The erythropoietin receptor in normal and cancer tissues

Critical Reviews in Oncology/Hematology 67 (2008) 39–61

The erythropoietin receptor in normal and cancer tissues Wolfgang Jelkmann a,∗ , Julia Bohlius b , Michael Hallek b , Arthur J. Sytkowski c b

a Institute of Physiology, University of Luebeck, Ratzeburger Allee 160, D-23538 Luebeck, Germany Cochrane Haematological Malignancies Group (CHMG), Department I Internal Medicine, University of Cologne, D-50937 Cologne, Germany c Division of Hematology and Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA

Accepted 19 March 2008

Contents 1. 2.

3.

4.

5.

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structure and function of the erythropoietin receptor (EPO-R) in erythroid tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Identification of the EPO-R . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. The EPO-R gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. The structure of the EPO-R: a member of the cytokine receptor superfamily . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.1. Overall structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.2. The extracellular portion of the EPO-R . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.3. The cytoplasmic portion of the EPO-R . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Signal transduction pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.1. Phosphorylation of the EPO-R . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.2. EPO’s signal transduction cascade . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Function of the EPO-R in non-malignant non-hemopoietic tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Heart and blood vessels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Kidneys . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Central and peripheral nervous system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Expression and functionality of the EPO-R in tumor cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. The EPO-R in solid tumor biopsies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Human cancer cell cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Animal models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Clinical relevance of anemia in cancer patients and treatment options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Recombinant human ESAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1.1. Effectiveness and safety of ESA therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Clinical trials in cancer patients receiving ESAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.1. Evidence for improved tumor control or survival in patients receiving ESAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.2. Evidence for decreased tumor control or survival in patients receiving ESAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Thromboembolic events in cancer patients receiving ESAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.1. Methodological considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4. Implications for practice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Reviewers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biographies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract The hormone erythropoietin (EPO) is essential for the survival, proliferation and differentiation of the erythrocytic progenitors. The EPO receptor (EPO-R) of erythrocytic cells belongs to the cytokine class I receptor family and signals through various protein kinases and STAT ∗

Corresponding author. Tel.: +49 451 500 4150; fax: +49 451 500 4151. E-mail address: [email protected] (W. Jelkmann).

1040-8428/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.critrevonc.2008.03.006

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transcription factors. The EPO-R is also expressed in many organs outside the bone marrow, suggesting that EPO is a pleiotropic anti-apoptotic factor. The controversial issue as to whether the EPO-R is functional in tumor tissue is critically reviewed. Importantly, most studies of EPO-R detection in tumor tissue have provided falsely positive results because of the lack of EPO-R specific antibodies. However, endogenous EPO appears to be necessary to maintain the viability of endothelial cells and to promote tumor angiogenesis. Although there is no clinical proof that the administration of erythropoiesis stimulating agents (ESAs) promotes tumor growth and mortality, present recommendations are that (i) ESAs should be administered at the lowest dose sufficient to avoid the need for red blood cell transfusions, (ii) ESAs should not be used in patients with active malignant disease not receiving chemotherapy or radiotherapy, (iii) ESAs should be discontinued following the completion of a chemotherapy course, (iv) the target Hb should be 12 g/dL and not higher and (v) the risks of shortened survival and tumor progression have not been excluded when ESAs are dosed to target Hb <12 g/dL. © 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: Anemia; Cancer; Erythropoiesis stimulating agents; EPO receptor

1. Introduction The glycoprotein erythropoietin (EPO) is essential for the survival, proliferation and differentiation of the erythrocytic progenitors in the bone marrow. Insufficient EPO production is the main cause of the anemia in patients with chronic kidney disease (CKD) as circulating EPO is mainly of renal origin. In contrast to the anemia of CKD, the etiology of the anemia of chronic disease (ACD) in patients with cancer is multifactorial and only partly due to a lack of EPO [1]. Other pathogenetic factors in ACD are the inhibition of the proliferation of the erythrocytic progenitors by immunomodulatory peptides, the insufficient iron availability, the increased hemolysis and, possibly, bleeding. In addition, myelosuppressive therapeutics inhibit erythropoiesis. Nevertheless, the administration of recombinant human EPO (rhuEPO) and its analogues to anemic patients with cancer receiving chemotherapy can reduce the proportion of patients who require red blood cell (RBC) transfusions. In 1993, the use of rHuEPO was approved by the FDA for the treatment of anemia in patients with solid tumors and non-myeloid malignancies undergoing chemotherapy. In 2002, the hyperglycosylated rHuEPO mutein Darbepoetin alfa was also licensed for the treatment of anemia in cancer patients receiving chemotherapy. Additional recombinant erythropoiesis stimulating agents (ESAs) have recently entered the European market for use in nephrology, oncology and surgery. Evidence has accumulated during the past 20 years that the EPO receptor (EPO-R) is not only expressed by erythropoietic cells but by a number of other tissues including the cardiovascular system and the brain. While these findings promise beneficial effects of endogenous EPO and its therapeutic analogues as tissue-protective factors, for example in ischemic and degenerative heart and brain diseases [2], fear has also arisen that EPO may promote tumor cell survival and stimulate tumor growth [3,4]. The first part of this article reviews the structure and function of the EPO-R of the erythrocytic progenitors. The second part describes the alleged role of the EPO-R outside the bone marrow. In particular, the controversial preclinical studies on the action of EPO on tumor cells are considered. Finally, the recent developments in the use of rhuEPO and

its analogues in cancer patients receiving chemotherapy are outlined.

2. Structure and function of the erythropoietin receptor (EPO-R) in erythroid tissues 2.1. Identification of the EPO-R The initial identification of the erythropoietin (EPO) receptor (EPO-R) proved more difficult than would have been expected. Although a method of EPO purification had been published in detail [5], the striking shortage of starting material – the urine of anemic humans – made it virtually impossible for the feat to be repeated. Secondly, the radioiodination of EPO using the then popular chloramine T method resulted in inactivation of the hormone, thus making it useless for receptor studies. Lastly, a highly enriched population of cells bearing the EPO-R was not generally available. EPO was first radiolabeled for receptor studies by incorporation of tritiated thymidine into its sialic acid residues. Using this radiolabeled EPO, specific binding sites (“EPO-R”) were demonstrated on erythroid cells [6]. Later, radioiodination in the presence of IodogenTM , which appears to label only one of four available tyrosyl residues in EPO, resulted in a labeled EPO with preserved biologic activity, useful for receptor binding studies [7,8]. Once rHuEPO became available, numerous investigators began to study the EPO-R on a variety of erythroid cell types, both normal and transformed, and receptor numbers ranging from 34/cell [9] to approximately 3000/cell [10] were reported. Highly enriched erythroid colony forming cells, which are predominantly CFU-E, reportedly express approximately 1000 EPO-R/cell [11]. Interestingly, studies of the thermodynamics of binding frequently revealed two different affinity classes of EPO-R (binding sites) with the higher affinity class ranging from KD ≈ 90–900 pM and the lower affinity from KD ≈ 200–9000 pM [8,12]. Other studies identified only a single class of receptors [13,14]. There is no clear molecular explanation for these different observations. There was also some evidence that differential glycosylation of the receptor was responsible for the two affinity classes, but it was later disproven [15].

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Various crosslinking studies were also carried out, usually by incubating 125 I labeled EPO with cells (to permit binding to the EPO-R) followed by addition of a homobifunctional crosslinking reagent like disuccinimidyl suberate (DSS). After dissolution, cell proteins were subjected to SDSPAGE and autoradiography. Using such methods, numerous investigators reported radiolabeled crosslinked species of 85–150 kDa, and frequently more than one radiolabeled species was seen [7,12,16,17]. It was suggested that differences in the size of the EPO/EPO-R complex might be due to altered posttranslational processing of a single EPO-R protein [18]. Also, a receptor heterodimer was proposed [19]. These observations have not been explained fully in light of the apparent molecular mass of the EPO-R (62–66 kDa) observed by Western blotting. It is possible to modulate cell surface EPO-R expression and the biologic response to EPO in erythroid cells. Pretreatment of an EPO sensitive erythroid cell line (Rauscher murine erythroleukemia [20,21]) with dimethyl sulfoxide or other polar/planar compounds led to a marked amplification of the biologic response to EPO, including an increase in number of cells responding, increase in rate of response and a markedly left shifted EPO dose/response curve, consistent with greater sensitivity to the hormone [22]. Binding studies showed that DMSO treatment increased EPO-R density from 3000/cell to over 20,000/cell with no significant change in receptor mRNA levels [23]. Interestingly, thermodynamic analysis revealed a Scatchard curve that was upwardly convex, indicative of positive cooperativity with a Hill coefficient, nH, of 6.75, indicating the presence of receptor oligomers or clusters comprising several EPO-R molecules and, potentially, other accessory proteins. 2.2. The EPO-R gene D’Andrea et al. [24] isolated a cDNA encoding a predicted 507 amino acid murine EPO-R by using an expression library constructed from Friend erythroleukemia cells. Unexpectedly, whereas radiolabeled EPO binding studies of the original Friend cells revealed a single affinity class of EPO-R, expression of the cDNA in COS cells resulted in two affinity classes. A 508 amino acid human EPO-R cDNA was constructed from a partial cDNA and an exon sequence [24] and, independently, from OCIM1 erythroleukemia cells and from fetal liver by screening with murine EPO-R cDNA [25]. The human gene is localized to chromosome 19pter-q12 [26]. The EPO-R gene is approximately 6 kb in length and comprises eight exons [27–29]. Exons 1–5 encode the extracellular (exoplasmic) domain, exon 6 encodes the transmembrane domain and exons 7 and 8 encode the cytoplasmic domain. Importantly, EPO-R mRNA and/or EPO-R protein have/has been detected in several non-hematopoietic tissues including brain, kidney, placenta, endothelial cells, myocardiocytes, macrophages, retinal cells, cells of the adrenal cortex as well as numerous human cancers. The non-hematopoietic actions of EPO and non-hematopoietic EPO-R are discussed below.

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Although the EPO-R is transcribed continuously [30], hypoxia and anemia may upregulate EPO-R expression in hematopoietic tissue and brain [31]. EPO-R mRNA splicing is more efficient in erythroid cells than in brain [31]. Extrinsic regulators of EPO-R gene expression also include stem cell factor [32] and interleukin-1␣ [33], which are stimulators, and interferon-␥ [34], ionomycin and phorbol ester PMA [34], which are inhibitors. The EPO-R gene prime 5 flanking region contains GATA1 [35] and SP1 [35] regulatory elements. There is also a negative CCACC motif located in the +79 to +135 fragment of the human EPO-R gene promoter [36]. 2.3. The structure of the EPO-R: a member of the cytokine receptor superfamily 2.3.1. Overall structure The unprocessed human EPO-R protein consists of 508 amino acids with a predicted molecular mass of approximately 56 kDa. It should be noted that crosslinking and photo-affinity labeling experiments have resulted in higher molecular mass estimates of 65–105 kDa [19,25,37]. When the murine EPO-R was expressed in COS cells followed by Western blotting, multiple EPO-R bands of 62, 64, and 66 kDa were detected [38]. The endogenous EPO-R of erythroid cells, though of somewhat higher apparent molecular weight, also appeared as more than one size. Possible explanations include glycosylation (there is one glycosylation site), ubiquitination and other modifications or interactions with other proteins. There is a 24 amino acid signal peptide, which is cleaved upon processing. The extracellular portion of the human EPO-R contains 225 amino acids with 22 amino acids in the transmembrane domain and 236 amino acids in the cytoplasmic portion. The EPO-R, along with the IL-2R beta chain, is a founding member of the cytokine receptor superfamily [39-41], which includes an array of other family members. Each family member has a single hydrophobic transmembrane spanning region, a variable cytoplasmic region and an extracellular region with an overall 15–35% homology. The extracellular portion contains conserved cysteine residues and a “WSXWS” motif [40–43]. This motif in the EPO-R is critical for ligand binding, internalization and signal transduction [44,45]. None of the cytokine receptor superfamily members exhibit any kinase or other enzymatic domain in the cytoplasmic region. There are limited homologies in the membrane proximal regions and the so-called Box 1/proline rich motif and Box 2 motif [46–48], which support the mitogenic function of the receptor. 2.3.2. The extracellular portion of the EPO-R The 225 amino acids of the extracellular portion of the human EPO-R comprise two fibronectin type III-like domains [49]. Each domain has seven beta strands, and these seven beta strands are connected by six loops containing amino acid residues involved in EPO binding. Solution

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and X-ray crystallographic studies of the interaction of non-glycosylated, mutated EPO with the extracellular portion of the EPO-R (the so called soluble EPO-R; sEpoR) demonstrated that one EPO molecule binds to two EPO-R although, at least in solution, with markedly different affinities [49]. These two non-identical binding sites on the EPO molecule have been designated Site 1 (KD = 0.2 nM) and Site 2 (KD = 2.1 ␮M) [50]. Of course, the affinity of each site for the EPO-R when it is in the plasma membrane or when it exists as a preformed dimer has not been thoroughly addressed. The carboxy-terminal portion of the EPO-R extracellular region contains the dimerization domain. Binding of EPO to the EPO-R or to preformed EPO-R dimers results in a conformational change that triggers intracellular signaling, like it is seen commonly among receptors of the cytokine superfamily. The growth hormone receptor is the prime example of dimerization in the cytokine receptor superfamily [51]. 2.3.3. The cytoplasmic portion of the EPO-R The 236 amino acid cytoplasmic portion of the EPO-R lacks a kinase domain. The membrane proximal region contains the Box 1 and Box 2 motifs with limited homology within the superfamily [46–48]. The Box 1 motif, IWPGIPSP, and its flanking regions are necessary for the binding of JAK2 kinase to the receptor and for JAK2 activation [52–54], although a carboxy-terminal truncated EPO-R with only the Box 1 motif is sufficient to induce EPO dependent cell proliferation [55]. A region between Box 1 and Box 2, but not Box 2 itself, may be required for EPO dependent cell proliferation [56]. Other portions of the cytoplasmic domain are necessary for receptor internalization, p70 S6 kinase phosphorylation and activation [57]. 2.4. Signal transduction pathways 2.4.1. Phosphorylation of the EPO-R EPO’s binding to the EPO-R dimer induces a conformational change in the receptor pair that leads to activation of numerous kinases and other signaling molecules. Among these are JAK2 kinase, phosphatidylinositol 3-kinase (PI3K), protein kinase C (PKC), Lyn kinase [58] and Tec kinase [59]. Activated JAK2 and Lyn then phosphorylate the EPO-R [58,60]. A detailed, up-to-date description of the numerous states of the EPO-R and the protein-protein interactions and signaling elements associated with it may be found in [61]. The murine and human EPO-R cytoplasmic domains contain eight and nine tyrosine residues, respectively. Phosphorylation of the eight homologous residues (Tyr285 of the human EPO-R does not appear to have been studied) results in docking of SH2 domain containing proteins, such as Grb2, STAT5, Shc and CrkL, coupling EPO-R to its signal transduction pathways. Numerous studies in a variety of cell systems have attributed specific functions to several of these tyrosines, both collectively and individually. For example, Tyr343 and/or Tyr401 of the murine receptor participates in STAT5 activation and in phosphorylation of the docking

protein Gab2 [62–65]. Other studies can be cited that specify functions for each of these tyrosines [66]. Despite this work, there is at least one intriguing study that suggests that the tyrosine residues of the EPO-R may not be required for growth and terminal differentiation of human CD34+ erythroid progenitors [67]. 2.4.2. EPO’s signal transduction cascade The literature on EPO signal transduction in hematopoietic cells is very extensive, and numerous kinases, adapter proteins and other molecules have been shown to be phosphorylated in response to EPO-R activation. However, not all studies agree on the pattern of these phosphorylation events. They may be very dependent upon the cell background used. Most detailed studies have not been carried out in erythroid cells expressing endogenous EPO-R, although there are some exceptions. The group of proteins that have been reported to be phosphorylated in response to EPO-R activation include JAK2 kinase, protein kinase C, PKB (Akt), MAP kinase kinase (MEK), MAP kinase (ERK1/2), hematopoietic protein tyrosine phosphatase PTP1C, Gab1 and Gab2. Others include GSK-3b, phospholipase C-␥1 [68], GAP [69], Raf-1 [70], Ras [69], Shc [71], Vav [72], c-fps/fes [73], Lyn [74] and c-Cbl [75]. It is clear that these and other molecules point to numerous signaling pathways as part of EPO’s signal transduction cascade, leading to targets in the cytoplasm, nucleus and mitochondrion. 2.4.2.1. The JAK2-STAT5 pathway. JAK2 is a protein tyrosine kinase (PTK) [76] that is key to signaling of the EPO-R. The binding of EPO to the EPO-R results in autophosphorylation of JAK2 and its activation [52]. Activated JAK2 phosphorylates the EPO-R and other proteins including STAT1 and STAT5 [77,78]. These STAT proteins homodimerize, are translocated to the nucleus and direct gene transcription by binding to specific DNA elements [79,80]. There is, however, some debate about the precise role of STAT proteins in erythropoiesis, including findings that suggest that STAT5 is not essential for erythroid cell differentiation [81]. The JAK2/STAT5 pathway appears to be anti-apoptotic in erythropoiesis, since EPO induces Bcl-xL through STAT5 [79], and STAT5 interacts with PI-3K, which is required for cell cycle progression [44]. 2.4.2.2. Ras-Raf-MAP kinase pathway. There is great complexity in the Ras-Raf-MAP kinase pathway induced by EPO, and there is significant disagreement due to results that have been obtained using different cell backgrounds, both transformed and normal. The classic pathway is as follows: Receptor → Shc → Grb2 → Sos1 → Ras → Raf-1 → MEK → MAP kinase A series of reports indicate that EPO can induce activation of all of these signaling molecules. The EPO dependent tyrosine phosphorylation of Ship1 (SH2 inositol 5-phosphatase

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1) results in its binding to the EPO-R and the recruitment of Shc and Grb2 to the receptor [82]. It is far from clear that EPO activates this pathway by following the classic sequence in all cells, since JAK2 can associate with and phosphorylate Raf-1 [83]. Studies carried out in one of the authors’ laboratories showed that EPO stimulation of the EPO-R resulted in phosphorylation of Raf-1, MEK and ERK1/2. However, inhibition of Raf-1 activation did not alter the phosphorylation of Myc [84]. The role of the Ras-Raf pathway in erythropoiesis is incompletely understood. In human erythroid colony forming cells, the MAP kinase pathway appears to be essential for erythropoiesis [85]. However, in the Friend murine erythroleukemia cell line, MAP kinase inhibition induced erythroid differentiation [86]. Furthermore, studies of the truncated EPO-R demonstrated EPO induced proliferation without activation of MAP kinases [87]. 2.4.2.3. The PI-3K pathway. Activated PI-3 kinases phosphorylate phosphatidylinositol 4,5-bisphosphate (PI-(4,5)P2 ) to yield phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)-P3 ), which then activates PDK1 leading to phosphorylation of Akt (protein kinase B: PKB) which is a serine threonine kinase. Phosphorylated Akt then phosphorylates and inactivates BAD (a member of the Bcl-2 family), forkhead transcription factor and caspase 9 [88,89], thereby inhibiting apoptosis. Furthermore, Akt phosphorylates and activates glycogen synthase kinase 3 (GSK-3) [90], which has a role in the regulation of cyclin D1 and in the regulation of c-myc. Akt also phosphorylates and inactivates Raf at serine 259, identifying it as a regulator of the Raf/MEK/ERK signaling pathway [91]. Activation of the EPO-R leads to its association with the SH2 domains of the PI-3K regulatory subunit p85 [62,92–94]. The phosphorylation of insulin receptor substrate-2 (IRS-2) leads to its interaction with p85 as well [95]. p85 docks with phosphorylated Gab1 and Gab2 after EPO stimulation, as does SHP2 [96]. The phosphorylation of Vav protein after EPO stimulation may result in its association with the EPO-R as well as with p85 [72]. Work in one of the authors’ laboratories has demonstrated that PI-3K activity is required for EPO induced initiation of c-myc transcription whereas the MAP kinase pathway is required for its elongation [84]. It has been shown that PI-3K participates in cell survival during erythropoiesis and in activation of p70 S6 kinase, leading to phosphorylation of ribosomal protein S6 [97]. 2.4.2.4. The protein kinase C pathway. The protein kinase C family of serine/threonine kinases includes approximately 13 members [98–101] divided into several classes based on structure and cofactor regulation. 1) The conventional PKCs include ␣, ␤, ␤2 and ␥, which require calcium and diacylglycerol for their activation. 2) The novel PKCs include ␦, ␧, ␩, ␪ and ␮, which are calcium independent but diacylglycerol dependent.

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3) The atypical PKCs include ␨, ␫ and ␭, which are not activated by calcium or by diacylglycerol. 4) The recently described ␯, which may be grouped with ␮ based on structural homology. EPO induces tyrosine phosphorylation of phospholipase C (PLC)-␥, increasing diacylglycerol and calcium in human BFU-E derived erythroblasts [102-104] and activating PKC in Rauscher erythroleukemia and normal murine erythroid cells [105,106]. Since activated PKC is a serine/threonine kinase, it can phosphorylate Raf-1 [107,108], presumably at the cell membrane. However, studies from one of the authors’ laboratories suggest that PKC might not be upstream of Raf1, at least in the BaF3-EPO-R hematopoietic cell system [84]. PKC inhibitors such as H7 block formation of CFUE colonies suggesting that activation of PKC is required for normal erythropoiesis [109]. Additionally, it has been reported that PKC ␣ plays a role in differentiation of CD34+ human bone marrow progenitor cells [110]. PKC has been shown to be important for gene regulation in erythroid cells [105,106,111,112]. EPO dependent upregulation of c-myc requires the PKC ␧ isoform in Rauscher erythroleukemia cells [113]. Furthermore, the PKC ␧ signal is required for EPO induced DNA synthesis (growth signal) but not for EPO induced globin gene expression (differentiation signal). Other proliferation related genes that are regulated by EPO through one or another PKC mediated pathways include c-fos, c-jun, Bcl-3, GATA-2 and Bcl-xL [84,114,115]. 2.4.2.5. Other EPO dependent signaling events. Additional signaling events triggered by the EPO-R continue to be reported. For example, the adaptor protein CrkL is phosphorylated in response to EPO-R activation, resulting in its association with Shc and c-Cbl [116,117]. It also complexes with the guanidine nucleotide exchange factor C3G leading to activation of Rap1 GTPase. The beta2 subunits of heterotrimeric GTP binding proteins are released from the EPO-R upon binding of EPO [118].

3. Function of the EPO-R in non-malignant non-hemopoietic tissues EPO is a tissue-protective factor with much more pleiotropic potential than previously thought. EPO-R mRNA, EPO binding sites and EPO-R signaling have been detected in a variety of non-hemopoietic tissues such as heart, blood vessels, kidneys, liver, gastrointestinal tissues, pancreatic islands, testis, female reproductive tract, placenta and, operating separately from the other parts of the body, the brain [119–121]. 3.1. Heart and blood vessels EPO-R mRNA and EPO-R protein have been demonstrated in cultures of human cardiomyocyte cell lines [122]

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and in freshly drawn samples of human heart [123]. EPO treatment reduces myocardial infarct size, protects against ischemia-reperfusion injury and promotes ventricular remodeling in experimental animals subjected to coronary ligation [124–126]. EPO exerts anti-apoptotic effects on myocytes and fibroblasts in the heart [127,128]. The therapeutic potential of EPO for myocardial protection is under clinical investigation. Anagnostou et al. [129,130] discovered that human endothelial cells express EPO-R mRNA. EPO mobilizes endothelial progenitor cells [131,132], stimulates the proliferation of endothelial cells [129] and promotes neovascularization [133,134]. High doses of EPO (≥100 U/mL) have been shown to reduce the incidence of apoptosis in bovine pulmonary artery endothelial cultures treated with bacterial lipopolysaccharide [135]. Vascular smooth muscle cells are also EPO-responsive. EPO induces their contraction [136] and vasoconstriction [137] when added in high concentrations to isolated smooth muscle cells in primary culture or to isolated vessels, respectively. EPO increases oncogenes promoting DNA replication and smooth muscle cell growth [138]. Both the PI-3K/Akt [139] and the MAPK [140] pathways appear to play important roles in these reactions. 3.2. Kidneys EPO-R transcripts have been detected in tubular and mesangial cells of human and rodent kidneys [141]. High concentrations of EPO (200 U/mL) protect human proximal tubular epithelial cells (PTEC) in primary culture from hypoxia-induced apoptosis [142]. EPO (≥10 U/mL) prevents apoptotic cell death in response to oxidative stress in the human PTEC line HK-2 [143]. Similarly high dosed EPO lowers sodium excretion in isolated perfused rat kidneys [144]. EPO treatment reduces renal injury and dysfunction caused by ischemia/reperfusion [142–146], and it accelerates renal tubular regeneration in rodents with cisplatin-induced acute renal failure [147]. Recent evidence suggests that EPO exerts its nephroprotective effect by inducing the accumulation of hypoxia-inducible factor 1 (HIF-1) in the kidneys [146]. 3.3. Central and peripheral nervous system EPO-R mRNA and EPO-R protein are detectable in distinct areas of the mammalian brain, including cortex, hippocampus, capsula interna and midbrain [148]. The EPO-R is expressed by neurons, astrocytes and endothelial cells in brain. The functional consequences of the fact that brain capillary endothelial cells transcribe two splice forms of EPO-R mRNA are not known [149]. Importantly, the EPO-R is also expressed in peripheral nerves [150]. EPO exerts both neurotrophic and neuroprotective effects (for a review, see [151]). It stimulates the proliferation and differentiation of neuronal stem and progenitor cells [152,153]. EPO promotes the survival of septal choliner-

gic neurons in rats with fimbria-fornix transsections [154]. EPO infused into the lateral brain ventricles of rodents prevents ischemia-induced learning disability and rescues hippocampal neurons from death [155], while EPO infused into the cerebral ventricles reduces ischemia-induced place navigation disability and cortical infarction [156]. Locally administered EPO upregulates the expression of Bcl-xL in the hippocampal CA1 field in gerbils with experimental cerebral ischemia [157]. The neuroprotective action of EPO on anoxic primary hippocampal neuronal cell cultures involves the PI-3K/Akt pathway which is necessary to maintain the mitochondrial membrane potential [158]. Systemically administered EPO appears to enter the brain in very small amounts. It has been claimed that EPO may cross the blood–brain barrier via EPO-R-mediated transfer [159], but the mechanisms of this transfer still need to be clarified in more detail. Investigators have reported that the systemic administration of high doses of EPO reduces the volume of infarction [160] and mortality [161] in experimental animals with middle cerebral artery occlusion. In hypoxic brain recombinant EPO reduces inflammatory reactions [162]. Carbamoylated EPO derivatives that do not stimulate erythropoiesis still confer neuroprotection [163]. Possibly, these compounds exert their effect by binding to heterodimers formed by one EPO-R molecule and by ␤cR [164], which is the common cytokine receptor monomer shared by the receptors for GM-CSF, IL-3 and IL-5.

4. Expression and functionality of the EPO-R in tumor cells 4.1. The EPO-R in solid tumor biopsies Knowledge that EPO has pleiotropic functions has prompted the critical question of whether tumor cells express the EPO-R and whether EPO can induce or promote tumor growth [165]. Since EPO-R mRNA is detectable in cancer tissue [166–170] – like it is in non-malignant tissues – it was important to investigate whether the EPO-R genomic locus is amplified in tumors compared with non-oncogenic loci. To answer this Sinclair et al. [171] have studied ∼1000 tumor samples by microarray analysis and 68 by RT-PCR with probes flanking either side of the EPO-R locus. The authors obtained no evidence for amplification of the EPO-R gene in tumor tissues above the normal two copies. A microarray analysis based on several different probes for EPO-R revealed that EPO-R transcript levels were not elevated above the one in equivalent normal tissues in breast, brain, colon, kidney, lung, prostate and lymphoma tumor samples [171]. It is not clear whether tumors translate EPO-R protein in significant amounts. Most reports claiming the presence of EPO-R protein were based on studies with the commercial polyclonal antibody C-20 (Santa Cruz) that has been raised against the 20 carboxy-terminal amino acids of the human EPO-R [166–168,172–186]. However, the results of

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such investigations are difficult to interpret because the C-20 antibody interacts with many proteins other than the EPOR [187]. C-20 binds to proteins of 35, 66 and 100 kDa that are clearly unrelated to the EPO-R [187–189]. In all likelihood, the 66 kDa band seen on Western blotting is heat shock protein HSP70 [187,188,190], and the 100 kDa band is HSP90 as judged from amino acid sequence analysis [189]. Also, the published immunohistochemical staining patterns do not enable one to see whether the stain is localized to the cell membranes, considered a prerequisite for EPO-R function. The interpretation of findings with the C20 antibody is further complicated by the observation that there are major batch-to-batch differences in the staining patterns with this product [191]. Another commonly used polyclonal antibody, M-20 (Santa Cruz), which is raised against the 20 carboxy-terminal amino acids of murine EPOR, reacts with both the human and the mouse EPO-R on immunoblotting, but also with several other proteins [187]. The C-20 and M-20 antibodies falsely produce stain signals in tissues of EPO-R knockout mouse tissues [187]. Other antibodies tested have included H-194 (polyclonal anti-human EPO-R; Santa Cruz), 07–311 (polyclonal anti-murine EPOR; Upstate) and MAB307 (monoclonal anti-human EPO-R; R&D Systems). Unfortunately, these antibodies also crossreact with multiple proteins of sizes differing from that predicted for the EPO-R [187,189]. Although MAB 307 blocks signaling through EPO-R, it is unsuitable for Western blotting [192]. All of these facts seriously call into question the significance of the published immunohistochemical studies of EPO-R protein in biopsies from human tumors. 4.2. Human cancer cell cultures EPO-R mRNA is usually detectable in cultures of human cancer cells, either in primary culture or on establishment as permanent lines [167,170,173,176–178,193–201]. In fact, a recent reinvestigation of various human cancer cell lines (SHSY5Y, MCF-7, HepG2, U2-OS, HeLa, HEK293T, RCC4, HCT116, 7860 wt and SW480) performed in one of the authors’ laboratories by real-time RT-PCR has confirmed the expression of EPO-R mRNA in all cultures under study [189]. However, neither hypoxia exposure nor EPO treatment was found to alter the level of EPO-R mRNA expression in this study, whereas other investigators measured increased EPO-R mRNA levels in SiHa cervical [176] and MCF7 breast cancer cells [175,202] immediately after hypoxia exposure. In looking for molecular mechanisms it turns out that hypoxia-response DNA elements in control of the Epo-R gene remain to be shown. EPO mRNA is also detectable in several cancer cell lines, and stimulation of EPO production under hypoxic conditions has been suggested as a paracrine mechanism favouring the survival of cancer cells [167,168,174–177,183,199,201,203]. In addition, it has been proposed that tumor cell-derived EPO may promote angiogenesis in vivo [185,186].

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Due to the lack of specific anti-EPO-R antibodies it is not clear to which extent cultured tumor cells express the EPO-R protein. Sinclair et al. [204] have recently provided a nice overview of the methodological deficiencies in studies aimed at demonstrating EPO-R in tumor tissue. Several investigators used the C-20 antibody for immunodetection of the EPO-R on Western blots of cell extracts [167,170,176–178,195,199,205], but this antibody was subsequently shown to be unsuitable for such studies [187–189]. When the EPO-R gene was knocked out by small interfering RNA technique (RNAi) in EPO-R positive human osteosarcoma cells, only the 60 kDa protein was lost on staining with C-20 [189]. This finding excludes the possibility that the other proteins that react with the C-20 antibody are generated by alternative RNA splicing or by posttranslational processing. EPO-R binding studies utilizing 125 I-labeled EPO failed to detect specific EPO binding sites on human MDA-MB-231 and MCF-7 breast carcinoma cells [206]. There are many reports stating that erythropoiesis stimulating agents did not induce EPO-R signaling and did not stimulate proliferation of cancer cells in culture [172,189,194,197,206–213], even when very high doses of the drugs were applied. The possibility has been considered that EPO may exert other effects than growth promotion. For example, in cultures of human neuroblastoma cells (h-NMB) EPO inhibits growth, while it stimulates differentiation [210]. In contrast to the majority of reports, in some in vitro studies growth stimulating effects of EPO on cancer cells were seen [173–175,195], at least when very high doses were applied which greatly exceeded the concentrations measured in untreated healthy humans (∼0.02 U/mL plasma) or after s.c. administration of the recombinant drugs in clinical practice (increase by ∼0.15 U/mL plasma on administration of 150 U rHuEPO per kg body weight; [214]). As an example, 250 U rHuEPO per mL culture medium was required to show significant effects on protein phosphorylation, DNA synthesis, proliferation and migration in breast cancer cell cultures [175,202]. EPO concentrations of ≥10 U/mL were necessary to stimulate the release of angiogenic growth factors in pediatric tumor cell lines [177]. On the other hand, relatively low concentrations of EPO (0.5–1 U/mL) sufficed to promote the growth of distinct human renal [195] and prostate cancer cell lines [215]. EPO treatment (1 U/mL) has been reported to increase cyclooxygenase2 and ␤-casein mRNA levels in papillary thyroid cancer cells (line NPA), while gene array studies indicated that EPO does not alter the expression of 96 genes that support angiogenesis [170]. Other investigators have found that EPO (≥30 U/mL) lowers the level of hypoxia-inducible transcription factor 1␣ (HIF-1␣) and of secreted vascular endothelial growth factor (VEGF) in SK-OV-3 ovarian cancer cells [205]. Dunlop et al. [216] have reported activation of key signaling pathways (STAT5, Akt, ERK) in the nonsmall cell lung carcinoma cell line H838 on EPO treatment, while there was no proliferative response. Further studies showed that the EPO-R was not ubiquitinated and protea-

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somally degraded following EPO stimulation of H838 cells [217]. Provided tumor cells in culture possess functional EPO-R molecules one explanation for the different outcomes of the studies could relate to the differences in culture conditions, for example the presence or lack of fetal calf serum. In fact, a synergism on cell proliferation has been considered between EPO and other growth factors such as insulin or insulin-like growth factor-I [195]. However, a look at the above cited literature reveals that the negative and positive effects of EPO on tumor cell lines cannot be correlated with the use of either serum-containing or serum-free media. In addition, a study designed to improve protocols for removal of cancer cells from autologous stem cell preparations has shown that the addition of a mixture of EPO + IL-1␤ + IL-3 + IL6 + stem cell factor (SCF) does not influence the survival (under serum-free conditions) or proliferation (on serum supplementation) of primary breast cancer cells in culture [218]. Because most in vitro investigations on tumor cell growth were performed with permanent cell lines one may also raise the critical question as to the clinical relevance of reports of response to EPO in one laboratory but not in another. Here, a typical example is the divergent results obtained with the neuroblastoma cell line SH-SY5Y [189,219]. Hence, of major importance are the seminal studies by Bauer et al. [220] who investigated the effects of EPO (up to 400 U/mL) on the in vitro clonal growth of 53 human primary tumor specimens (mainly of renal or colorectal origin) in a soft agar cloning system. In this study, EPO treatment was without effect in 47 specimens, while stimulation was observed in 2 and inhibition in 5 specimens [220]. Kokhaei et al. [221] investigated effects of Epoetin alfa, Epoetin beta and Darbepoetin alfa on the in vitro proliferation of enriched tumor cells from eight patients with B-CLL, three patients with MCL and four MM patients. No ESA induced proliferation was observed with any of the preparations. Thus, neither cancer nor lymphoid malignant or myeloma cells in primary culture are generally responsive to EPO. Liu et al. [222] have considered the possibility that EPO may not influence the basal viability of tumor cells but may protect the cells from the cytotoxic effect of drugs like cisplatin. However, studies of the combined effects of EPO and chemotherapeutics on cancer cells in culture are not conclusive. Gewirtz et al. [223] have reported that EPO treatment fails to interfere with adriamycin, taxol and tamoxifen in parent MCF-7 or in p53 mutant MDA-MB231 breast cancer cell lines. Renal carcinoma cells (RCC) were found to undergo a higher degree of apoptosis on treatment with a combination of daunorubicin or vinblastine and EPO than with either of these agents alone [224]. In contrast to such chemosensitization, EPO was found to cause resistance to cisplatin in HeLa [176], U87 glioma and HT100 cervical cancer cells [225], and to dacarbazine in melanoma cells [199]. In order to demonstrate these effects extremely high EPO concentrations were necessary, being actually about 1000-fold higher

than the plasma concentration reached in cancer patient on rHuEPO treatment. Unphysiologically high in vitro levels of EPO can induce tyrosine phosphorylation of the ␤-chain of the GM-CSF receptor (␤cR), as shown in UT-7 cells [226]. On the other hand, at least in SH-SY5Y and PC-12 neuronal tumor cells EPO may exert cytoprotective effects, although ␤cR is not detectable in these cell lines [227]. A very recent study from one of the authors’ laboratory has shown that the long-term treatment of ovarian cancer cells (line A2780) with EPO results in the development of a phenotype exhibiting both enhanced EPO signaling and increased resistance to paclitaxel [228]. This effect is drug specific, since no change in cisplatin or carboplatin sensitivity was observed. The longterm treatment with EPO proved to lower the expression of the pro-apoptotic proteins Bcl-2 and Bcl-10 in A2780 cells [228]. 4.3. Animal models Because extrapolations from cancer cell culture studies in vitro to cancer growth in vivo bear great risks of uncertainty, preclinical in vivo studies of the effects of EPO on tumor growth are of major significance. Yasuda et al. [196] have first shown that the application of anti-EPO-antibody or of soluble EPO-R produces a reduction in tumor size of the transplants of uterine or ovarian tumors in nude mice. Similarly, inhibition of tumor angiogenesis and tumor cell viability was observed on i.p. administration of a synthetic EPO-R blocking peptide (EMP9) in mice with subcutaneous xenografts of human stomach choriocarcinoma (SHC) or melanoma (P39), while application of an EPO mimetic peptide (EMP1) was found to promote tumor angiogenesis and tumor cell survival [198]. These findings have led to the concept that EPO may stimulate tumor growth through its anti-apoptotic effect on the endothelium. Along these lines, the application of either anti-EPO-antibody, soluble EPO-R or an inhibitor of JAK2 proved to produce a delay in tumor growth in a tumor-Z chamber model with rat syngeneic mammary adenocarcinoma cells [173]. Obviously, the direct effects of endogenous EPO on tumor growth deserve to be carefully investigated. Hardee et al. [229] have recently applied a dorsal skin-fold window chamber technique in mice, which allowed the investigators to study tumorigenesis by intravital microscopy. Tumor angiogenesis and tumor growth were inhibited when either soluble EPO-R or anti-EPO antibody was co-injected with mammary carcinoma cells in the window chambers, or when an EPO-antagonist protein (R103A-EPO) was expressed by the tumor cells. In contrast, expression of a constitutively active EPO-R (EPOR-R129C) in tumor cells significantly stimulated tumor angiogenesis and growth [229]. Thus, EPO may be required to maintain the integrity of the endothelium in tumors. The therapeutic implications of these effects are still to be clarified, because the increased vessel surface of tumors may improve the local delivery of anti-cancer drugs, as has been shown in 5-fluorouracil treated mice bearing human cancer xenografts [213].

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The primary goal of the therapy with rHuEPO or its analogues in cancer patients is to reduce the need for red blood cell transfusion. In addition, the correction of anemia may improve intratumoral oxygenation and, thus, the sensitivity towards chemo- and radiotherapy. Studies in tumor-bearing rats have shown that rHuEPO treatment can improve tumor oxygenation independently from the increase in Hb concentrations [230]. Thews et al. [231] have demonstrated that the cytotoxic efficacy of cyclophosphamide is greater in rats treated with carboplatin, if anemia is prevented by the administration of rHuEPO. Other authors have reported that maintenance of Hct by rHuEPO therapy leads to sensitization of the tumors to cisplatin cytotoxicity in immunodeficient mice bearing human ovarian cancer [232]. rHuEPO has been reported to suppress the growth of Lewis lung carcinoma in mice in synergy with cisplatin, though not with mitomycin C or cyclophosphamide [233]. The long-acting rHuEPO analog Darbepoetin alfa has been also proven to prevent anemia, and to increase tumor oxygenation and cisplatin delivery in the murine model of Lewis lung carcinoma [212]. Darbepoetin alfa treatment reduces tumor mass in mice under chemotherapy, but it does not modulate tumor growth directly [212]. The increase in anti-tumor efficacy of 5-flurouracil in immunodeficient mice transplanted with human A431 squamous cell or HT25 colorectal carcinomas has also been explained by an improvement in tumor perfusion and, thus, drug delivery on rHuEPO therapy [213]. In another treatment study rHuEPO alone or in combination with paclitaxel had no effect on the growth of breast carcinoma xenografts in nude mice [206]. Increased tumor sensitivity to radiotherapy on alleviation of anemia by rHuEPO treatment has been shown in nude mice engrafted with human glioblastoma [234,235]. EPO per se did not appear to affect the growth of non-irradiated tumors in this model [234], nor in a similar one in rats [236]. Recent careful investigations of rats transplanted with R3230 rat mammary adenocarcinomas and of mice transplanted with either CT-26 mouse colon carcinomas, HCT-116 human colon carcinomas or FaDu human head and neck tumors have confirmed that rHuEPO does not stimulate tumor growth and angiogenesis in vivo even when administered for several weeks at high doses (2000 U/kg thrice per week) [237]. Mittelman et al. [238] have reported that rHuEPO treatment induces complete tumor regression in 30–60% of mice with syngeneic myelomas. This regression has been related to a T cellmediated tumor-specific immune response to the myeloma cells [238]. Subsequent studies in two lymphoproliferative murine models (MOPC-315 MM and BCL1 B-cell leukemia/lymphoma) have confirmed that rHuEPO treatment reduces tumor cell growth [239]. Indeed, EPO augments B-cell responses, manifested by stimulation of immunoglobulin production and LPS-induced proliferation of splenocytes [240]. The action of EPO as an immunomodulator is not completely straightforward, however. The positive effect of EPO therapy on the survival of mice carrying a subclone of colon 26 adenocarcinoma has been related to a decrease

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in the production of the cachexia-inducing cytokine IL-6 [241]. With a view to the neuroprotective potential of EPO, Bianchi et al. [242] have recently studied the effect of EPO therapy on cisplatin induced peripheral neurotoxicity by measurements of nerve conduction velocity and the nociceptive threshold to heat. rHuEPO dose-dependently prevented neurotoxicity. Separate experiments showed that rHuEPO treatment did not alter the growth of 13,762 rat mammary carcinoma in the animals [242]. This study is of principal interest, because it indicates that rHuEPO therapy may exert advantageous effects in tumor patients on chemotherapy irrespective of its effects on erythropoiesis and tumor control. Along these lines, earlier studies have shown that rHuEPO treatment enhances the recovery from cisplatin-induced acute renal failure in rats, irrespective of its effect on red cell mass [147]. rHuEPO also exerts angioprotective renal effects in rats treated with cyclosporine A [243]. Furthermore, EPO has been proven to protect the heart against doxorubicininduced cardiomyocyte atrophy and degeneration, loss of contractile proteins and inflammatory cell infiltration in mice [244]. Finally, it has been shown that EPO inhibits apoptosis of organ cultures of human hair follicles treated with cyclophosphamide [245].

5. Clinical relevance of anemia in cancer patients and treatment options Anemia, defined as a deficiency in the concentration of Hb-containing RBC, is a widely prevalent complication among cancer patients. The National Cancer Institute and others have agreed to use the following classification for anemia based on hemoglobin (Hb) values [246]: Grade 0, within normal limits (Hb values are 12.0–16.0 g/dL for women and 14.0–18.0 g/dL for men), Grade 1, mild (10.0 g/dL ≤ Hb < 12.0 for women, 14.0 for men), Grade 2, moderate (8.0 ≤ Hb < 10.0 g/dL), Grade 3, serious/severe (6.5 ≤ Hb 8.0 g/dL), Grade 4, life threatening (Hb < 6.5 g/dL). About 32% of cancer patients present with anemia at diagnosis and about 54% of initially non-anemic cancer patients develop anemia during treatment [247,248]. The latter is caused by either the cancer itself or by cytotoxic treatment [246]. For the affected patients anemia can be a debilitating problem; it negatively influences their quality of life (QoL) [249], and it is associated with shorter overall survival times [250]. Anemia is the primary indication for RBC transfusion. Before rHuEPO was available, blood transfusion was the only treatment option for severe cancer-related anemia. Homologous blood transfusion is the fastest method to alleviate symptoms; however, short-term and long-term risks exist [251]. Potential complications associated with blood transfusion are transmission of infectious diseases, transfusion

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reactions, alloimmunization, lung injury, over-transfusion and immune modulation with possible adverse effects on tumor growth. The risks of transfusion-related transmissions are 1:180,000 per unit of blood transfused for hepatitis B virus, 1:1,600,000 for hepatitis C virus and 1:1,900,000 for HIV in the US [251]. Recombinant ESAs provide an alternative to RBC transfusions. 5.1. Recombinant human ESAs Three different originator brands of ESAs are available to date for treatment of cancer patients: Epoetin alfa (Procrit® , Eprex® , Erypo® , Johnson & Johnson/Ortho Biotech; Epogen® , Amgen), Epoetin beta (NeoRecormon® , Hoffmann-La Roche) and Darbepoetin alfa (Aranesp® , Amgen). Epoetin alfa and Epoetin beta both consist of 165 amino acids that are identical to those of endogenous human EPO but exhibit minor differences in their carbohydrate structures [252]. Darbepoetin alfa, a hyperglycosylated rHuEPO analogue produced by means of site-directed mutagenesis, has a longer half-life (24–26 h) compared to Epoetin alfa and beta (6–9 h, i.v. values). All three established ESAs have similar clinical efficacy [253,254]. Since the patents for the Epoetins that are engineered in Chinese hamster ovary (CHO) cell cultures transfected with EPO cDNA have expired in the European Union (EU), “Biosimilar” products have recently received approval by the European Medicines Evaluation Agency (EMEA). The approved indications of these rHuEPOs, which have been given either the International Non Proprietary Name (INN) Epoetin alfa (Abseamed® , Medice; Binocrit® , Sandoz; Epoetin alfa HEXAL® , HEXAL) or Epoetin zeta (Silapo® , cellpharm/Stada; Retacrit® , Hospira Enterprises), include the anemias of chronic kidney disease (CKD; approved for i.v. administration only) and of chemotherapy for solid tumors, malignant lymphoma or multiple myeloma [255]. Other novel ESAs, namely the long-living, pegylated, form of Epoetin beta (C.E.R.A., Continuous Erythropoiesis Receptor Activator, Mircera® , Roche Pharma) and Epoetin delta (Dynepo® , Shire), which is produced in human fibrosarcoma cell cultures of the line HT1080 transfected with a cytomegalo-virus (CMV) promoter, are presently used for the treatment of CKD patients (s.c. and i.v.) only in the EU. In addition to the standard regimen of 10,000 IU (total dose) or 150 IU/kg thrice weekly, Epoetin alfa and Epoetin beta can also be administered once weekly [256,257]. The recommended dose for Darbepoetin alfa is 2.25 ␮g/kg per week [258]. However, a commonly used regimen is Darbepoetin alfa in a fixed dose of 200 ␮g every two weeks [259]. 5.1.1. Effectiveness and safety of ESA therapy Multiple studies and subsequent meta-analyses [260–265] have demonstrated that ESA treatment successfully increases hemoglobin (Hb) levels and reduces the likelihood of transfusion for a proportion of treated patients.

In a meta-analysis including 42 studies with 6510 patients the relative risk to receive RBC transfusions was 0.64 [95% confidence interval (CI) 0.60, 0.68] [263]. The most serious adverse events associated with ESAs include pure red blood cell aplasia in CKD patients [266], increased mortality associated with cardiovascular and thromboembolic events, and the potential for tumor growth promotion. An increased risk for thromboembolic events has been consistently observed in various patient populations [267–269]. Opposing mechanisms on the effects of EPO on tumor growth have been proposed. Tumor tissue is often hypoxic and increasing Hb levels with ESA may increase tumor oxygenation and thus improve the effectiveness of radiation and chemotherapy and subsequently improve survival (see Section 4.3). By contrast, it has also been hypothesized that EPO might directly stimulate tumor cell growth (see Section 4.2). Either endogenously produced or exogenously administered EPO could theoretically promote the proliferation and survival of EPO-R expressing cancer cells. 5.2. Clinical trials in cancer patients receiving ESAs 5.2.1. Evidence for improved tumor control or survival in patients receiving ESAs Randomized controlled studies in cancer patients undergoing chemotherapy have supported the notion that ESAs might improve tumor control or overall survival. Littlewood et al. [270] investigated the efficacy of Epoetin alfa on RBC transfusion needs in 375 patients suffering from solid or non-myeloid hematological tumors treated with a non-platinum-based chemotherapy. Patients with an initial Hb level ≤10.5 g/dL or with a decrease in Hb of ≥1.5 g/dL per chemotherapy cycle received subcutaneous Epoetin alfa (150 IU/kg three times weekly) or placebo given for 12–24 weeks during chemotherapy and then for an additional 4 weeks. Median survival in the Epoetin alfa group was 17 months versus 11 months for patients receiving placebo, there was no statistically significant difference for survival at 12 months (p = 0.13). Disease progression was assessed as adverse events only, however, both treatment arms showed similar results (18% in the Epoetin alfa versus 22% in the control group). Since this study was not adequately designed to evaluate survival or disease progression, these results are not readily interpretable. Antonadou et al. [271] investigated tumor response in 385 participants with pelvic malignancies undergoing radiotherapy with or without rHuEPO therapy. The investigators showed a statistically significantly improved disease-free survival at 4 years for participants treated with rHuEPO (85.3%) compared with the control group (67.2%, p < 0.001). Blohmer et al. [272] have investigated the impact of Epoetin alfa in patients with high-risk carcinoma of the uterine cervix (n = 257) treated with sequential chemoradiotherapy. An interim report suggests an improvement in relapse-free survival after 229 weeks for patients receiving Epoetin alfa 10,000 IU three times a week (tiw), with Hb target 13.0 g/dL, compared to controls

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(19 events versus 31 events, p = 0.034). There was no difference in overall survival. Both studies have not been fully published to date. An early meta-analysis including the Littlewood study has supported these findings [273]. In this meta-analysis the data of 19 randomized controlled trials including 2805 patients published before May 2002 were assessed; results indicated a survival benefit for patients receiving rHuEPO (adjusted data: hazard ratio [HR] 0.81 [95% CI 0.67, 0.99]; unadjusted data: HR 0.84 [95% CI 0.69, 1.02]). However, none of the studies included was designed to detect differences in survival. 5.2.2. Evidence for decreased tumor control or survival in patients receiving ESAs These promising results were contradicted for the first time in 2003 by two large randomized controlled trials [268,274]. In these studies (Table 1), patients receiving ESAs had an increased rate of tumor progression and increased mortality compared to controls. The unexpected results of these and another three studies, which had to be closed prematurely because of increased rates of thromboembolic and cardiovascular events [275–277] prompted an Oncologic Drugs Advisory Committee hearing at the FDA in May 2004 [278] where the safety of ESAs was discussed. Since then, another three randomized controlled trials showed detrimental effects on overall survival for patients receiving ESAs [279–282] and one ongoing trial showed shortened progression free survival in patients receiving ESAs [283]. The reasons for the observed detrimental effects are not fully understood to date. Hypotheses discussed to explain these results include both methodological limitations of the studies and biological factors relating to thromboembolic complications and tumor growth stimulation by ESAs. 5.2.2.1. Clinical trials in head and neck cancer patients. In the first study (ENHANCE), published by Henke and co-workers [268], 351 patients undergoing definitive or postoperative radiotherapy for advanced head and neck cancer (T3, T4, or nodal involvement) were randomized to receive Epoetin beta or placebo given in parallel with radiotherapy. The primary endpoint was loco-regional progression-free survival. Reported Hb targets were Hb > 14 g/dL in women and >15 g/dL in men. The patients received a relatively high dosage of Epoetin beta (300 IU/kg three times a week). Survival in patients receiving Epoetin beta was significantly lower compared with patients receiving placebo (relative risk of death 1.39 [95% CI 1.05, 1.84], p = 0.02). The relative risk for loco-regional tumor progression was also higher in patients receiving Epoetin beta with a relative risk of 1.69 ([95% CI 1.16, 2.47], p = 0.007). There were more episodes of hypertension, hemorrhage, thrombosis and pulmonary embolism in patients receiving Epoetin beta compared with placebo (11% versus 5%) and more patients died of cardiac disorders in the treatment group (5.6% versus 3%). A second study [283], with a similar study design compared to the ENHANCE study [268], was presented in

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December 2006 by the Danish Head and Neck Cancer Group (DAHANCA). In this ongoing phase III, open label study (DAHANCA 10) patients with head and neck cancer (T1–T4, any N) undergoing radiotherapy were randomized to receive 150 ␮g of Darbepoetin alfa every week until completion of radiotherapy [283]. Primary endpoint was loco-regional control rates at 5 years. At baseline patients had to have Hb < 14.5 g/dL. Darbepoetin had to be stopped when Hb level reached 15.5 g/dL. The study was terminated in November 2006 after a planned interim analysis including 522 of planned 600 patients. An updated analysis presented at the 14th Conference of the European Cancer Organisation in 2007 showed a poorer outcome in 5 years local regional control (RR 1.44 [95% CI 1.06, 1.86]) for patients receiving Darbepoetin, a statistically significant difference in 5 years disease specific survival (RR 1.38 [95% CI 1.01, 1.88]) and shorter but not statistically significant overall survival (RR 1.28 [95% CI 0.98, 1.68]) [283]. Since the interim analysis did not provide evidence for a potential benefit for the patients receiving Darbepoetin the investigators decided to stop recruitment of patients into the study, however, the study is ongoing until end of 2008 when more definite results are awaited. Another three studies in patients with head and neck cancer patients did not report significant differences in terms of tumor control and overall survival [284–287]. RTOG 99-03 was a randomized controlled, open label, US cooperative trial in patients with stage I–IV non-metastasis squamous cell carcinoma of the head and neck [284,285]. Patients received treatment with radiation therapy with or without chemotherapy. The specific radiotherapy schedule was dependent on disease stage and the planned chemotherapy. Patients were randomized to receive Epoetin alfa plus iron or no Epoetin alfa and no iron supplementation. rHuEPO was given at a dose of 40,000 IU once a week for 8–9 weeks or until completion of radiotherapy. Baseline Hb levels were 9–13.5 g/dL for male and 9–12.5 g/dL for female patients. rHuEPO had to be withheld at Hb levels of 16 g/dl in men and 14 g/dL in women. The study was planned to include 372 patients. However, the study was stopped after an unplanned interim analysis performed in October 2003 after the results of the ENHANCE study [268] had been published. The interim analysis showed a non-significant trend towards poorer tumor control and survival in the rHuEPO arm and the study was terminated at that point. Results for the 148 patients included until suspension of the study showed locoregional control rates at 1 year of 63% in the group receiving rHuEPO and 70% in control patients (HR 1.18 [95% CI 0.67, 2.09]) [285]. Overall survival at one year was 70% for patients receiving rHuEPO compared to 81% in the control arm (HR 1.57 [95% CI 0.76, 3.27]) [285]. In the recently published full text manuscript a longer follow up was reported, overall survival at 3 years was 56% for patients receiving rHuEPO compared to 57% in the control arm (HR 1.17 [95% CI 0.72, 1.89]) [284]. The differences between treatments group with respect to tumor control and overall survival were not statis-

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Table 1 Clinical trials on the use of erythropoiesis stimulating agents (ESAs) in head and neck cancer, breast cancer, lung cancer and no concomitant cancer treatment patients Study (Author, year, ref. no.) Head and neck cancer Henke et al., 2003 [268] (ENHANCE) Overgaard et al., 2007 [283] (DAHANCA 10) EPO-GBR-7, 2004 [286] Machtay et al., 2007 [284,285] (RTOG 99-03) Rosen et al., 2003 [287] Breast cancer Leyland-Jones et al., 2003 and 2005 [274,267] (BEST) Moebus et al., 2007 [290]

No. of patients

Tumor (stage)

Hb at baseline (mean, g/dL)

ESA

Anti-neoplastic therapy

Doubleblind

Tumor control

Overall survival [95% confidence interval]

351

Head and neck (T3, T4, nodal involvement)

11.8

Epoetin-␤

RT

Yes

RR 1.39 [1.05–1.84]

522

Inclusion criterion: Hb < 14.5 g/dL 13.5

DARB-␣

RT + Nimorazole

No

301

Head and neck (T1–T4, nodal involvement) Head and neck (I–IV)

Epoetin-␣

RT

No

148

Head and neck (I–IV, M0)

12.1

Epoetin-␣

RT, some + Pb-CT

No

90

Head and neck (IV)

12.2

Epoetin-␣

RT and CT

No

Local regional progression RR 1.69 [1.16–2.47] Local regional control RR 1.44 [1.06–1.96] Local failure rate 25% vs. 29% Local regional control HR 1.20 [0.72–2.02] PFS, no difference, p = 0.35

939

Breast cancer (M1)

12.8

Epoetin-␣

CT

Yes

PFS HR 1.00, p = 0.98

641

Breast cancer (≥4 nodes+)

12.6

Epoetin-␣

CT

No

463 354

Breast cancer (M1) Breast cancer (I–IV)

11.4 11.3

Epoetin-␤ Epoetin-␣

CT CT

No No

5-year DFS 72% vs. 71%, p = 0.86 PFS 1.07 [0.89–1.30] NR

Survival at one year HR 1.37 [1.07-1.74] 5-year OS 81% vs. 83%, p = 0.89 OS HR 1.07 [0.87–1.33] p = 0.82

Advanced NSCLC

10.3

Epoetin-␣

Yes

NR

HR 1.84 [1.01–3.35]

Yes

PFS HR 0.80 [0.63–1.03] PFS HR 1.02 [0.86–1.21] Overall tumor response 60% and 56% Median time to progression 467 vs. 419 days

HR 0.80 [0.61–1.05]

RR 1.28 [0.98–1.68] Survival at one year 77% vs. 80% HR 1.17 [0.72–1.89] No difference, p = 0.57

Aapro et al., 2008 [291] (BRAVE) Chang et al., 2005 [288] (EPO-CAN-17) Lung cancer Wright et al., 2007 [292] (EPO-CAN-20) Vansteenkiste et al., 2002 [293]

320

SCLC, NSCLC (I–IV)

10.1

DARB-␣

None, RT, some + CT Pb-CT

Amgen 2001 0145, 2007 [296]

596

Extensive SCLC

12.0

DARB-␣

Pb-CT

Yes

Grote et al., 2005 [295] (N93-004) [294] EPO-CAN-15, 2004 [275]

224

SCLC

12.9

Epoetin-␣

Pb-CT

Yes

106

Limited SCLC

13.5

Epoetin-␣

RT + Pb-CT

Yes

349

HD, MM, NHL

9.6

DARB-␣

CT

Yes

PFS HR 1.02 [0.80-1.30]

HR 1.37 [1.02-1.83]

349 146

MM, NHL, CLL MM, NHL

9.3 9.5

Epoetin-␤ Epoetin-␤

Yes No

NR NR

HR 1.04 [0.80–1.36] Deaths 3% vs. 10%

144

MM, NHL, CLL

8.0

Epoetin-␤

CT CT, some patients none CT

Tumor progression 50% vs. 45%

Deaths 27% vs. 29%

989

9.5

DARB-␣

None

Yes

NR

HR 1.30 [1.07-1.57]

9.5

Epoetin-␣

None

Yes

NR

Deaths 20% vs. 22%

9.8

DARB-␣

None

Yes

NR

Deaths 5% vs. 0

285

Solid and hematological malignancies Solid and hematological malignancies Solid and hematological malignancies Active non-myeloid cancer

10.2

DARB-␣

None

No

NR

RR 1.4 [0.4–4.6]

220

Non-myeloid malignancies

10.2

DARB-␣

None

Yes

NR

Deaths 7% vs. 9%

100

Various solid tumors

10.1

Epoetin-␣

None

Yes

NR

Deaths 2% vs. 4%

Solid and hematological malignancies Pelvic malignancies

9.8

Epoetin-␣

CT

Yes

Hematological malignancies Hedenus et al., 2003 [281] (Amgen 2000 0161) [280] ¨ Osterborg et al., 2002 [300] Cazzola et al., 1995 [298] ¨ Osterborg et al., 1996 [299] No treatment Smith et al., 2008 [301] Amgen 2001 0103 [279] Abels, 1993 [302] Smith et al., 2003 [303] (Amgen 990111B&C) Charu et al., 2004 [304] (Amgen 2003 0219) Gordon et al., 2006 [305] (Amgen 2003 0204) Mystakidou et al., 2005 [306]

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124 86

Studies showing improved tumor control or survival 375 Littlewood et al., 2001 [270]

HR 0.93 [0.78–1.11] HR 1.17 [0.89–1.55] Deaths 40% vs. 19%

Disease progression Median OS 17 months 18% vs. 22% vs. 11 months, p = 0.13 Antonadou et al., 2001 [271] 385 NR NR RT No 4-year DFS 85.3% vs. NR 67.2%, p < 0.001 OS at 229 weeks 16 Blohmer et al., 2004 [272] 257 Cervical carcinoma 11.9 Epoetin-␣ RT-CT No RFS at 229 weeks 19 deaths vs. 23 deaths, events vs. 31 events, p = 0.20 p = 0.034 Abbreviations: CT: chemotherapy, RT: radiotherapy, NR: not reported, Pb-CT: platinum based chemotherapy, HR: hazard ratio, RR: relative risk, CI: confidence interval, SCLC: Small cell lung cancer, NSCLC: nonsmall cell lung cancer, HD: Hodgkin ’s disease, NHL: non-Hodgkin’s lymphoma, MM: multiple myeloma, CLL: chronic lymphatic leukemia, DARB: Darbepoetin, RFS: relapse-free survival, PFS: progression-free survival.

tically significant. One fatal pulmonary embolism and two fatal myocardial infarctions were reported in the Epoetin alfa arm [284]. The study EPO-GBR-7 was conducted in patients with stage I to IV head and neck cancer [286]. Patients were undergoing radiotherapy with curative intent at a dose of 60–70 Gy and were randomized in a phase III, open label study to receive Epoetin alfa or standard care. At base-

line patients had to have Hb levels <15 g/dL. rHuEPO was given at a dose of 10,000 IU three times per week (tiw) for patients with Hb < 12.5 g/dL and 4000 IU tiw for patients with Hb > 12.5 g/dL. Treatment was aiming to maintain Hb between 12.5 and 15 g/dL. There were no differences in local failure rates within radiation fields (rHuEPO 25% versus control 29%) and similar overall response rates (99% in both arms). Overall survival at one year was similar in both treat-

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ment groups (rHuEPO 77% versus control 80%). About 3% of patients in the ESA group experienced thromboeombolic events compared to 1% in the control group. In the study published by Rosen et al. [287], 90 patients with stage IV head and neck cancer undergoing treatment with paclitaxel, fluorouracil, hydroxyurea and concomitant radiation were randomized to receive 40,000 IU rHuEPO once weekly plus iron or RBC transfusions only. There were no differences in terms of progression free survival (p = 0.35) or overall survival (p = 0.57) between the treatment groups. 5.2.2.2. Clinical trials in breast cancer patients. In patients with breast cancer clinical trials also provided conflicting evidence. One of studies that prompted the first ODAC hearing in 2004 was conducted in patients with metastatic breast cancer [267,274]. This trial was a multinational, multicentre study including 939 women with metastatic breast cancer (BEST). Women were prospectively randomized to receive Epoetin alfa (40,000 IU once-weekly) or placebo. This study was terminated prematurely by an independent data monitoring committee based on the first 4 months of safety data. There was a significant survival difference between patients receiving Epoetin alfa (70%) and those in the placebo group (76%) at 12 months (HR 1.37 [95% CI 1.07, 1.74], p = 0.01). This difference was due to an increased mortality in the treatment arm in the first 4 months (41 deaths versus 16 deaths). In particular, mortality rates due to fatal thrombovascular events (1.1% versus 0.2%) and disease progression (6% versus 2.8%) were higher with Epoetin alfa than with placebo. Despite the higher number of deaths due to disease progression in patients receiving Epoetin alfa the duration of progression-free survival was similar in both study groups (HR 1.00, p = 0.98). At 19 months there was a convergence of survival curves. In the meantime several other large studies in both advanced and localized breast cancer patients have been reported. None of those studies reported statistically significantly increased risks for tumor progression or decreased overall survival in patients receiving ESAs. EPO-Can-17 was a phase III, open label study including 354 stage I–IV breast cancer patients undergoing chemotherapy who were randomized to receive 40,000 IU Epoetin alfa or supportive care for 16 weeks [288]. At baseline patients had to have Hb < 15 g/dL, the aim was to maintain Hb levels between 12 and 14 g/dL. The study demonstrated a statistically significant improvement of QoL (primary study endpoint), there was no significant difference in overall survival for patients receiving Epoetin and controls (p = 0.82) [289]. The overall response rate among 74 patients with stage IV breast cancer was similar in both treatment arms (37% versus 30%) [289]. The rate of thrombovascular events was 10.8% for patients receiving Epoetin alfa and 7.9% for patients in the supportive care group [288]. In a phase III open label trial presented by Moebus et al. [290] patients with lymph node positive breast cancer undergoing dose-intensive (every 2 weeks) treatment with

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sequential single agent courses of epirubicin, paclitaxel and cyclophosphamide (ETC) were randomized to receive Epoetin alfa or RBC transfusions. Clinical endpoints of the study included disease free survival and overall survival. Patients received 150 IU rHuEPO per kg body weight three times per week. rHuEPO had to be stopped at an Hb level of 13 g/dL. Based on 641 evaluated patients, there was no difference in 5 years disease free (72% versus 71%, p = 0.86) and overall survival (81% versus 83%, p = 0.89). The rate of thrombovascular events was 3.0% for patients receiving Epoetin alfa and 1.7% for patients in the supportive care group. In the BRAVE open label trial patients with metastatic breast cancer undergoing anthraycline and/or taxane containing chemotherapy with baseline Hb < 12.9 g/dL were randomized to receive 30,000 IU of Epoetin beta once weekly or standard care for 24 weeks [291]. Primary endpoint of the study was survival. Four hundred sixty-three patients were randomized and evaluated. After a follow-up of 18 months there was no difference in overall survival (HR 1.07 [95% CI 0.87, 1.33], p = 0.522) or progression-free survival (HR 1.07 [95% CI 0.89, 1.30], p = 0.448) between the two study groups. However, during the 24 weeks study period there were more deaths in the Epoetin beta group (21%) compared to the control group (15%) [291]. 5.2.2.3. Clinical trials in lung cancer patients. Furthermore, increased mortality but not increased tumor progression was reported in patients with non-small cell lung cancer (NSCLC) [282], multiple myeloma and lymphoma [280], and in patients with anemia of cancer and active disease not undergoing chemotherapy or radiotherapy [279]. Epo-Can-20 was a phase III, double-blind study to assess QoL in patients with advanced stage (IIIA, IIIB, IV or recurrent) NSCLC undergoing palliative chemoand/or radiotherapy with baseline Hb < 12 g/dL randomized to receive 40,000 IU Epoetin alfa per week to maintain Hb levels between 12 and 14 g/dL compared to supportive care [282,292]. The study was planned to include 300 patients but was closed after an unplanned interim analysis after 70 patients were enrolled. The interim analysis was conducted following the publication of detrimental effects of ESAs in the studies ENHANCE [268] and BEST [274]. Because the interim analysis demonstrated decreased survival in the rHuEPO group (median 63 days versus 129 days; HR 1.84 [95% CI 1.01, 3.35], p = 0.04), the study was stopped. The results of this study are in contrast to the results of a previous study including patients with NSCLC or small cell lung cancer (SCLC) [293]. This study served for the approval of Darbepoetin alfa for the treatment of chemotherapy induced anemia in cancer patients. Three hundred fourteen patients with previously untreated NSCLC or SCLC receiving platinum-containing chemotherapy for at least 12 weeks were randomized to receive Darbepoetin 2.25 ␮g/kg weekly or placebo. There was no evidence that Darbepoetin-treated patients with NSCLC or SCLC had decreased survival (HR 0.80 [95% CI 0.61, 1.05], p = 0.09) or progression free sur-

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vival (HR 0.80 [95% CI 0.63, 1.03], p = 0.09). However, the study was not designed to detect an impact on overall survival of a specified magnitude. Randomized controlled studies in patients with limited or extensive SCLC receiving etoposide/cisplatin chemotherapy and Epoetin alfa (n = 224) [294,295] or extensive stage SCLC receiving platinum-based chemotherapy and Darbepoetin alfa (n = 596) [296] did not detect decreased survival or shortened time to progression in patients receiving ESAs. 5.2.2.4. Clinical trials in patients with lymphoproliferative malignancies. In patients with lymphoproliferative malignancies increased mortality was shown in one study. The study published by Hedenus et al. [281] was a phase III, randomized, double-blind, placebo-controlled, multicentre study in anemic patients with lymphoproliferative malignancies (MM, NHL, CLL, HL, Waldenstrom macroglobulinemia) receiving chemotherapy. Patients with Hb < 11 g/dL at baseline were randomized to receive placebo or Darbepoetin alfa at a dose of 2.25 ␮g/kg weekly for 12 weeks. Darbepoetin alfa was titrated to maintain Hb values >14 g/dL in women and >15 g/dL in men. Patients receiving ESA experienced shorter overall survival compared to controls (HR 1.37 [95% CI 1.02, 1.83], p = 0.037). The adverse effect on survival was not observed in an earlier publication [281] but became apparent with updated data and longer follow up [280,297]. There was no significant difference in progression free survival between the two arms (HR 1.02 [95% CI 0.80, 1.30]). The incidence of thrombovascular events was higher (3.4% versus 0.6%) for patients receiving Darbepoetin [280]. In contrast, three previous randomized controlled studies in patients with lymphoma and multiple myeloma including n = 146 [298], n = 144 [299] and n = 349 patients [300] did not report increased mortality in patients receiving Epoetin beta [298-300]. 5.2.2.5. Clinical trials in patients not receiving anti-cancer treatment. One recent study reported increased mortality in patients receiving ESAs (Amgen 20010103) in cancer patients with active disease not undergoing chemo- or radiotherapy [279,301]. Study 20010103 was a phase III, randomized, double-blind, placebo-controlled multicentre study of Darbepoetin alfa in patients with anemia of cancer who were not receiving chemotherapy or myelosuppressive radiotherapy. Patients had to have baseline Hb < 11 g/dL and were randomized to receive Darbepoetin 6.75 ␮g/kg once every 4 weeks for 16 weeks or RBC transfusions. Hb had to be maintained between 12 and 13 g/dL. Nine hundred eighty-nine of planned 1000 patients have been included to date; this is the second largest study on ESAs in cancer patients. Overall survival was statistically significantly decreased in patients receiving Darbepoetin compared to controls (HR 1.30 [95% CI 1.07, 1.57], p = 0.008). At the same time, there were no significant reductions in RBC transfusion requirements (HR 0.85 [95% CI 0.62, 1.17]). Arterial and venous embolism and

thrombombolism was increased in patients receiving Darbepoetin compared to controls (3.1% versus 1.3%). ESAs are licensed for the treatment of chemotherapy associated anemia in cancer patients but not for patients with anemia of cancer not undergoing chemotherapy. However, there are several randomized controlled clinical trials in patients with anemia of cancer not receiving chemotherapy or radiotherapy [292,302–306]. Apart from the study by Wright et al. ([292]; see above), none of these studies reported statistically significantly increased mortality in patients receiving ESAs. In summary, three studies showed detrimental effects on tumor progression or more deaths due to tumor progression [267,268,274,283] and five studies showed decreased survival in patients receiving ESAs [267,268,274,279–282,292]. It remains unclear whether the increased rates of deaths due to tumor progression in both the BEST [267,274] and the ENHANCE [268] study can be explained by tumor growth stimulation, since this observation has not been seen consistently across trials as shown above. Some causes of death still remain unclear despite intensive re-evaluation of the BEST study through an independent data monitoring team. The DAHANCA study is ongoing until the end of 2008, and no mature data are available to date. A direct relationship between the presence of EPO-R on tumor cells and tumor proliferation in response to exogenous ESAs has not been established. Overall, the evidence from clinical trials is insufficient to draw firm conclusions whether ESAs can promote tumor proliferation or not. 5.3. Thromboembolic events in cancer patients receiving ESAs It is of major importance to consider whether thromboembolic complications can be triggered by higher Hb levels in patients receiving ESAs. As described above, in the Henke et al. study [268] more patients receiving rHuEPO experienced thrombovascular events and died due to cardiac disorders. However, very high Hb levels were achieved at the end of that study (male participants, 15.4 g/dL, S.D. 1.7), which might have substantially contributed to the high number of thrombovascular events and cardiac deaths. In studies evaluating the effects of ESAs on Hct in patients with endstage renal failure who had pronounced cardiovascular risk factors, patients with high Hct had an increased mortality due to thrombovascular events [307,308]. Thus, it was concluded that studies targeting Hb levels above 12 g/dL, thereby aiming beyond the mere correction of anemia as suggested in the ASCO/ASH guidelines [309], were associated with a higher risk of thrombovascular events. ESAs might also have a thrombogenic potential independent of Hb levels. A retrospective case–control study in 147 consecutive cervical carcinoma patients undergoing concurrent chemotherapy, radiation and rHuEPO therapy revealed a statistically significant association between ESA treatment and thromboembolic complications (odds ratio compared with

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women not receiving ESA (10.3 [95% CI 2.3, 46.2]) [310]. No association was found between the mean or maximum Hb level and risk of thromboembolic complications, suggesting that ESAs might have a genuinely thrombogenic potential. This notion is supported by a study that demonstrated augmented platelet reactivity and endothelial activation in rHuEPO treated healthy volunteers [311]. Based on these findings it has been proposed that an increase in circulating E-selectin may enhance the risk of thromboembolism on rHuEPO therapy, particularly in patients with atherosclerosis [311]. 5.3.1. Methodological considerations Another major limitation in the interpretation of the studies is methodological insufficiencies of the clinical trials conducted to far. One potential problem are baseline imbalances reported in both the BEST [267,274] and the ENHANCE [268] study which might explain the detrimental effects observed. Other limitations include suboptimal cancer treatment. In the study published by Henke et al. [268], patients with head and neck cancer stage III and IV underwent surgery and received radiotherapy, as this was the standard treatment at the time of protocol development for this study. Treatment for this patient group, however, has evolved over time and would nowadays include chemotherapy as well. After a rigorous revision of several studies for the ODAC hearing in 2007 the FDA reviewers concluded that most of the studies were not optimally designed to assess tumor response or progression [297]. The limitations in the study designs pointed out by the FDA reviewers included lack of prospective documentation of prognostic factors [267,274], study populations including heterogeneous patient populations with mixed tumor types, disease status, and prognostic factors receiving various chemotherapy regimens [267,279,281], a lack of uniform and rigorous radiographic staging at baseline [283,286,288] and during follow up in order to assess tumor response or progression [280,282,285,286,288,290,292]. Often, tumor biopsies were not done to diagnose recurrence [283,286]. Most studies did not monitor adequately for possible thrombotic/cardiovascular events [267,269,274,276,277,279,286,290]. Many of the studies did not follow approved ESA regimens. Studies were using either dose schedules or Hb targets that were outside the license indication [279,281,286,288,290,292] or included patient populations that were not consistent with the approved indication, i.e. patients receiving radiotherapy [268] or no anti-cancer treatment [279]. In addition, many of the studies discussed above have only been published as abstracts [271,272,277,290] or parts of ODAC hearing briefing materials [286] to date. These limitations hamper the interpretation of the studies discussed. 5.4. Implications for practice After the first hearing in May 2004 the FDA concluded that the target Hb concentration should not exceed 12 g/dL

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[278]. Package inserts in the USA were amended to include this recommendation. In March 2007 a black box warning was added to the package inserts in the USA. This warning was updated in November 2007 to recommend that (1) ESAs should be used at the lowest dose that will gradually increase the Hb concentration to the lowest level sufficient to avoid the need for RBC transfusions, (2) ESAs should not be used in patients with active malignant disease not receiving chemotherapy or radiotherapy, (3) ESAs should be discontinued following the completion of a chemotherapy course, (4) the target Hb should be 12 g/dL and not higher and (5) the risks of shortened survival and tumor progression have not been excluded when ESAs are dosed to target Hb < 12 g/dL [256,258].

Reviewers The reviewers of this manuscript are: Professor Peter Vaupel, Director, Johannes Gutenberg University, Institute of Physiology and Pathophysiology, Duesbergweg 6, D-55128 Mainz, Germany. Dr. Kodetthoor B. Udupa, Ph.D, University of Arkansas for Medical Sciences and Central Arkansas Veterans Healthcare System Reynolds Institute on Aging, Dept of Geriatrics, Physiology and Biophysics, 4300 West Seventh Street Little Rock, AR 72205, United States. Professor Stephen T. Sawyer, Professor of Pharmacology and Toxicology, School of Medicine, Virginia Commonwealth University, Molecular Biology and Genetics, P.O. Box 980678, Richmond, VA 23298-0678, United States.

Acknowledgement Thanks are due to Ms. Lisa Zieske for the expert secretarial support in the preparation of the manuscript.

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Biographies Wolfgang Jelkmann is the Director of the Institute of Physiology, University of L¨ubeck. He received his medical degree from the Medical School of Hannover in 1974. He undertook his post-graduate training in physiology and pharmacology at the University of Regensburg, the Tulane University, New Orleans, and the Medical School of L¨ubeck. In 1990, he became a Full Professor of Physiology at the University of Bonn before he moved to L¨ubeck in 1995. His research interests include the production and action of inflammatory cytokines and hemopoietic growth factors with emphasis on erythropoietin, thrombopoietin, and vascular endothelial growth factor, oxygen supply to tissues, high altitude physiology and renal physiology. He has authored over 140 original publications and 90 review articles and book chapters. Julia Bohlius is oncologist at the Department of Internal Medicine I at the University Hospital in K¨oln. Michael Hallek is Professor of Medicine and the Director of the Department of Internal Medicine I at the University Hospital in K¨oln. Arthur Sytkowski is Associate Professor of Medicine at Harvard University and Director of the Laboratory for Cell and Molecular Biology, Division of Hematology and Oncology at Beth Israel Deaconess Medical Center in Boston.