Chris P. Miller, PhD, and Nicholas Burwick, MD
Dr. Miller is Acting Instructor, Division of Hematology, University of Washington School of Medicine
Dr. Burwick is Hematology/Oncology Fellow, University of Washington, Fred Hutchinson Cancer Research Center
The discovery of the adverse effects of erythropoiesis-stimulating agents (ESAs) on mortality and/or tumor progression in eight clinical trials and recent meta-analyses has led to substantial revisions in the guidelines for ESA therapy in chemotherapy-induced anemia.1 Furthermore, the United States Food and Drug Administration has installed a Risk Evaluation and Mitigation Strategy to ensure the safe use of ESAs. This program mandates that prescribers of ESAs must participate in the “Assisting Providers and Cancer Patients with Risk Information for the Safe use of ESAs” (APPRISE) program, provide patients with a medication guide discussing the benefits and risks of ESAs, and obtain written informed consent upon each new course of treatment. Yet beyond venous thromboembolism, which is a well-recognized risk of ESA therapy, large gaps persist in our understanding of the mechanisms by which ESAs can promote deleterious effects in cancer patients. At issue is whether expression levels of erythropoietin receptor (EpoR) on the surface of tumor cells or tumor blood vessels are sufficient to transduce signals that impart significant biologic effects in response to Epo stimulation.
Using a novel, specific, and sensitive monoclonal anti-EpoR antibody and a semi-quantitative western immunoblot assay, scientists at Amgen recently reported that total EpoR expression in a panel of 66 human tumor cell lines ranged from undetectable (28 lines) to as high as 1,600 to 3,200 EpoR dimers per cell (four lines), corresponding to 8.3 percent to 20 percent of the EpoR level in erythroid progenitor cells.2 Using an alternative specific monoclonal anti-EpoR antibody, our group similarly found that cell surface EpoR levels in a panel of 29 human tumor cell lines ranged from 1.2 percent to 25.2 percent (mean 8.3%) of the EpoR level in Epo-dependent UT7EPO cells.3 Thus, EpoR is clearly present at significant levels in at least certain cancer cell lines. However, disparities exist among reports from different laboratories regarding the ability of Epo to stimulate signal transduction and impart cellular responses,4 in some cases even among the same cell lines,2,5,6,7 and a clear threshold level of expression at which EpoR is functional has not been defined. Moreover, it has been difficult to establish animal models that recapitulate the clinically observed adverse effects of exogenous ESAs.4,8
The discrepancies and lack of consensus from pre-clinical studies in cell lines and animal models highlights the notion that definitive confirmation or denial of a significant role for tumor-expressed EpoR must come from clinically focused studies using primary tumor samples. Correlating tumor EpoR levels with tumor progression and survival outcomes in completed and ongoing clinical trials of ESAs offers a direct means of testing the EpoR-tumor hypothesis. For hematopoietic cancers, surface EpoR levels can be measured specifically by flow cytometry. For example, we recently discovered that EpoR levels on the surface of abnormal bone marrow plasma cells vary considerably among patients with multiple myeloma, whereas the corresponding lymphocytes from these patients were uniformly negative.9
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Measurements of EpoR levels in solid tumors are confounded by the lack of anti-EpoR antibodies that are sufficiently sensitive and specific for definitively detecting EpoR by immunohistochemistry.10,11 Even the most sensitive antibody to date (Amgen A82) was reported to exhibit some non-specific staining in negative control cells in this application.12EpoR mRNA represents a surrogate but must be tempered against technical challenges relating to the extensive degradation that characterizes RNA extracted from formalin-fixed archival tumors, concerns as to whether EpoR mRNA uniformly correlates with surface protein levels, and the potentially confounding effects of cell-type heterogeneity in tumor samples. Nevertheless, we recently optimized a sensitive and specific quantitative RT-PCR assay for EpoR mRNA in archival tumors and found that EpoR mRNA levels vary as much as 30-fold among breast cancer and head and neck cancer specimens.3 Moreover, laser capture microdissection studies in a panel of breast cancers revealed that variation of EpoR mRNA levels was greater among inter-tumor specimens than intra-tumor specimens (i.e., between tumor epithelial and endothelial cell fractions) (CPM, unpublished observation). Thus, despite the known heterogeneity in tumor vasculature, solid tumors can be segregated by their overall EpoR levels.
Ultimately, future investigation in this area must determine whether there is clinical utility in measuring EpoR protein and/or EpoR mRNA levels in tumor specimens prior to initiation of ESA therapy and whether the Epo/EpoR axis represents a target for cancer therapy.13 Well-designed clinical studies that incorporate corollary analyses of EpoR levels along with clinical outcomes data will be helpful to further our understanding of this important issue.
Acknowledgments The authors would like to thank Drs. C. Anthony Blau and Pam Becker for insightful discussions. Dr. Miller is supported by a mentored research scholar grant from the American Cancer Society [117682-MRSG-09-268-01-CCE]. Dr. Burwick is supported by a T32 training grant in hematology from the National Institutes of Health.
- NCCN Clinical Practice Guidelines in Oncology. NCCN guidelines: cancer- and chemotherapy- induced anemia. Version 2.2011. www.nccn.org/professionals/physician_gls/ PDF/anemia.pdf
- Swift S, Ellison AR, Kassner P, et al. Absence of functional EpoR expression in human tumor cell lines. Blood. 2010;115:4254-4263.
- Miller CP, Lowe KA, Valliant-Saunders K, et al. Evaluating erythropoietin-associated tumor progression using archival tissues from a phase III clinical trial. Stem Cells. 2009;27:2353-2361.
- Arcasoy MO. Erythropoiesis-stimulating agent use in cancer: preclinical and clinical perspectives. Clin Cancer Res. 2008;14:4685-4690.
- Dunlop EA, Percy MJ, Boland MP, et al. Induction of signalling in non-erythroid cells by pharmacological levels of erythropoietin. Neurodegener Dis. 2006;3:94-100.
- U m M, Gross AW, Lodish HF. A “classical” homodimeric erythropoietin receptor is essential for the antiapoptotic effects of erythropoietin on differentiated neuroblastoma SH-SY5Y and pheochromocytoma PC-12 cells. Cell Signal. 2007;19:634-645.
- Shi Z, Hodges VM, Dunlop EA, et al. Erythropoietin-induced activation of the JAK2/ STAT5, PI3K/Akt, and Ras/ERK pathways promotes malignant cell behavior in a modified breast cancer cell line. Mol Cancer Res. 2010;8:615-626.
- Miller CP, Valliant-Saunders K, Blau CA. Limitations of a murine transgenic breast cancer model for studies of erythropoietin-induced tumor progression. Transl Oncol. 2010;3:176-180.
- Becker PS, Miller CP, Wood BL, et al. Expression of erythropoietin receptors by plasma cells from patients with multiple myeloma: potential relevance to pharmacological use of erythropoietin. J Clin Oncol. (ASCO Meeting Abstract). 2010;28:8124.
- Elliott S, Busse L, Bass MB, et al. Anti-Epo receptor antibodies do not predict Epo receptor expression. Blood. 2006;107:1892-1895.
- Brown WM, Maxwell P, Graham AN, et al. Erythropoietin receptor expression in non-small cell lung carcinoma: a question of antibody specificity. Stem Cells. 2007;25:718-722.
- Elliott S, Busse L, McCaffery I, et al. Identification of a sensitive anti-erythropoietin receptor monoclonal antibody allows detection of low levels of EpoR in cells. J Immunol Methods. 2010; 352:126-139.
- Miller CP, Blau CA. Using gene transfer to circumvent off-target effects. Gene Ther. 2008;15:759-764.
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