
`F
`F
`F
`F
`F
`M
`M
`F
`F
`M
`M
`M
`M
`M
`F
`M
`F
`F
`M
`M
`F
`F
`F
`M
`F
`M
`F
`F
`M
`
`Caryotype
`46XY
`46XX
`nv.
`n.v.
`46XX
`46XX
`45XX, 7
`47XX, +8
`46XX
`46XY
`46XY
`46XX
`46XX
`46XY
`46XY
`46XY
`46XY
`46XY
`46XX
`n.v.
`46XX
`46XX
`46XY
`46XY
`46XX, 5q
`46XX
`nv.
`46XY
`nv.
`46XY
`47XX, +8
`46XX
`46XY
`
`%Blasts
`3.5
`2
`2
`3.75
`2
`1.75
`2.25
`1
`1
`4.25
`5
`3
`3.5
`0.25
`1.25
`2.25
`1
`1.25
`3.75
`1.5
`3.5
`11.25
`7
`13
`6
`6
`13.75
`17
`6.75
`20.25
`5.75
`28
`26.75
`
`F female; M male; RA refractory anemia; RAEB refractory
`anemia with excess of blasts; RAEBt
`refractory anemia with excess
`of blasts in transformation; RARS refractory anemia with ring
`sideroblasts.
`
`
`The Hematology Journal
`
`NTBIlevels
`
`Peripheral blood serum was obtained by centrifugation
`at 3000 r.p.m. Serum aliquots were stored at —20°C
`until
`their NTBI
`levels were evaluated using the
`chromatographic method.” Briefly, 450 ul of serum
`was added to 50 yl of nitrilotriacetic acid (NTA)
`800 mM (pH 7.0) and allowed to stand for 20 min. The
`solution was then ultrafiltered using Amicon Centricon
`30 microconcentrator units (Amicon Millipore Cor-
`poration, Bedford, MA, USA), and the ultrafiltrate
`(20 yl) was injected directly into the HPLC system
`using a Perkin Elmer Series 200 IC titanium pump.
`The chromatographic conditions were the following:
`flow rate 1.5 ml/min; mobile phase isocratic containing
`20% acetonitrile and 80% sodium phosphate buffer,
`5 mM (pH 7.0) containing 3 mM CP22; visible detec-
`tion, 450 nm. A standard curve was generated by
`injecting different concentrations of iron prepared in a
`100-fold excess of NTA. The standards were routinely
`run at 0 to 10 uM, although absorbance waslinear up
`to 40 uM. Under these conditions, the 0 uM standard
`corresponds to 80 mM of NTA.The addition of 80 mM
`of NTA to the serum of normalindividuals always
`results in negative NTBI values. These values are less
`than the 0 uM standard, presumably because some iron
`is donated from NTAtotransferrin.
`The NTBIlevel was considered relevant at concen-
`trations of +1 uM or higher.”!
`
`MDA assay
`
`Free MDA wasevaluated by meansof a colorimetric
`assay for lipid peroxidation using a Bioxytech LPO-586
`kit (Oxis International Inc, Portland, OR, USA).
`
`Clonogenic assay
`
`The BM samples were collected in preservative-free
`heparin. The BM mononuclear cells (BMMNC) were
`separated by means of gradient centrifugation using
`Ficoll Lymphoprep (Nicomed Pharma As, Oslo, Nor-
`way), and 1 x 10° cells/ml in IMDM werecultured in
`1 ml of a mixture containing 20% FCS, 0.3% agar, GM-
`CSF 200 U/ml, IL-3 100 U/ml and SCF 8 U/ml (Gen-
`zyme, Cambridge, MA, USA). The plates were incub-
`ated in humidified air with 5% CO, at 37°C for 14 days.
`Aggregates containing > 50 cells were scored as colonies,
`whereas those containing <50 cells were scored as
`clusters. All of the cultures were set up in quadruplicate.
`
`Definition of growth pattern
`
`The agarcolony assay was used on the fresh BMMNCin
`order to classify the MDS patients into two groups
`(leukemic or non-leukemic) according to their pattern of
`clonal growth:” (1) normal;
`(2) absent or reduced
`growth;
`(3) small number of colonies with a large
`number of leukemic clusters; (4) normal or large number
`of colonies with a large number of leukemic clusters.
`Patterns 1 and 2 were considered non-leukemic, while
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`patterns 3 and 4 with a colony/cluster ratio of <5 were
`considered typical of a leukemic growth.”
`
`Results
`
`NTBI levels in myelodysplastic syndromes
`A Cortelezzi et al
`
`155
`
`NTBIlevels and FAB classification
`
`MDSserum NTBIlevels (Figure 1) were significantly
`higher than those observed in the normal controls
`(+0.8140.26 vs —0.63+0.15 uM, P<0.01). NTBI
`
`
`
`
`
`
`
`
`E75
`N (10)
`
`aT}
`=
`TROD RR tee
`MDS(33)
`
`ar
`
`¥'
`x
`3
`LR (21)
`
`Ei
`HR(12)
`
`_+Cs
`1,2
`1
`0,8
`S 06
`04
`:"
`a
`0
`4 -0,2°
`-0,4
`-0,6
`-0,8 4
`
`S =
`
`Apoptosis
`
`Apoptosis was evaluated by means of a TdT/dUTP assay
`using the commercially available “In Situ Cell Death
`Fluorescein Detection Kit” (Roche Diagnostic, Mann-
`heim, Germany). Briefly, 1—2 x 10° cells were fixed in
`cold ethanol 70% in PBS, and stored at 4°C until use.
`Before staining, cells were washed in PBS, permeabilized
`with 100 ul Triton X-100 0.1% in sodium citrate 0.1%
`for 2 min on ice, and again washed twice in PBS. After
`incubation for 1 h at 37°C with dUTP FITC,with and
`without TdT,
`cells were washed again in PBS,
`resuspended in 500 ul of PBS and made ready for
`cytofluorimetric analysis. A total of 1 x 10* events were
`analyzed.
`
`Statistical analysis
`
`The between-group correlations and differences were
`respectively evaluated using Spearman’s Rank correla-
`tion coefficient and the Mann-Whitney test. A P value
`of <0.05 was considered statistically significant.
`
`Figure 1 NTBI levels in samples from normal individuals and
`MDS patients. **P value <0.01 vs normals; °°P value <0.01 vs
`LR.
`
`Table 2 Hematological parameters of MDSpatients
`NTBI
`M/E
`Hb
`Ferritin
`Reticulocytes
`Fe
`
`Dioagnosis
`Patient
`uM
`ratio
`g/dl
`pg/l
`x1
`ug/dl
`Trf sat.%
`RA
`1
`2.09
`0.3
`12.7
`215
`17.1
`138
`57
`2
`1.71
`2
`9
`602.9
`25.2
`126
`47
`3
`0.43
`1.9
`14
`198.2
`14.1
`224
`36
`4
`1.53
`1.9
`12.1
`13.5
`12.3
`112
`24
`5
`0.81
`3.9
`11.7
`24.8
`8.6
`79
`23
`6
`1
`1.1
`10
`316
`13.2
`94
`39
`7
`2.9
`1.2
`11.4
`480
`19
`47
`17
`8
`0.41
`6.8
`11.4
`8.5
`18.7
`75
`20
`9
`0.49
`1.5
`10
`323
`25.9
`140
`48
`10
`3.94
`1.07
`12.4
`243
`9.6
`96
`37
`11
`0.89
`3.8
`12.1
`121.6
`23.7
`105
`36
`12
`0.27
`0.6
`10.5
`80.8
`14.4
`101
`32
`13
`4.16
`0.3
`8.5
`703.3
`12.9
`92
`40
`14
`3.81
`0.9
`8.9
`562
`16
`81
`40
`15
`0.54
`0.3
`11.2
`231
`16.6
`119
`51
`16
`0.99
`0.7
`9.2
`459
`10.8
`92
`45
`17
`1.26
`0.52
`11.2
`1160.1
`8.5
`183
`82
`18
`3.38
`0.7
`8.1
`676
`8
`156
`59
`19
`0.27
`1.7
`10.6
`792.2
`10.6
`103
`46
`20
`1.22
`1.1
`15.5
`394
`12.7
`124
`54
`21
`1.85
`0.8
`10.6
`460
`11.7
`115
`54
`22
`0.53
`4
`10.1
`378.7
`15.2
`50
`24
`23
`0.47
`0.66
`8.3
`275.7
`15
`81
`30
`24
`1.05
`3.2
`9.2
`750
`41
`115
`53
`25
`0.02
`1.7
`12
`103.3
`11.9
`104
`45
`26
`0.88
`2.3
`11.3
`218.4
`11.2
`67
`26
`27
`0.35
`0.7
`12.1
`267.7
`18.4
`106
`41
`28
`1.07
`0.6
`11
`393
`18.6
`99
`34
`29
`0.27
`1.3
`12.7
`100
`10.1
`119
`39
`30
`0.27
`1.2
`10.8
`347.8
`15.8
`131
`47
`31
`0.67
`3.4
`13.9
`407.2
`8.4
`85
`36
`32
`0.28
`10.8
`9.1
`61.7
`36
`71
`26
`33
`0.81
`1.24
`8.5
`109
`9.3
`87
`58
`
`RARS
`
`RAEB
`
`RAEB t
`
`M/E ratio was defined as number of leucocytes (without granulocytes)/number of erythroblasts. M/E myeloid/erythroid; NTBI non
`transferrin bound iron. For other abbreviations see Table 1.
`
`
`The Hematology Journal
`Dr. Reddy’s Laboratories, Inc. v. Celgene Corp.
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`

`NTBIlevels in myelodysplastic s
`A Cortelezzi et al
`
`was particularly high in the LR subgroup and
`significantly higher than those in the HR subgroup
`(+ 1.3640.34 vs —0.16+0.19 uM, P<0.01). Only four
`out of 21 LR patients had negative NTBI values,
`whereas none of the HR patients had NTBIlevels of
`>1 uM.
`NTBI did not correlate with hemoglobin (Hb),
`reticulocyte
`count,
`serum iron (Fe),
`ferritin or
`transferrin (Trf) saturation (Table 2).
`
`significantly greater in the MDSpatients as a whole
`than in the normal controls (2.65+0.41 vs 1.49+0.27,
`P<0.05), and was also greater in the LR thanin the
`HRsubgroup (3.04+0.54 vs 1.6+0.39, P<0.05).
`Patients with high NTBI levels (>1 4M) showed
`increased apoptosis in comparison with those with low
`NTBI
`levels,
`although
`this difference was not
`statistically
`significant
`(3.4+0.99
`vs
`2.25+0.35,
`P=0.4) (Figure 5).
`
`NTBI
`
`Figure 3 NTBIlevels and pattern of growth. NL non leukemic
`growth; L_
`leukemic growth. *P value <0.05.
`
`MDSpatients had significantly higher levels of free
`
`MDA (Figure normal_subjects4) than the
`
`
`
`(0.95+0.08 vs 0.4+0.08 um, P<0.01), but
`there
`was no significant difference between the HR and
`LR_
`subgroups
`(0.86+0.1 vs 1+0.11 uM, P=0.7).
`Moreover, in samples from these patients there was
`no correlation between MDA levels, NTBI or M/E
`ratio.
`
`NTBIlevels and bone marrow M/E ratio
`
`There wasa significant correlation (P<0.05) between
`NTBI and the myeloid/erythroid (M/E) ratio among
`the MDSsamples. Patients with NTBI levels of >1 uM
`had a M/E ratio of 0.99+0.15, whereas those with
`NTBI levels of <1 uM had a M/Eratio of 2.44+0.54
`(P<0.05) (Figure 2).
`
`NTBIlevels and pattern of growth
`
`Patients with an in vitro leukemic growth pattern had
`significantly lower levels of NTBI than those with a non-
`leukemic pattern (0.5+0.38 vs 1.18+0.33, P<0.05)
`(Figure 3). All four LR patients with NTBI levels of
`<1 uM hadanin vitro leukemic growth pattern.
`
`Serum MDA values
`
`NTBIlevels and apoptosis
`
`Apoptosis was evaluated in 24 MDS and 10 normal
`BMMNC samples. Apoptosis was
`found
`to be
`
`M/Eratio
`
`NTBI>t
`
`NTBIK1
`
`pmol/L
` ee
`%apoptosis NTBIxI
`
`}
`HR(12)
`~ MDS (33)
`N (10)
`Figure 4 MDAlevels in samples from normal individuals and
`MDSpatients.
`
`LR (21)
`
`NTBI>1
`
`Figure 2 M/E ratio in patients with low (<1 uM) and high
`Figure 5 Apoptosis in patients with low (<1 um) and high
`(>1pM)levels of NTBI.
`(>1 uM)levels of NTBI.
`
`The Hematology Journal
`
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`
`

`

`Discussion
`
`The multifactorial pathogenesis of MDS has not yet
`been fully clarified, but many authors have suggested
`that apoptosis is one of the most relevant pathogenetic
`events that
`takes place in MDS hemopoietic pre-
`cursors. The oxidative DNA damage induced by free
`radicals is one of the possible mechanisms leading to
`apoptosis in MDS, as is suggested by the fact that
`MDS CD34*cells have an increased level of oxidized
`pyrimidine nucleotides.'? NTBI, a low molecular weight
`iron fraction, has recently been reported as showing
`pro-oxidant activity that induces free radicals produc-
`tion.** In thalassemia patients, NTBI levels do not
`correlate with iron overload but seem to be linked to
`the severity of the ineffective erythropoiesis.2! No data
`are
`available
`concerning NTBI
`levels
`in other
`conditions where ineffective erythropoiesis and iron
`overload are present and due to different causes from
`those in thalassemia. The role of NTBI was therefore
`evaluated in MDSpatients, where dyserythropoiesis is
`associated with apoptotic cell damage.
`NTBI was high in MDSpatients as a whole, but
`particularly so in the LR subgroup. High NTBIlevels
`correlated with a low M/E ratio, thus suggesting that
`ineffective erythropoiesis may play a central role in
`increasing the NTBI deriving from cytoplasmic iron by
`dismissal
`from the labile pool. The relationship
`between NTBI and erythropoiesis suggests that high
`NTBIlevels after chemotherapy may be a consequence
`of the interruption of erythropoietic activity and iron
`removal from transferrin by the erythron.** NTBIlevels
`were low in HR MDS, whose M/E ratio was higher
`and apoptosis less than in the LR subgroup. In this
`context, preliminary results concerning BM apoptosis
`in 24 MDSpatients indicate a trend towards a
`correlation between high NTBI levels and increased
`apoptosis. NTBI could also have a negative effect on
`apoptosis in erythroid precursors by inducing the
`generation of oxidative damage.
`
`References
`
`N
`
`1 Brittenham GM, Adams PC, Gordeuk VR, Rouault TA.
`Diagnosis and treatmentofiron overload in the practice
`of hematology. American Society of Hematology,
`Education Programme: 234, 1998.
`Jensen PD, Heickendorff L, Pedersen B, Bendix Hansen
`K, Jensen FT, Christensen T, Boesen AM, Ellengaard J.
`The effect of iron chelation on haemopoiesis in MDS
`patients with transfusional iron overload. British Journal
`of Haematology 94: 288, 1996.
`Grootveld M, Bell JD, Halliwell B, Aruoma OI, Bomford
`A, Sadler PJ. Non transferrin bound iron in plasma or
`serum from patients with idiopathic hemochromatosis.
`Journal of Biological Chemistry 264: 4417, 1989.
`Breuer W, Greenberg E, Cabantchik ZI. Newly delivered
`transferrin iron and oxidative cell injury. FEBS Letters
`403: 213, 1997.
`
`we
`
`>
`
`NTBI levels in myelodysplastic syndromes
`A Cortelezzi et al
`157
`
`The high MDAvalues observed in both the LR and
`HR subgroups is not surprising because MDA is
`generally regarded as a marker of the peroxidative
`damage induced in cell membranes by both physical
`and chemical oxidative stress.~ It
`is
`therefore an
`indirect index of the enhanced oxidative distress that
`occurs either in peripheral or MDS CD34* cells.”
`The presence of leukemic growth pattern in MDSis
`prognostic of an evolution towards leukemia.”* Low
`NTBIlevels correlated with leukemic growth pattern in
`our patients, thus suggesting that the presence of the
`leukemic clone may play an important
`role in
`sequestering iron, which is
`indispensable
`for
`the
`proliferation of blast cells.7”* Confirmation of this
`hypothesis comes from the negative NTBI values
`observed in the four LR patients with a leukemic
`growth. Leukemic growth,
`together with a relative
`reduction in the erythroid compartment,
`therefore
`seems to be responsible for the lower levels of NTBI
`in MDS, whereas high NTBI
`levels are found in
`patients showing non-leukemic growth in vitro and/or
`an expandederythron.
`The evidence of potentially toxic-free iron in the
`presence of dyserythropoiesis may explain the good
`results obtained with iron chelation therapy in
`transfused MDS patients and suggests that
`it may
`also be useful in untransfused MDSpatients. Further
`in vitro studies are being made to evaluate the effect of
`desferrioxamine and other iron chelators on dysery-
`thropoiesis by reducing NTBI levels and,
`therefore,
`oxidative damage.
`
`Acknowledgments
`This paper was partly supported by MURSTto G Fiorelli
`and MD Cappellini, by Associazione Italiana contro le
`Leucemie (sezione di Milano) and by intramural funds of
`IRCCS, Ospedale Maggiore, Milan.
`
`5 Al Refaie FN, Wickens DG, Wonke B, Kontoghiorghes
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`6 Brittenham GM, Olivieri NF, Roualt TA. Iron physiol
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`in myelodysplastic syndromes. Leukemia 7: 144, 1993.
`
`~
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`The Hematology Journal
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`IPR2018-01509
`Exhibit 2017, Page 6
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