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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
`ª 2000 The European Haematology Association All rights reserved 1466 – 4680/00 $15.00
`www.nature.com/thj
`
`Non-transferrin-bound iron in myelodysplastic syndromes: a marker of
`ine€ective erythropoiesis?
`
`Agostino Cortelezzi*,1,4, Chiara Cattaneo1,4, Silvia Cristiani1, Lorena Duca2, Barbara Sarina1,
`Giorgio L Deliliers3, Gemino Fiorelli2 and Maria Domenica Cappellini2
`
`1Servizio Autonomo di Ematologia Diagnostica, Ospedale Maggiore, IRCCS, Milano, Italy; 2Centro Anemie Congenite,
`Dipartimento di Medicina Interna, Ospedale Maggiore, IRCCS, Milano, Italy; 3Centro Trapianti di Midollo, Ospedale Maggiore,
`IRCCS, Milano, Italy
`
`Iron overload is usually observed in patients (even untransfused) with
`Introduction:
`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 MDS patients and we tried to correlate
`them with ine€ective 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 MDS patients as a whole than in normal controls. The bone marrow
`progenitor cells in the MDS patients with high NTBI levels showed a higher degree of
`apoptosis, but this di€erence was not statistically significant. Patients with a leukemic growth
`pattern had lower NTBI levels than those with a non-leukemic pattern.
`ine€ective
`Conclusion: These data suggest
`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 blast cells.
`The Hematology Journal (2000) 1, 153 – 158
`
`Keywords: non-transferrin-bound iron; myelodysplastic syndromes; dyserythropoiesis; apoptosis
`
`Introduction
`
`ine€ective
`Iron overload, due to the presence of
`erythropoiesis and to the increased absorption follow-
`ing the anemic state,
`is usually observed in patients
`a€ected by myelodysplastic syndromes (MDS), even
`untransfused.1 Jensen et al.2 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
`ine€ective 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
`4A 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.3,4
`NTBI
`is more
`readily taken up by cells
`than
`transferrin-bound iron is,5 and contributes towards
`increasing the labile pool of cytoplasmic iron (LIP).6
`The degree of cell damage induced by NTBI can be
`deduced from the level of lipid peroxidation, which is
`known to be a consequence of an increase in LIP.3
`Increased bone barrow (BM) cell apoptosis is a
`common finding in MDS, particularly in patients at
`low risk of
`leukemic evolution.7 – 10 Although many
`factors are likely to be involved in this process, such as
`an increased ratio of pro- vs anti-apoptotic proteins
`(bcl2 family),11 the real etiopathogenesis of MDS is far
`from being completely understood. It has recently been
`reported that the high serum levels of tumor necrosis
`factor-a (TNF-a) observed in MDS patients may lead
`to apoptosis by inducing the generation of
`free
`radicals, which can induce oxidative DNA damage.12 – 18
`In particular, Peddie et al.19 have demonstrated that
`
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`NTBI levels
`
`Peripheral blood serum was obtained by centrifugation
`at 3000 r.p.m. Serum aliquots were stored at 7208C
`until
`their NTBI
`levels were evaluated using the
`chromatographic method.20 Briefly, 450 ml of serum
`was added to 50 ml 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 ml) 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 bu€er,
`5 mM (pH 7.0) containing 3 mM CP22; visible detec-
`tion, 450 nm. A standard curve was generated by
`injecting di€erent concentrations of iron prepared in a
`100-fold excess of NTA. The standards were routinely
`run at 0 to 10 mM, although absorbance was linear up
`to 40 mM. Under these conditions, the 0 mM standard
`corresponds to 80 mM of NTA. The addition of 80 mM
`of NTA to the serum of normal
`individuals always
`results in negative NTBI values. These values are less
`than the 0 mM standard, presumably because some iron
`is donated from NTA to transferrin.
`The NTBI level was considered relevant at concen-
`trations of +1 mM or higher.21
`
`MDA assay
`
`Free MDA was evaluated by means of 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 16105 cells/ml in IMDM were cultured 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% CO2 at 378C for 14 days.
`Aggregates containing 450 cells were scored as colonies,
`whereas those containing 550 cells were scored as
`clusters. All of the cultures were set up in quadruplicate.
`
`Definition of growth pattern
`
`The agar colony assay was used on the fresh BMMNC in
`order to classify the MDS patients into two groups
`(leukemic or non-leukemic) according to their pattern of
`clonal growth:22
`(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
`
`154
`
`NTBI levels in myelodysplastic syndromes
`A Cortelezzi et al
`
`MDS CD34+ cells have an increased level of oxidized
`pyrimidine nucleotides.
`On the basis of these observations, the levels of
`NTBI and malondyaldehyde (MDA), an index of lipid
`peroxidation, were evaluated in serum taken from
`untransfused MDS patients, with the aim of revealing
`their possible role in ine€ective 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 were classified accord-
`ing to the FAB criteria as having refractory anemia
`(RA, 12 cases), RA with ring sideroblasts (RARS, nine
`cases), RA with excess of blasts (RAEB, 10 cases), and
`RAEB in transformation (RAEB-t, two cases). Patients
`with RA or RARS were 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
`
`Patient
`
`Sex
`
`Caryotype
`
`%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 1
`
`3
`
`6 6
`
`13.75
`17
`6.75
`20.25
`5.75
`28
`26.75
`
`46XY
`46XX
`n.v.
`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
`n.v.
`46XY
`n.v.
`46XY
`47XX, +8
`46XX
`46XY
`
`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
`
`1 2 3 4 5 6 7 8 9
`
`10
`11
`12
`13
`14
`15
`16
`17
`18
`19
`20
`21
`22
`23
`24
`25
`26
`27
`28
`29
`30
`31
`32
`33
`
`RA
`
`RARS
`
`RAEB
`
`RAEB-t
`
`F=female; M=male; RA=refractory anemia; RAEB=refractory
`anemia with excess of blasts; RAEB-t=refractory anemia with excess
`of blasts in transformation; RARS=refractory anemia with ring
`sideroblasts.
`
`The Hematology Journal
`
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`

`

`patterns 3 and 4 with a colony/cluster ratio of 55 were
`considered typical of a leukemic growth.23
`
`Results
`
`NTBI levels in myelodysplastic syndromes
`A Cortelezzi et al
`
`155
`
`NTBI levels and FAB classification
`
`MDS serum NTBI levels (Figure 1) were significantly
`higher than those observed in the normal controls
`(+0.81+0.26 vs 70.63+0.15 mM, P50.01). NTBI
`
`Figure 1 NTBI levels in samples from normal individuals and
`MDS patients. **P value 50.01 vs normals; 88P value 50.01 vs
`LR.
`
`Ferritin
`mg/l
`
`Reticulocytes
`6103
`
`Fe
`mg/dl
`
`Trf sat.%
`
`215
`602.9
`198.2
`13.5
`24.8
`316
`480
`8.5
`323
`243
`121.6
`80.8
`703.3
`562
`231
`459
`1160.1
`676
`792.2
`394
`460
`378.7
`275.7
`750
`103.3
`218.4
`267.7
`393
`100
`347.8
`407.2
`61.7
`109
`
`17.1
`25.2
`14.1
`12.3
`8.6
`13.2
`19
`18.7
`25.9
`9.6
`23.7
`14.4
`12.9
`16
`16.6
`10.8
`8.5
`8
`10.6
`12.7
`11.7
`15.2
`15
`41
`11.9
`11.2
`18.4
`18.6
`10.1
`15.8
`8.4
`36
`9.3
`
`138
`126
`224
`112
`79
`94
`47
`75
`140
`96
`105
`101
`92
`81
`119
`92
`183
`156
`103
`124
`115
`50
`81
`115
`104
`67
`106
`99
`119
`131
`85
`71
`87
`
`57
`47
`36
`24
`23
`39
`17
`20
`48
`37
`36
`32
`40
`40
`51
`45
`82
`59
`46
`54
`54
`24
`30
`53
`45
`26
`41
`34
`39
`47
`36
`26
`58
`
`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 – 26106 cells were fixed in
`cold ethanol 70% in PBS, and stored at 48C until use.
`Before staining, cells were washed in PBS, permeabilized
`with 100 ml 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 378C with dUTP FITC, with and
`without TdT,
`cells were washed again in PBS,
`resuspended in 500 ml of PBS and made ready for
`cytofluorimetric analysis. A total of 16104 events were
`analyzed.
`
`Statistical analysis
`
`The between-group correlations and di€erences were
`respectively evaluated using Spearman’s Rank correla-
`tion coe(cid:129)cient and the Mann-Whitney test. A P value
`of 50.05 was considered statistically significant.
`
`Table 2 Hematological parameters of MDS patients
`
`Hb
`g/dl
`
`12.7
`
`9 1
`
`4
`12.1
`11.7
`10
`11.4
`11.4
`10
`12.4
`12.1
`10.5
`8.5
`8.9
`11.2
`9.2
`11.2
`8.1
`10.6
`15.5
`10.6
`10.1
`8.3
`9.2
`12
`11.3
`12.1
`11
`12.7
`10.8
`13.9
`9.1
`8.5
`
`NTBI
`mM
`
`2.09
`1.71
`0.43
`1.53
`0.81
`1
`2.9
`0.41
`70.49
`3.94
`70.89
`70.27
`4.16
`3.81
`0.54
`0.99
`71.26
`3.38
`70.27
`1.22
`1.85
`0.53
`0.47
`71.05
`0.02
`70.88
`70.35
`71.07
`0.27
`0.27
`70.67
`70.28
`0.81
`
`M/E
`ratio
`
`0.3
`2
`1.9
`1.9
`3.9
`1.1
`1.2
`6.8
`1.5
`1.07
`3.8
`0.6
`0.3
`0.9
`0.3
`0.7
`0.52
`0.7
`1.7
`1.1
`0.8
`4
`0.66
`3.2
`1.7
`2.3
`0.7
`0.6
`1.3
`1.2
`3.4
`10.8
`1.24
`
`Dioagnosis
`
`Patient
`
`1 2 3 4 5 6 7 8 9 1
`
`0
`11
`12
`13
`14
`15
`16
`17
`18
`19
`20
`21
`22
`23
`24
`25
`26
`27
`28
`29
`30
`31
`32
`33
`
`RA
`
`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
`
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`

`156
`
`NTBI levels in myelodysplastic syndromes
`A Cortelezzi et al
`
`was particularly high in the LR subgroup and
`significantly higher than those in the HR subgroup
`(+1.36+0.34 vs 70.16+0.19 mM, P50.01). Only four
`out of 21 LR patients had negative NTBI values,
`whereas none of the HR patients had NTBI levels of
`41 mM.
`NTBI did not correlate with hemoglobin (Hb),
`reticulocyte
`count,
`serum iron (Fe),
`ferritin or
`transferrin (Trf) saturation (Table 2).
`
`significantly greater in the MDS patients as a whole
`than in the normal controls (2.65+0.41 vs 1.49+0.27,
`P50.05), and was also greater in the LR than in the
`HR subgroup (3.04+0.54 vs 1.6+0.39, P50.05).
`Patients with high NTBI levels (41 mM) showed
`increased apoptosis in comparison with those with low
`NTBI
`levels,
`although this di€erence was not
`(3.4+0.99
`2.25+0.35,
`statistically
`significant
`vs
`P=0.4) (Figure 5).
`
`NTBI levels and bone marrow M/E ratio
`
`There was a significant correlation (P50.05) between
`NTBI and the myeloid/erythroid (M/E) ratio among
`the MDS samples. Patients with NTBI levels of 41 mM
`had a M/E ratio of 0.99+0.15, whereas those with
`NTBI levels of 51 mM had a M/E ratio of 2.44+0.54
`(P50.05) (Figure 2).
`
`NTBI levels 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, P50.05)
`(Figure 3). All four LR patients with NTBI levels of
`51 mM had an in vitro leukemic growth pattern.
`
`Serum MDA values
`
`MDS patients had significantly higher levels of free
`MDA (Figure
`4)
`than
`the
`normal
`subjects
`(0.95+0.08 vs 0.4+0.08 mM, P50.01), but
`there
`was no significant di€erence between the HR and
`(0.86+0.1 vs 1+0.11 mM, P=0.7).
`LR subgroups
`Moreover,
`in samples from these patients there was
`no correlation between MDA levels, NTBI or M/E
`ratio.
`
`NTBI levels and apoptosis
`
`Apoptosis was evaluated in 24 MDS and 10 normal
`BMMNC samples. Apoptosis was
`found to be
`
`Figure 3 NTBI levels and pattern of growth. NL=non-leukemic
`growth; L=leukemic growth. *P value 50.05.
`
`Figure 4 MDA levels in samples from normal individuals and
`MDS patients.
`
`Figure 2 M/E ratio in patients with low (51 mM) and high
`(41 mM) levels of NTBI.
`
`Figure 5 Apoptosis in patients with low (51 mM) and high
`(41 mM) levels of NTBI.
`
`The Hematology Journal
`
`Dr. Reddy’s Laboratories, Inc. v. Celgene Corp.
`IPR2018-01504
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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.19 NTBI, a low molecular weight
`iron fraction, has recently been reported as showing
`pro-oxidant activity that induces free radicals produc-
`tion.3,4 In thalassemia patients, NTBI levels do not
`correlate with iron overload but seem to be linked to
`the severity of the ine€ective erythropoiesis.21 No data
`are
`available
`concerning NTBI
`levels
`in other
`conditions where ine€ective erythropoiesis and iron
`overload are present and due to di€erent causes from
`those in thalassemia. The role of NTBI was therefore
`evaluated in MDS patients, where dyserythropoiesis is
`associated with apoptotic cell damage.
`NTBI was high in MDS patients as a whole, but
`particularly so in the LR subgroup. High NTBI levels
`correlated with a low M/E ratio, thus suggesting that
`ine€ective 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
`NTBI levels after chemotherapy may be a consequence
`of the interruption of erythropoietic activity and iron
`removal from transferrin by the erythron.24 NTBI levels
`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 MDS patients
`indicate a trend towards a
`correlation between high NTBI levels and increased
`apoptosis. NTBI could also have a negative e€ect on
`apoptosis in erythroid precursors by inducing the
`generation of oxidative damage.
`
`References
`
`1 Brittenham GM, Adams PC, Gordeuk VR, Rouault TA.
`Diagnosis and treatment of iron overload in the practice
`of hematology. American Society of Hematology,
`Education Programme: 234, 1998.
`2 Jensen PD, Heickendor€ L, Pedersen B, Bendix-Hansen
`K, Jensen FT, Christensen T, Boesen AM, Ellengaard J.
`The e€ect of iron chelation on haemopoiesis in MDS
`patients with transfusional iron overload. British Journal
`of Haematology 94: 288, 1996.
`3 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.
`4 Breuer W, Greenberg E, Cabantchik ZI. Newly delivered
`transferrin iron and oxidative cell injury. FEBS Letters
`403: 213, 1997.
`
`NTBI levels in myelodysplastic syndromes
`A Cortelezzi et al
`
`157
`
`The high MDA values 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.25 It
`is therefore an
`indirect index of the enhanced oxidative distress that
`occurs either in peripheral or MDS CD34+ cells.19
`The presence of leukemic growth pattern in MDS is
`prognostic of an evolution towards leukemia.26 Low
`NTBI levels 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.27,28 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 expanded erythron.
`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
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`in vitro studies are being made to evaluate the e€ect of
`desferrioxamine and other iron chelators on dysery-
`thropoiesis by reducing NTBI levels and, therefore,
`oxidative damage.
`
`Acknowledgments
`This paper was partly supported by MURST to G Fiorelli
`and MD Cappellini, by Associazione Italiana contro le
`Leucemie (sezione di Milano) and by intramural funds of
`IRCCS, Ospedale Maggiore, Milan.
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`Exhibit 2017, Page 6
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