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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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`‘lhe Hen-«flog Jami (200m 1. 153 158
`(I)
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`mnanlemm/Ihj
`
`Non-transferrin-bound iron in myelodysplastic syndromes: a marker of
`ineffective erythropoiesis?
`
`Agostino Cortelezzi*"", Chiara Cattaneo”, Silvia Cristiani', Lorena Ducal, Barbara Sarina',
`Giorgio L Deliliers’, Gemino Fiorelli2 and Maria Domenica Cappellini2
`
`lServizio Autonomo di Ematologia Diagnostica, Ospedale Maggiore, IRCCS, Milano, Italy; zCenIro Anemie Congenile,
`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 MDS patients and we tried 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 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 difference was not statistically 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 blast cells.
`The Hematology Journal (2000) l, 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.l 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
`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, Servin'o 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 Cortelezn' 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}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)!s
`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.’
`Increased bone barrow (BM) cell apoptosis is a
`common finding in MDS, particularly in patients at
`low risk of leukemic evolution.7 '° Although many
`factors are likely to be involved in this process, such as
`an increased ratio of pro- vs anti-apoptotic proteins
`(bc12 family)," 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-az) observed in MDS patients may lead
`to apoptosis by inducing the generation of free
`radicals, which can induce oxidative DNA damage.'2 '8
`In particular, Peddie et al.'9 have demonstrated that
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`NTBI levels In myolodylplxlc syntomes
`
`ACorteIezzi et at
`
`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 of lipid
`peroxidation, were evaluated in serum taken from
`untransfused MDS patients, 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
`connols 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
`RA
`
`RARS
`
`RAEB
`
`RAEBt
`
`Patient
`1
`2
`3
`4
`5
`6
`7
`8
`9
`10
`11
`12
`13
`14
`15
`16
`17
`18
`19
`I)
`21
`22
`23
`24
`75
`26
`27
`28
`B
`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
`
`Caryatype
`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, Sq
`46XX
`n.v.
`46XY
`n.v.
`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; RAEB t
`refractory anemia with excess
`of blasts in transformation; RARS refractory anemia with ring
`sideroblasts.
`
`
`The My Journal
`
`NTBI levels
`
`Peripheral blood serum was obtained by centrifugation
`at 3000 rpm. Serum aliquots were stored at —20°C
`until
`their NTBI
`levels were evaluated using the
`chromatographic method?" Briefly, 450 pl of serum
`was added to 50 ul 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
`(20111) 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/rnin; 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 ”M, although absorbance was linear up
`to 40 ”M. Under these conditions, the 0 uM 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 uM standard, presumably because some iron
`is donated from NTA to transferrin.
`The NTBI level was considered relevant at concen-
`
`trations of +1 ”M or higher.”
`
`MDA assay
`
`Free MDA was evaluated by means of a colorimetric
`assay for lipid peroxidation using a Bioxytech LPG-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 1x105 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% 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 quadmplimte.
`
`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 growt
`: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
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`

`mu levels In myolodyqlallc epitome:
`A Cortelezzi er a!
`
`(I)
`155
`
`patterns 3 and 4 with a colony/cluster ratio of < 5 were
`considered typical of a leukemic growth.”
`
`Results
`
`Apoptosis
`_
`1‘9on315 was evaluated by means ofaTdT/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 [11 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 [11 of PBS and made ready for
`cytofluorimetric analysis. A total of 1 x 10‘ events were
`analyzed.
`
`NTBI levels and FAB classification
`
`MDS serum NTBI levels (Figure 1) were significantly
`higher than those observed in the normal controls
`+
`1+ 2
`_
`+ 1
`M P
`1 NTBI
`( 0'8 ‘0' 6 vs
`063—0' 5 fl
`’
`(0'0 )-
`
`
`
`
`
`
`
`
`1.4
`1,2
`I
`i 0’3
`0 g:
`g 0:2
`3
`0
`[—
`2 _0’2_
`-0,4
`
`Statistical analysis
`
`The between-group correlations and differences were
`rFspeawdy, evaluated “8mg Speannan 8 Rank correla'
`"on coeffic‘cnt and_the Mann:wmtney_tcs_t- A P value
`of <0.05 was consrdered statistically Significant.
`
`4’“
`..
`y
`.
`
`-0.8 .
`.
`..
`;
`,.
`,
`.
`.
`-
`1
`V
`_.
`..
`V
`1110803)
`LR (21)
`H1102)
`N(10)
`Figure l 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 MDS patients
`NTBI
`M/E
`Hb
`Ferritin
`Reticuloc res
`Fe
`
`Diaagnosis
`Patient
`[w
`ratio
`g/dl
`ng/l
`x l
`ug/d!
`th 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
`1 5
`81
`30
`24
`1.05
`3.2
`9.2
`750
`41
`1 15
`53
`25
`0.02
`1.7
`12
`103.3
`1 1.9
`104
`45
`26
`0.88
`2.3
`1 1.3
`218.4
`1 1.2
`67
`26
`27
`0.35
`0.7
`12.1
`267.7
`18.4
`106
`41
`28
`1.07
`0.6
`1 l
`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 l.
`
`
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`III'BI larch II myolodyaplalc syntomac
`A Cottelezzi et al
`
`was particularly high in the LR subgroup and
`significantly higher than those in the HR subgroup
`(+1.36i034 vs —0.16i0.19 uM, P<0.01). Only four
`out of 21 LR patients had negative NTBI values,
`whereas none of the HR patients had NTBI levels of
`>1 uM.
`NTBI did not correlate with hemoglobin (Hb),
`reticulocyte
`count,
`serum iron (Fe),
`ferritin or
`transferrin (Trt) saturation (Table 2).
`
`significantly greater in the MDS patients as a whole
`than in the normal controls (2.65 1-0.4] vs 1.49:0.27,
`P<0.05), and was also greater in the LR than in the
`HR subgroup (3.04i0.54 vs 1.6 $0.39, P<0.05).
`Patients with high NTBI levels (>1uM) showed
`increased apoptosis in comparison with those with low
`NTBI
`levels,
`although
`this difference was not
`statistically
`significant
`(3.4i0.99
`vs 2.25i0.35,
`P=0.4) (Figure 5).
`
`NTBI levels and bone marrow M[E ratio
`
`There was a significant correlation (P<0.05) between
`NTBI and the myeloid/erythroid (M/E) ratio among
`the MDS samples. Patients with NTBI levels of >1 [1M
`had a M/E ratio of 09910.15, whereas those with
`NTBI levels of <1 [1M had a M/E ratio of 2.44:0.54
`(P<0.05) (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 (0.5i0.38 vs US$0.33, P<0.05)
`(Figure 3). All four LR patients with NTBI levels of
`<1 uM 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
`(0.95i0.08 vs 0.4i0.08 uM, P<0.01), but
`there
`was no significant difference between the HR and
`LR subgroups
`(0.86:0.1 vs
`l-l_-0.11 uM, P=0.7).
`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
`
`NTBI
`
`umol/l.
`
`Figure 3 NTBI levels and pattern of growth NL non leukemic
`growth; L leukemic growth. ‘1’ value <0.05.
`
`
`
`MDAumol/L
`
`mm)
`mar)
`» MDS(33)
`mm)
`Figure 4 MDA levels in samples from normal individuals and
`MDS patients.
`
`M/Eratio
`
`NTBl>I
`
`NTBI<1
`
`%apoptosis
`
`Nin
`2
`
`,5
`
`l
`
`NTBl>l
`
`N'I'Bkl
`
`Figure 2 M/E ratio in patients with low (<1 um) and high
`(>1 m) levels of NTBI.
`
`Figure 5 Apoptosis in patients with low ( <1 11M) and high
`(>1 uM) levels of NTBI.
`
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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 pathogenen'c
`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.” 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 thalassernia. 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
`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
`NTBI levels after chemotherapy may be a consequence
`of the interruption of erythropoietic activity and iron
`removal from transferrin by the erythron." 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 effect on
`apoptosis in erythroid precursors by inducing the
`generation of oxidative damage.
`
`References
`
`l 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, Heickendorff L, Pedersen B, Bendix Hansen
`K, Jensen Ff, Christensen T, Boesen AM, Ellengaard I.
`The effect 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, Anioma OI, Bomford
`A, Sadler 1’]. Non transferrin bound iron in plasma or
`serum from patients with idiopathic hemochromatosis.
`Journal of Biological Chemistry 264: 44”, 1989.
`4 Breuer W, Greenberg E, Cabantchik Zl. Newly delivered
`transferrin iron and oxidative cell injury. FEBS Letters
`403: 2l3, 1997.
`
`mm levels II myelodysplastlc symiornos
`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.” 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 MDS is
`prognostic of an evolution towards leukemia.26 Low
`NTBI levels correlated with leukemic grth 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?” 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
`also be useful in untransfused MDS patients. 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 MU RST to G Fiorelli
`and MD Cappellini, by Associazione ltaliana contro 1e
`Leueemie (sezione di Milano) and by intramural funds of
`lRCCS, Ospedale Maggiore, Milan.
`
`5 A] Refaie FN, Wickens DG, Wonke B, Kontoghiorghes
`GJ, Hoffbrand AV. Semm non transferrin bound iron in
`beta thalassaernia major patients treated with desferriox
`amine and L1. British Journal of Haematology 82: 43l,
`1992.
`6 Brittenham GM, Olivieri NF, Roualt TA. lron physiol
`ogy and iron overload. American Society of Hematology,
`Education Programme: 177, 1996.
`7 Yoshida Y. Hypothesis: apoptosis may be the mechanism
`responsible for the premature intramedullary cell death
`in myelodysplastic syndromes. Leukemia 7: 144, I993.
`
`
`
`I'll Ilsa-tum Journal
`Dr. Reddy’s Laboratories, Inc. v. Oelgene Corp.
`IPR2018-01509
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`ml levels In myelodylplslc synaomcs
`A Cortelezzi et at
`
`8
`
`10
`
`ll
`
`12
`
`l3
`
`14
`
`15
`
`l6
`
`17
`
`Raza A, Gezer S, Mundle S, Gao XZ, Alvi S, Borok R,
`Rifkin S, Iftikhar A, Shetty V, Parcharidou A, Loew J,
`Marcus B, Khan Z, Chaney C, Showel J, Gregory S,
`Preisler H. Apoptosis in bone marrow biospy samples
`involving stromal and hematopoietic cells in 50 patients
`with myelodysplastic syndromes. Blood 86: 168, 1995.
`Yoshida Y, Anzai N, Kawabata H. Apoptosis in
`myelodysplasia: a paradox or paradigm? Leukemia
`Research 19: 887, I995.
`Rajapaksa R, Ginzton N, Rott LS, Greenberg PL.
`Altered oncoprotein expression and apoptosis
`in
`myelodysplastic syndrome marrow cells. Blood 88:
`4275, 1996.
`Parker JE, Fishlock KL, Mijovic B, Czepulkowski B,
`Pagliuca A, Mufti GJ. “Low risk” myelodysplastic
`syndrome is associated with excessive apoptosis and an
`increased ratio of pro versus anti apoptotic be] 2 related
`proteins. British Journal of Haematology 103: 1075, I998.
`Verhoef GE, De Schouwer P, Ceuppens JL, Van Damme
`J, Goossens W, Boogaerts MA. Measurement of serum
`cytokine levels in patients with myelodysplastic syn
`dromes. Leukemia 6: 1268, 1992.
`Gersuk GM, Beckham C, Loken MR, Kiener P,
`Anderson JE, Farrand A, Troutt AB, Ledbetter JA,
`Deeg HJ. A role for tumor necrosis factor alpha, Fas and
`Fas ligand in marrow failure associated with myelodys
`plastic syndrome. British Journal of Haematology 103:
`176, 1998.
`Reza S, Shetty V, Dar S, Qawi H, Raza A. Tumor
`necrosis factor alpha levels decrease with anticytokine
`therapy in patients with myelodysplastic syndromes.
`Journal of Interferon and Cytokine Research 18: 871,
`1998.
`
`R323 A, Mundle S, Shetty V, Alvi S, Chopra H, Span L,
`Parcharidou A, Dar S, Venugopal P, Borok R, Gezer S,
`Showel J, Loew J, Robin E, Rifkin S, Alston D,
`Hernandez B, Shah R, Kaizer H, Gregory S. Novel
`insights into the biology of myelodysplastic syndromes:
`excessive apoptosis and the role of cytokines. Interna
`tional Journal of Hematology 63: 265, 1996.
`Ram A, Mundle S, Shetty V, Alvi S, Chopra H, Span L,
`Parcharidou A, Dar S, Venugopal P, Borok R, Gezer S,
`Showel J, Loew J, Robin E, Rifkin S, Alston D,
`Hernandez B, Shah R, Kaizer H, Gregory S, Preisler H.
`A paradigm shift in myelodyslastic syndromes. leukemia
`10: 1648, 1996.
`Shetty V, Mundle S, Alvi S, Showel M, Broady Robinson
`L, Dar S, Borok R, Showel J, Gregory S, Rifkin S, Gezer
`S, Parcharidou A, Venugopal P, Shah R, Hernandez B,
`Klein M, Alston D, Robin E, Dominquez C, Raza A.
`Measurement of apoptosis, proliferation and three
`cytokines in 46 patients with myelodysplastic syn
`dromes. Leukemia Research 20: 891, 1996.
`
`18
`
`19
`
`20
`
`21
`
`23
`
`24
`
`25
`
`26
`
`27
`
`28
`
`Shoji Y, Uedono Y, lshikura UH, Takeyama N, Tanaka
`T. DNA damage induced by tumor necrosis factor a in
`L929 cells is mediated by mitochondrial oxygen radical
`formation. Immunology 84: 543, 1995.
`Peddie CM, Wolf CR, McLellan Ll, Collins AR, Bowen
`DT. Oxidative DNA damage in CD34 + myelodysplastic
`cells is associated with intracellular redox changes and
`elevated plasma tumor necrosis factor a concentration.
`British Journal of Haematology 99: 625, 1997.
`Porter JB, Abeysinghe RD, Marshall L, Hider LC, Singh
`S. Kinetics of removal and reappearance of non
`transferrin bound plasma iron with desferoxamine
`therapy. Blood 88: 705, 1996.
`Cappellini MD, Duca L, Tavazzi D, Marelli S, Fargion S,
`Zatelli S, Cerino M, Graziadei G, Fiorelli G. Non
`transferrin bound plasma
`ion,
`iron overload and
`ineffective erythropoiesis in thalassemia major and
`interrnedia. Blood (Suppl. 92) 1: 3138, I998.
`Sawada K, Ieko M, Notoya A, Tarumi T, Koizumi K,
`Kitayama S, Nishio H, Fukada Y, Yasukouchi T,
`Tamagichi M, Katoh S, Koike T. Role of cytokines in
`leukemic type growth of myelodysplastic CD34+ cells.
`Blood 88: 319, 1996.
`Cortelezzi A, Cattaneo C, Cristiani S, Sarina B, Pomati
`M, Silvestris I, lbatici A, Maiolo AT. Low plasma stem
`cell factor levels correlate with in vitro leukemic growth
`in myelodysplastic syndromes. Leukemia Research 23:
`271, I999.
`Bradley SJ, Gosriwitana l, Srichairatanakool S, Hider
`RC, Porter JB. Non transferrin bound iron induced by
`myeloablative chemotherapy. British Journal of Haema
`tology 99: 337, I997.
`Lamarino G, Di Pierro D, Tava- B, Cerroni L,
`Giardina B. Simultaneous separation of malondialde
`hyde, ascorbic acid and adenine nucleotide derivatives
`from biological samples by ion paining high perfor
`mance liquid chromatography. Analytical Biochemistry
`197: 191, 1991.
`Greenberg PL. In vitro marrow culture studies in the
`myelodysplastic syndromes. Seminars in Oncology 19: 34,
`I992.
`Leardi A, Caraglia M, Selleri C, Pepe S, Pi- C, Notaro
`R, Febbrocini A, De Lorenzo S, Musico M, Abruzzese A,
`Bianco AR, Tagliaferri P. Desferrioxamine increases iron
`depletion and apoptosis induced by ara C of human
`myeloid leukaemic cells. British Journal of Haematology
`102: 746, 1998.
`Hileti D, Panayotidis P, Hoflbrand V. Iron chelators
`induce apoptosis in proliferating cells. British Journal of
`Haematology 89: 181, 1995.
`
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`IPR2018-01509
`Exhibit 2017, Page 7
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