Hematopoietic Growth Factors David T. Bowen

Contents

10.1 Introduction . . . . 99 10.2 Mechanism of Anemia . . . 100 10.2.1 Ineffective Erythropoiesis . . . 100

10.2.1.1 Clonogenic Erythroid

Growth . . . 100 10.2.1.2 Erythroid Apoptosis . . . . 100 10.2.1.3 Incriminating the Mito-

chondria . . . 101 10.2.2 Additional Contributors to Anemia

in MDS . . . 101 10.2.3 Myelodysplasia Subtypes

with Preferential Erythroid Lineage Involvement . . . 101 10.2.3.1 Sideroblastic Anemia

with Single-lineage

Involvement . . . 101 10.2.3.2 Refractory Anemia with

Single-lineage Involvement 101 10.2.3.3 5q± Syndrome . . . 102 10.3 Treatment of Anemia with Hemato-

poietic Growth Factors . . . 102 10.3.1 Recombinant Erythropoietin

Therapy . . . 102 10.3.1.1 Which Patients Will

Respond? . . . 102 10.3.1.2 How Prolonged a

Therapeutic Trial? . . . 105 10.3.1.3 How Durable Are Erythroid

Responses to Growth

Factors? . . . 105 10.3.1.4 Quality of Life . . . 105

10.3.1.5 Dosing Schedule . . . . 106 10.3.1.6 Mechanism of Action? . . 106 10.3.2 Erythroid Response to Other Growth

Factors and Combination Therapy 106 References . . . . 106

10.1 Introduction

The first report of recombinant hematopoietic growth factor therapy in myelodysplastic syndrome (MDS) pa- tients was published in 1987, in a landmark study of eight patients with predominantly high-risk MDS treated with recombinant granulocyte-macrophage col- ony-stimulating factor (GM-CSF) (Vadhan-Raj et al.

1987). An increase in mature leukocytes (neutrophils, monocytes, eosinophils, and lymphocytes) was ob- served in all patients, with an erythroid response in three subjects. Since these early studies, the focus has shifted to stimulants of erythropoiesis with the avail- ability of recombinant erythropoietins (EPO; alpha and beta), and recently the longer-acting derivative er- ythropoietin compounds. The currently available hema- topoietic growth factors have no clear role in the routine management of neutropenia or thrombocytopenia in MDS patients, although low-dose granulocyte colony- stimulating factor (G-CSF) therapy is sometimes advo- cated for patients with recurrent infections and severe neutropenia (Negrin et al. 1992). Despite the vast litera- ture and multitude of clinical studies, the precise role of hematopoietic growth factors in the management of MDS patients remains to be clearly defined. Definitive clinical trials powered to demonstrate improved quality of life and improved survival are still required.

Hematopoietic Growth Factors

David T. Bowen

The most prevalent clinical problem for patients with myelodysplastic syndromes is anemia. Eighty per- cent of patients are anemic at presentation (Sanz et al.

1989) and the majority require red cell transfusion at some stage of the disease. Although transfusion remains an appropriate intervention for many patients, increas- ing understanding of the optimal use of therapeutic agents, including the availability of several new drugs, will lead to an increasing proportion of patients treated with the intention of eliminating the transfusion need.

10.2

Mechanism of Anemia

As predicted from the clinical presentation, in vitro stud- ies of clonogenic growth have confirmed erythropoiesis as the lineage expressing the most prominent defect (Backx et al. 1993; Sawada et al. 1995). While the anemia of MDS can often be multifactorial (Fig. 10.1), the most prominent pathological processes are ineffective and hy- poproliferative erythropoiesis. Ineffective erythropoiesis is more commonly seen in refractory anemia (RA), re- fractory anemia with ring sideroblasts (RARS) and re- fractory anemia with excess blasts (RAEB)(usually

<10% blasts), while patients with >10% blasts have more hypoproliferative erythropoiesis (Cazzola et al. 1982).

10.2.1

Ineffective Erythropoiesis

Morphological abnormalities of erythroid precursors are one of the diagnostic hallmarks of MDS (Bennett et al. 1982). These include nuclear abnormalities such as megaloblastoid changes and nuclear irregularity.

Bone marrow from patients with RARS tends to show less nuclear irregularity, an increase in proerythroblasts and the classical iron deposition in mitochondria dem- onstrated on staining with Perl's reagent. The morpho- logical similarities with megaloblastic anemia suggest a fundamental defect in DNA synthesis as the pathologi- cal process underpinning ineffective erythropoiesis in MDS, although the nature of this defect remains elusive.

10.2.1.1 Clonogenic Erythroid Growth

Committed erythroid progenitor growth is reduced in most patients (May et al. 1985). MDS bone marrow is relatively deficient in erythroid (compared with mye- loid) clonogenicity as demonstrated both by replating of blast colonies (Backx et al. 1993) or by early CD34

cell lineage commitment (Sawada et al. 1995). Erythroid progenitor growth in semi-solid culture can be partially augmented in vitro by a variety of survival-augmenting (anti-apoptotic) strategies. These include increased con- centrations of early-acting hematopoietic growth factors such as stem cell factor (Backx et al. 1992) and granulo- cyte-colony stimulating factor (Schmidt-Mende et al.

2001), antioxidants (amifostine) (List et al. 1997), gluco- corticoids (Koeffler et al. 1978) and caspase inhibitors (Hellstrom-Lindberg et al. 2001). Residual non-clonal erythroid progenitors can be identified in some patients (Asano et al. 1994), and may be preferentially stimulated by in vivo hematopoietic growth factor therapy (Rigolin et al. 2002).

10.2.1.2 Erythroid Apoptosis

The paradox of a morphologically expanded bone mar- row erythron with peripheral anemia is explained by an increased rate of intramedullary erythroid cell death, most likely by augmented apoptosis (Hellstrom-Lind- berg et al. 1997a; Lepelley et al. 1996; Raza et al.

1996). While augmented erythroid apoptosis has been demonstrated by a variety of techniques, conflicting data cloud the clarification of the pathological processes that induce this apoptosis.

Fig. 10.1. Mechanisms of hematopoietic progenitor/precursor cell death in MDS. Extrinsic factors may include inhibitory cytokines (e.g., TNF-a, IFN-c and autoimmune attack (?HLA restricted). Intrinsic abnormalities include genetic (chromosome anomalies/gene mu- tations) or epigenetic phenomena (e.g., promoter methylation).

Mitochondrial dependent and independent pathways are impli- cated. TNF-a tumor necrosis factor-a, IFN-c interferon-c, Th1 subset of T-helper lymphocytes, HLA human leucocyte antigen, RS ring sideroblasts

Apoptosis and DNA fragmentation can clearly be demonstrated in the progenitor-enriched bone marrow CD34

cells (Parker et al. 2000; Peddie et al. 1997) as well as in erythroid precursors (Matthes et al. 2000;

Raza et al. 1995; Suarez et al. 2004). The final common effector pathway of apoptosis via caspase-3 is augment- ed in mononuclear cells (Boudard et al. 2000; Hell- strom-Lindberg et al. 2001) and erythroid cells (Hell- strom-Lindberg et al. 2001), predominantly in RA and RARS, and inhibition of caspase-3 promotes in vitro er- ythroid colony growth (Boudard et al. 2000). Caspase-3 can be activated through mitochondrial-dependent pathways (via Caspase-9), and also mitochondria-inde- pendent signals initiated via caspase-8. Erythroid pre- cursors from MDS patients are more sensitive to Fas- mediated cell death, but the evidence for release of a Fas-mediated erythroid block by in vitro inhibition of the Fas pathway is conflicting (Boudard et al. 2002;

Claessens et al. 2002; Dror 2003; Hellstrom-Lindberg et al. 2001). Clarification of the role of ªdeath receptorº pathway proteins (including Fas, FADD) may further elu- cidate the emerging contribution of extrinsic inhibitory and autoimmune processes to ineffective erythropoiesis.

10.2.1.3 Incriminating the Mitochondria

Several strands of evidence support a pathological role for mitochondrial defects in low-risk MDS. The patho- logical accumulation of ferritin in mitochondria is the basis for the diagnosis of the MDS subtypes with side- roblastic changes. This ferritin represents almost exclu- sively the recently described mitochondrial ferritin (Cazzola et al. 2003), which appears to preferentially ac- cumulate in RARS and congenital X-linked sideroblastic anemia, although the pathological relevance has yet to be established. While iron accumulation occurs only in sideroblastic mitochondria, augmented release of cy- tochrome c, activation of caspase-9 (Tehranchi et al.

2003), and loss of mitochondrial membrane potential (Matthes et al. 2000; Michalopoulou et al. 2004) incrim- inate the mitochondrion as at least an intermediary, and perhaps the primary perpetrator of premature erythroid apoptosis in both RARS and RA. Mitochondrial DNA mutations at loci encoding critical mitochondrial genes have recently been described in MDS patients (Gatter- mann 2004; Gattermann et al. 2004; Shin et al. 2003), but these may equally represent expansion of a clone with a coincident random heteroplasmic mutation, or a pathologically irrelevant abnormality.

10.2.2

Additional Contributors to Anemia in MDS Peripheral red cell destruction/loss may also produce anemia in MDS. Red cell loss may result from bleeding associated with thrombocytopenia or platelet functional defects. The red cell lifespan is shortened in some MDS patients, and this may be due to hemolysis (often with a positive direct antiglobulin test) (Sokol et al. 1989) or hy- persplenism. Many diverse red cell abnormalities are also described in MDS, though their clinical significance is less clear (Chalevelakis et al. 1991; Higgs et al. 1983;

Lintula 1986). Many patients with long-standing transfu- sion therapy show progressively increasing transfusion needs, possibly due to multiple mechanisms.

10.2.3

Myelodysplasia Subtypes with Preferential Erythroid Lineage Involvement

10.2.3.1 Sideroblastic Anemia with Single-line- age Involvement

RARS patients as defined by the French-American-Brit- ish (FAB) classification (Bennett et al. 1982) can be di- vided into two groups on the basis of single lineage (er- ythroid) vs. multilineage dysplasia and degree of non- erythroid lineage cytopenias. This is now recognized in the World Health Organization (WHO) classification (Bennett 2000) with the single lineage subtype renamed RARS, and the multilineage subtype refractory cytope- nia with multilineage dysplasia (RCMD)-RS. RARS has a better prognosis (77% vs. 56% 3-year survival) with no cases of leukemic transformation in a recently re- ported study (Germing et al. 2000a). However, myeloid colony growth may also be severely reduced in typical RARS patients, indicating a stem cell origin of this form of MDS (Hellstrom-Lindberg et al. 2001).

10.2.3.2 Refractory Anemia with Single-lineage Involvement

As for sideroblastic anemia the WHO classification re- cognizes two forms of the old FAB subtype refractory anemia (RA). The single-lineage subtype is renamed RA, and the multilineage involved subtype RCMD.

The prognosis and AML transformation rates are better for RA than for RCMD, but the difference is not as marked as for sideroblastic anemia (Germing et al.

2000b).

a

10.2.3.3 5q± Syndrome

The 5q± syndrome occurs predominantly in elderly women and is characterized by anemia, frequently thrombocytosis, and an isolated deletion of the long arm (or part thereof) of chromosome 5. Overall, 46%

of patients have erythroid hypoplasia, with only 13%

showing hyperplastic erythropoiesis (Giagounidis et al. 2004). The recent observation that the karyotypic de- fect is present in precursors of myeloid and lymphoid lineages as well as erythroid cells poses some funda- mental mechanistic questions as to why the clinical pic- ture predominates in the erythroid lineage (Nilsson et al. 2000). It should be noted that not all patients with a 5q± abnormality have a typical 5q± syndrome.

10.3

Treatment of Anemia with Hematopoietic Growth Factors

The aim of interventional therapy for anemia is to im- prove quality-of-life benchmarked against either the baseline untreated state, or against best supportive care in the form of red cell transfusions. These interventional therapies have the potential to produce sustained in- creases in hemoglobin concentration and to thus avoid the up-and-down lifestyle accepted by so many regu- larly transfused patients and their physicians.

10.3.1

Recombinant Erythropoietin Therapy The therapeutic efficacy of recombinant Erythropoietin (EPO) (Table 10.1), alone or combined with granulocyte colony-stimulating factor (G-CSF) (Table 10.2) in the treatment of anemia is now well established for selected patients with MDS. Cohort studies have clearly demon- strated responses, and two randomized studies have confirmed the superior erythroid response rates com- pared with either best supportive care plus placebo (ver- sus EPO alone) (Italian Cooperative Study Group for rHuEpo in Myelodysplastic Syndromes 1998), or best supportive care alone (versus EPO + G-CSF) (Casadevall et al. 2004). EPO therapy is generally well tolerated with the most common side effects being flu-like symptoms and occasional splenic pain and enlargement. Throm- bocytopenia can be accentuated in non-responders to EPO, but rarely with adverse clinical consequences (Hellstrom-Lindberg et al. 1997b).

The role of EPO therapy remains to be precisely de- fined and several key questions remain unanswered:

10.3.1.1 Which Patients Will Respond?

Early indications of response predictors to EPO therapy alone were identified in a meta-analysis covering trials of EPO for MDS patients up to 1994, and including 205 patients from 17 trials (Hellstrom-Lindberg 1995).

Overall response rate was 16%, using 100% reduction of transfusion need as minimal response criteria. Fac- tors predictive of response were non-RARS subtype, pre-treatment serum Erythropoietin concentration of less than 200 units/l, and absent need for red cell trans- fusion. Patients with RARS responded less well to Ery- thropoietin therapy alone with an overall response rate of 8% (Hellstrom-Lindberg 1995). The only double blind, randomized, placebo-controlled study of Eryth- ropoietin treatment in MDS showed an overall benefit for Erythropoietin over placebo (p=0.007) in MDS pa- tients with <10% bone marrow myeloblasts. However, analysis of patient subgroups showed a significant effect of treatment only in non-transfused patients and in pa- tients with RA. Again baseline serum Erythropoietin levels of less than 200 units/l predicted for response (Italian Cooperative Study Group for rHuEpo in Myelo- dysplastic Syndromes 1998). Taking these studies to- gether, it is likely that RARS with transfusion need re- sponds poorly to Erythropoietin as monotherapy.

The synergistic therapeutic effect of G-CSF added to

EPO has now been convincingly demonstrated (Hell-

strom-Lindberg et al. 1998; Negrin et al. 1996; Remacha

et al. 1999). This effect was most pronounced in patients

with RARS, who have shown the best response rate to

the combination (~50%). The combination therapy

was well tolerated. Using pre-treatment Erythropoietin

as a ternary variable (<100, 100±500 or >500 u/l) and

RBC transfusion requirement (< 2 or ³2 units per

month) as a binary variable, a predictive model for re-

sponse was developed from the data of 98 patients

treated in two multi-center studies (Hellstrom-Lindberg

et al. 1997b). Three groups were identified with pre-

dicted response rates of 74%, 23% and 7%. The model

remained predictive of response in a prospective valida-

tion study, although response rates were predictably

lower at 61% and 14% in the ªgoodº and ªintermediateº

predictive groups, respectively (Hellstrom-Lindberg et

al. 2003). It is important to emphasize that this predic-

tive model was derived for patients treated with a ther- apeutic trial of 12-week duration only. Relatively low se- rum EPO concentration has also been predictive of re- sponse in other studies (Terpos et al. 2002), although the above predictive model (EPO concentration plus transfusion need) was not helpful in a recently reported trial (Mantovani et al. 2000). The response criteria used for different studies have varied in their stringency, and those using the most stringent criteria (complete re-

sponse = achievement of Hb >11.5 g/dl and transfusion independence; partial response = >2 g/dl increment in Hb concentration and independence from transfusion) (Casadevall et al. 2004; Hellstrom-Lindberg et al.

2003) will be most likely to identify durable responses.

Despite the availability of a validated predictive model for response prediction, 39% of patients with a high predictive score still fail to respond. Improved pre- dictors of response or earlier indicators of therapeutic

a

Table 10.1. Trials of erythropoietin alone in MDS

Number of patients Results Comments

Meta analyses

Hellstrom-Lindberg (1995) 205 from 17trials 16% overall response** Higher response rates if:

Serum EPO <200 U/l Non-RARS FAB type Non-transfusion dependent Rodriguez et al. (1994) 115 from 10 studies 23.5% response Higher response for RAEB (abstract only; Spanish

language)

No relationship to EPO level or transfusion requirement Post-meta analyses (larger studies)

Terpos et al. (2002) 281 45% overall at 26 weeks

(18% at 12 weeks)*

Prolonged therapy increased re- sponse rates especially for RARS Italian Cooperative Study

Group for rHuEpo in Myelodysplastic Syndromes

87 14/38 vs. 4/37responders;

(p ( =0.007)

Randomized double-blind place- bo-controlled study of EPO in low-risk MDS

(1998) In favor of EPO* Response assessed at 8 weeks

Response predictors = FAB type RA, basal EPO level <200 U/l, non-transfused

Stasi et al. (1997a) 43 16.7% (CR+PR)* ±

Stasi et al. (1997b) 25 4 CR, 5PR* Responders had lower serum

concentration of TNF-/*

Di Raimondo et al. (1996) 12 with RA only, normal WBC and platelets and short duration of disease

7 (58.3%) CR, 2PR* Highly selected, mild cases of RA

Rose et al. (1995) 116 28%* Serum EPO <100 Mu/ml predicted

for response (54% of RA with low EPO responded)

** Less stringent CR/PR criteria: for example CR=increase in Hb ³20 g/l and elimination of transfusion need; PR = increase in Hb of 10±20 g/l in non-transfused patients, or a reduction of transfusions by 50%

** CR/PR criteria per Hellstrom-Lindberg: CR=Hb >115 g/l and no transfusion need; PR=rise in Hb ³15 g/l in on-transfused patients, or elimination of transfusion need with stable Hb in previously transfused patients

Table 10.2. Trials of erythropoietin PLUS G-CSF in MDS

Number of patients Erythroid response Comments Casadevall et al.

(2004)

60 (randomized 1:1) 42% in EPO/G-CSF group** No improvement in QOL in treat- ment group; only 3/30 patients still responding at 12 months Hellstrom-Lindberg

et al. (1997b)

53 61%, good predictive

group**

Prospective study validating predic- tive model

14%, intermediate predic- tive group

Median response duration

=29 months (CR) vs. 5.5 months (PR)

Mantovani et al.

(2000)

33 61% erythroid response at

12 weeks*

Twelve of 17responders at 12 weeks and 14/20 responders at 20 weeks had ªgood erythroid responseª, namely independent of red cell transfusion/sustained increase in Hb >2 g/dl 80% erythroid response at

36 weeks

Thirteen responders maintained re- sponse on treatment for 2 years Remacha et al.

(1999)

32 Erythroid response in 50%

(12 patients CR and 4 PR)*

±

Hellstrom-Lindberg et al. (1997b)

Development of pre- dictive model from (Hellstrom-Lindberg et al. 1998) and (Negrin et al. 1996)

36% response** In multivariate analysis, serum EPO levels and initial transfusion need were significant predictors of re- sponse

Median duration of response=11±24 months

Predictive score for response devel- oped by log-likelihood and logistic High predictive group = 74%

probability of response

regression analyses.

Int. predictive group = 23%

probability of response Low predictive group = 7%

probability of response Hellstrom-Lindberg

et al. (1998)

50 in randomized study

Overall response rate was 38%**

In randomized study:

Response rates in the two arms were identical

Arm A=G-CSF for 4 weeks followed by combination for 10 weeks Median survival = 26 months,

leukemic transformation 28%

Arm B= EPO for 8 weeks followed by the combination for 10 weeks Median duration of response

in 20 long-term maintenance patients = 24 months

Response rates for RA, RARS, RAEB were 20%, 46%, 37%, respectively

response are clearly required. Early indications are that these may include relatively simple parameters such as those reflected in the WHO classification (Howe et al.

2004).

10.3.1.2 How Prolonged a Therapeutic Trial?

Two recent studies with less stringent response criteria than the Scandinavian trials have demonstrated an in- creased response rate with prolonged growth factor treatment. Responses to therapy with EPO + G-CSF in- creased from 61% at 12 weeks to 80% at 36 weeks (Man- tovani et al. 2000), while responses to EPO therapy alone increased from 18% at 12 weeks to 45% at 26 weeks in another cohort (Terpos et al. 2002). Both studies in- dicate that RARS (FAB classification) patients benefit most from these prolonged therapeutic trials, but the quality of response was not described.

10.3.1.3 How Durable Are Erythroid Responses to Growth Factors?

Quality of response determines durability with a med- ian response duration for complete responders (achieve- ment of Hb >11.5 g/dl and transfusion independence) of 29 months versus 5.5 months for partial responders (>2 g/dl increment in Hb concentration and indepen- dence from transfusion) (Hellstrom-Lindberg et al.

2003). Prolonged responses are reported with similar durability in some cohorts (Hast et al. 2001; Mantovani et al. 2000), but not others (Casadevall et al. 2004).

10.3.1.4Quality of Life

No randomized studies have been conducted with suffi- cient statistical power to demonstrate differences in quality of life (QOL). One small cohort study identified an increase in global QOL (EORTC QLQ-C30 instru- ment) in responding patients (Hellstrom-Lindberg et

a

Table 10.2 (continued)

Number of patients Erythroid response Comments

Negrin et al. (1996) 55 53 (96%) had a neutrophil

response

Response predicted by low serum EPO level, higher absolute basal 44 patients evaluated for an

erythroid response and 21(48%) had a response

reticulocyte counts and normal cytogenetics at study entry

81% of these responders maintained their response during an 8-week mainte- nance phase*

Imamura et al. (1994) 10 No responses in erythroid or platelets following 10 weeks of treatment

One delayed erythroid response following cessation of treatment

80% had a neutrophil re- sponse*

Considerably higher doses of G-CSF (intravenous) than in other studies Hellstrom-Lindberg

et al. (1993)

22 8 (38%) showed a significant

response in Hb**

Less-advanced pancytopenia, lower levels of serum EPO and ring sidero- blasts predicted for response

Negrin et al. (1993) 24 10 (42%) had an erythroid

response*

Low pre-treatment EPO levels only predictor for response

** Less stringent CR/PR criteria: for example, CR=increase in Hb ³20 g/l and elimination of transfusion need; PR = increase in Hb of 10±20 g/l in non-transfused patients, or a reduction of transfusions by 50%

** CR/PR criteria per Hellstrom-Lindberg: CR=Hb >115 g/l and no transfusion need; PR=rise in Hb ³15 g/l in on-transfused patients, or elimination of transfusion need with stable Hb in previously transfused patients

al. 2003), while a small randomized study failed to show any difference in QOL (Functional Assessment of Can- cer Therapy-anemia tool) between cohorts treated with EPO + G-CSF versus best supportive care (Casadevall et al. 2004).

10.3.1.5 Dosing Schedule

The vast majority of published studies have used dosing schedules for EPO of approximately 50,000±70,000 units/week in 3±5 divided doses for a minimum of 6 weeks. Small studies have indicated equivalent efficacy of once weekly dosing of EPO for MDS patients, usually at a total dose of approximately 40,000 U/week (Garypi- dou et al. 2003), but larger studies are required to con- firm this. Given the higher response rate of RARS pa- tients to combination therapy, it is reasonable to initiate treatment with EPO plus G-CSF for this group. If G-CSF is used, it should be added at a dose to normalize (and at least double) the neutrophil count if it is less than 1.5´ 10

/l or double the neutrophil count if it is more than 1.5´ 10

/l. As for all other patients on EPO treat- ment, functional iron deficiency has to be considered, though this has not been systematically studied as a cause for non-response to EPO in MDS.

10.3.1.6 Mechanism of Action?

A response to EPO + G-CSF therapy is morphologically associated with a reduction of bone marrow apoptosis, reduced (but more effective) bone marrow erythropoi- esis and, in RARS, a reduced number of ring sidero- blasts (Hellstrom-Lindberg et al. 1997a). In vitro, EPO + G-CSF reduces mitochondria-mediated pro-apoptotic pathway activation in erythroid culture systems from both RARS and RA patients (Tehranchi et al. 2003). A preferential stimulation of non-clonal hematopoietic cells in patients responding to EPO alone has also re- cently been reported (Rigolin et al. 2002).

10.3.2

Erythroid Response to Other Growth Factors and Combination Therapy

Several smaller cohort studies have indicated that re- sponse rates to the combination of EPO + granulocyte macrophage-colony stimulating factor (GM-CSF) are comparable to those with EPO + G-CSF (Economopou-

los et al. 1999; Hansen et al. 1993; Stasi et al. 1999). How- ever, the only randomized study of GM-CSF + placebo vs. GM-CSF + EPO showed low response rates (<10%

in each arm) and little difference between the two arms (Thompson et al. 2000). GM-CSF has more side effects than G-CSF and there is little evidence to recommend GM-CSF therapy in combination with EPO. Small stud- ies have also examined combinations of other agents (including growth factors) with EPO such as IL-3, 13- cis retinoic acid, cyclosporin-A, All-trans retinoic acid, or vitamin D, but none appear superior to EPO alone or in combination with G-CSF.

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