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Journal of Clinical Oncology, Vol 24, No 29 (October 10), 2006: pp. 4708-4713 © 2006 American Society of Clinical Oncology. DOI: 10.1200/JCO.2006.06.2737 Do Erythropoietin Receptors on Cancer Cells Explain Unexpected Clinical Findings?
From the Klinik für Strahlenheilkunde Universitätsklinikum; Pathologisches Institut, Universität Freiburg, Freiburg, Germany; Fred Hutchinson Cancer Research Center, University of Washington, Seattle, WA; Department of Radiation Oncology, David Geffen School of Medicine at UCLA, Los Angeles, CA Address reprint requests to Michael Henke, MD, Klinik für Strahlenheilkunde, Universitätsklinikum Robert Koch Strasse, 3 D-79106, Freiburg, Germany; e-mail: henke{at}uni-freiburg.de
PURPOSE: Recent reports suggest that cancer control may worsen if erythropoietin is administered. We investigated whether erythropoietin receptor expression on cancer cells may correlate with this unexpected finding. PATIENTS AND METHODS: Cancer tissue from patients with advanced carcinoma of the head and neck (T3, T4, or nodal involvement) and scheduled for radiotherapy was assayed retrospectively for erythropoietin receptor expression by immunohistochemistry. Patients were anemic and randomized to receive epoetin beta (300 U/kg) or placebo under double-blind conditions, given three times weekly starting 10 to 14 days before and continuing throughout radiotherapy. We administered 60 Gy following complete resection or 64 Gy subsequent to microscopically incomplete resection; 70 Gy were given following macroscopically incomplete resection or for definitive radiotherapy alone. We determined if the effect of epoetin beta on locoregional progression-free survival was correlated with the expression of erythropoietin receptors on cancer cells using a Cox proportional hazards regression model. RESULTS: We studied 154 of 157 randomly assigned patients; 104 samples were positive, and 50 were negative for receptor expression. Locoregional progression-free survival was substantially poorer if epoetin beta was administered to patients positive for receptor expression compared with placebo (adjusted relative risk, 2.07; 95% CI, 1.27 to 3.36; P < .01). In contrast, epoetin beta did not impair outcome in receptor-negative patients (adjusted relative risk, 0.94; 95% CI, 0.47 to 1.90; P = .86). The difference in treatment associated relative risks (2.07 v 0.94) was borderline statistically significant (P = .08). CONCLUSION: Erythropoietin might adversely affect prognosis of head and neck cancer patients if cancer cells express erythropoietin receptors.
Anemia has been consistently associated with poor outcome of cancer patients.1 Because of its potential to correct anemia and associated symptoms erythropoietin has been increasingly prescribed to these patients. However, two large multicenter trials recently reported decreased survival and disease control following erythropoietin treatment in patients with cancer of the breast2 and of the head and neck.3 Methodological issues may have played a role, but a "protective effect" of erythropoietin on cancer cells has also been discussed. In a subset of 154 patients from one clinical center of the head and neck trial,3 we explored whether the adverse effect of epoetin beta was restricted to patients with erythropoietin receptor (EpoR) expression on cancer.
Patients Head and neck cancer patients enrolled in a previously reported clinical trial3 treated at the Department of Radiooncology at the University Clinic, Freiburg, Germany, were included in this study. The trial was approved by the local ethics committee and performed in accordance with the revised Declaration of Helsinki and good clinical practice guidelines. Patient selection criteria, treatment parameters, follow-up schedule, and evaluation methods are described in detail in a former publication.3 Details of this subpopulation are specified elsewhere.4 Briefly, the study was double-blind, randomized and placebo-controlled. Main inclusion criteria were a decreased blood hemoglobin concentration at random assignment (< 13 g/dL in men; < 12 g/dL in women), T3 or T4 tumors or nodal involvement, and definitive radiation therapy alone or postoperative radiotherapy.
For the purpose of random assignment, patients were stratified according to resection status: stratum 1, postoperative radiation following complete (R0) resection; stratum 2, postoperative radiation of incompletely resected (R1 or R2) disease; and stratum 3, primary definitive radiotherapy. We administered 60 Gy following complete resection (R0), 64 Gy (allowable range for both, 56 to 64 Gy) to R1-resected tumors and 70 Gy (allowable range, 66 to 74 Gy) to macroscopically incompletely (R2) resected disease or for definitive treatment, respectively. Epoetin beta (300 U/kg) or placebo was administered three times per week and started 10 to 14 days before radiotherapy. Drug was continued throughout the radiotherapy course. Additionally, 200 mg iron (III) saccharate (Ferrum Hausmann, Vifor, St Gallen, Switzerland) was applied intravenously once weekly if the transferrin saturation was lower than 25%. Treatment was discontinued when target hemoglobin levels (
Immunohistochemistry Two independent reviewers unaware of all clinical data performed tissue processing and scoring of the slides. Differences between the two investigators were resolved by consensus. Applying a four-grade scale we evaluated semiquantitatively the expression intensity and proportion of positive-staining cells on the entire tissue section. Cytoplasmatic or membrane staining was considered positive. Missing staining of cancer cell was considered as score 0. Score 1 showed barely, score 2, moderate, and score 3, strong cellular staining. Any positive reaction required at least 10% of cancer cells to stain. For further analyses scores 0 and 1 were regarded as negative and scores 2 and 3 as positive (Fig 1).
Statistical Methods Locoregional progression-free survival was analyzed as primary end point. Progression was assumed if the tumor recurred or if its size increased by more than 25%. Standard censored failure time data techniques were used.5 Kaplan-Meier curves6 were calculated with event times being the time until locoregional progression or death, whichever occurred first. Subjects who neither died nor had progressing cancer within the irradiated volume were censored at time of their last follow-up. Comparisons between survival curves for epoetin beta treatment versus placebo were based on the stratified log-rank test with stratification for resection status. Additional stratification for American Joint Committee on Cancer stage (not shown here) produced similar results. Comparisons were done separately for patients, with cancer staining positive for EpoR and for patients whose cancers did not. Relative risks were calculated using the Cox proportional hazards regression model.7 Treatment was entered as a binary variable (1 for epoetin beta and 0 for placebo). A simple model including only treatment arm and stratified by resection status provided adjusted relative risks that correspond to the Kaplan-Meier analyses. This was done separately for receptor-positive and -negative patients. A model that included all patients in a single analysis evaluated treatment arm and receptor status together while including resection status as a three-level variable. A model with interaction term between receptor status and treatment arm allowed different treatment relative risks for receptor-positive versus -negative patients. Its log partial likelihood was compared with that of a model without interaction in order to test whether the treatment relative risks differed significantly with receptor status. The log partial likelihood ratio test was used.
Between March 1997 and April 2001 we randomly assigned 157 patients. Tissue samples were available for 154 patients. Samples stained positive for erythropoietin receptors in 104 patients and negative in 50 patients. Table 1 presents characteristics of patients by treatment assignment within the two groups of patients—receptor positive and receptor negative. For the most part, characteristics of patients assigned to treatment with epoetin beta were similar to those assigned placebo. There was some imbalance in regards to resection stratum. For receptor-positive patients, there were more high-risk patients (radiation treatment without surgery) on the epoetin beta arm, while for receptor negative patients, more high-risk patients were on the placebo arm. However, since our analysis stratified on resection status, this did not confound our results.
Consistent with the analysis of the entire trial, in the subset of patients enrolled from our center, treatment with epoetin beta was associated with decreased locoregional progression-free survival (adjusted relative risk, 1.58; P = .02). Figure 2 shows this effect for patients with receptor positive and negative cancer. The negative impact of epoetin beta appears to be restricted to patients whose cancers expressed EpoR. The resection status adjusted relative risks for loco-regional failure or death were 2.07 (P < .01) for receptor positive patients and 0.94 (P = .86) for receptor negative patients (treatment group v placebo). Note that any imbalances between treatment and placebo in resection status do not contribute to these relative risks as they have been adjusted for through stratification.
Table 2 presents the results of a Cox regression analysis that includes all patients and models the effects of resection status and receptor status as constants over time. Resection status is clearly associated with locoregional progression-free survival. The analysis confirms that after adjustment for the resection status, epoetin beta treatment is associated with increased risk of loco-regional progression or death for patients with receptor-positive cancers (relative risk, 1.4/0.7 = 2.0; P = .003) but not in those with receptor-negative cancers (relative risk, 0.98; P = .95). These relative risks are almost statistically significant (P = .08) based on the Cox partial likelihood ratio test.
Recently, a negative impact of erythropoietin on cancer control and survival was reported from two large multicenter trials.2,3 These observations have been questioned and concerns were raised about methodologic problems that may have contributed to the findings, but little attention has been given to possible biologic explanations.
Cancer cells are known to express EpoR,8-12 the expression of which seem to correlate to cellular dedifferentiation.8 Furthermore, erythropoietin may bind to EpoR in these cells and activate STAT5 and NF- It is of note that patients treated with placebo experienced different outcome depending on whether their cancer cells expressed EpoR or not. However, this was not statistically significant (P = .17) and may indicate possible effects of endogenous erythropoietin levels that show substantial variations.10 Further, though the small sample size may limit our conclusions, essential methodological pitfalls do not confound our observations: Clinical baseline characteristics were reasonably balanced, immunohistochemical processing was verified by adequate controls and the evaluation was unbiased because two independent researchers, blinded for all clinical parameters, scored the sections. It should be mentioned that, although we applied a simplified scoring system, our results could be confirmed when applying published scoring protocols23 (data not shown). The specificity of the antibody used in this study was recently questioned.24 In ours and other9,11,12,25,26 studies, it reliably detects cells known to express erythropoietin receptors in paraffin embedded tissue sections and Western blots identified the appropriate protein.8 Aside, however, from potential doubts on the molecular specificity of this antibody a correlation of immunohistochemical findings to clinical outcome suggests that biologic phenomena may explain the recently reported "negative erythropoietin trials." We conclude that EpoR is variably expressed on head and neck cancer cells and is associated with a detrimental effect of epoetin beta administration. A possible mechanism is that stimulation with recombinant erythropoietin protects residual tumor from radiation treatment. It might reassure that epoetin beta did not affect the clinical course of patients with EpoR-negative cancer. Further research should confirm our findings and elucidate the underlying mechanisms. All researchers contributed to design and implementation of the study and to data collection. D. Mattern and C. Bézay performed histopathological analyses. M. Pepe analyzed the data and M. Henke drafted the report. All researchers took part in the critical revision of the manuscript and approved its final version.
Although all authors completed the disclosure declaration, the following authors or their immediate family members indicated a financial interest. No conflict exists for drugs or devices used in a study if they are not being evaluated as part of the investigation. For a detailed description of the disclosure categories, or for more information about ASCO's conflict of interest policy, please refer to the Author Disclosure Declaration and the Disclosures of Potential Conflicts of Interest section in Information for Contributors.
Dollar Amount Codes (A) < $10,000 (B) $10,000-$99,999 (C)
Special thanks are due to Sabine Ahlers, Dagmar Eckert, and Ilse Liebhardt who, with particular dedication, gathered, documented, and verified the clinical data.
Terms in blue are defined in the glossary, found at the end of this article and online at www.jco.org. Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.
1. Grau C, Overgaard J: Significance of Hemoglobin Concentration for Treatment Outcome. Berlin, Germany, Springer, 2000 2. Leyland-Jones B, Semiglazov V, Pawlicki M, et al: Maintaining normal hemoglobin levels with epoetin alfa in mainly nonanemic patients with metastatic breast cancer receiving first-line chemotherapy: A survival study. J Clin Oncol 23:5960-5972, 2005 3. Henke M, Laszig R, Rube C, et al: Erythropoietin to treat head and neck cancer patients with anaemia undergoing radiotherapy: Randomised, double-blind, placebo-controlled trial. Lancet 362:1255-1260, 2003[CrossRef][Medline] 4. Schipper J, Henke M: [Erythropoietin in patients with head and neck carcinomas?]. Laryngorhinootologie 83:292-297, 2004[CrossRef][Medline] 5. Collet D: Modeling Survival Data in Medical Research. London, UK, Chapman Hall, CRC Press, 1994 6. Kaplan EL, Meier P: Nonparametric estimation from incomplete observation. J Am Stat Assoc 54:457-481, 1958[CrossRef] 7. Cox DR: Regression models and life table. J Royal Stat Soc B 34:187-220, 1972 8. Acs G, Acs P, Beckwith SM, et al: Erythropoietin and erythropoietin receptor expression in human cancer. Cancer Res 61:3561-3565, 2001 9. Arcasoy MO, Amin K, Chou SC, et al: Erythropoietin and erythropoietin receptor expression in head and neck cancer: Relationship to tumor hypoxia. Clin Cancer Res 11:20-27, 2005 10. Dagnon K, Pacary E, Commo F, et al: Expression of erythropoietin and erythropoietin receptor in non-small cell lung carcinomas. Clin Cancer Res 11:993-999, 2005 11. Hoogsteen IJ, Peeters WJ, Marres HA, et al: Erythropoietin receptor is not a surrogate marker for tumor hypoxia and does not correlate with survival in head and neck squamous cell carcinomas. Radiother Oncol 76:213-218, 2005[CrossRef][Medline] 12. Winter SC, Corbridge RJ, Cox GJ, et al: Hypoxia and anaemia in head and neck squamous cell carcinoma: Mechanisms of therapy failure and provision of new therapeutic targets. Clin Otolaryngol 30:99-104, 2005[CrossRef][Medline] 13. Feldman L, Wang Y, Rhim JS, et al: Erythropoietin stimulates growth and STAT5 phosphorylation in human prostate epithelial and prostate cancer cells. Prostate 66:135-145, 2006[CrossRef][Medline] 14. Lai SY, Childs EE, Xi S, et al: Erythropoietin-mediated activation of JAK-STAT signaling contributes to cellular invasion in head and neck squamous cell carcinoma. Oncogene 24:4442-4449, 2005[CrossRef][Medline] 15. Um M, Lodish HF: Antiapoptotic effects of erythropoietin in differentiated neuroblastoma SH-SY5Y cells require activation of both the STAT5 and AKT signaling pathways. J Biol Chem 281:5648-5656, 2006 16. Bittorf T, Buchse T, Sasse T, et al: Activation of the transcription factor NF-kappaB by the erythropoietin receptor: Structural requirements and biological significance. Cell Signal 13:673-681, 2001[CrossRef][Medline] 17. Siren AL, Fratelli M, Brines M, et al: Erythropoietin prevents neuronal apoptosis after cerebral ischemia and metabolic stress. Proc Natl Acad Sci U S A 98:4044-4049, 2001 18. Brines ML, Ghezzi P, Keenan S, et al: Erythropoietin crosses the blood-brain barrier to protect against experimental brain injury. Proc Natl Acad Sci U S A 97:10526-10531, 2000 19. Belenkov AI, Shenouda G, Rizhevskaya E, et al: Erythropoietin induces cancer cell resistance to ionizing radiation and to cisplatin. Mol Cancer Ther 3:1525-1532, 2004 20. Yasuda Y, Fujita Y, Masuda S, et al: Erythropoietin is involved in growth and angiogenesis in malignant tumours of female reproductive organs. Carcinogenesis 23:1797-1805, 2002 21. Lester RD, Jo M, Campana WM, et al: Erythropoietin promotes MCF-7 breast cancer cell migration by an ERK/mitogen-activated protein kinase-dependent pathway and is primarily responsible for the increase in migration observed in hypoxia. J Biol Chem 280:39273-39277, 2005 22. Pajonk F, Weil A, Sommer A, et al: The erythropoietin-receptor pathway modulates survival of cancer cells. Oncogene 23:8987-8991, 2004[CrossRef][Medline] 23. Acs G, Zhang PJ, Rebbeck TR, et al: Immunohistochemical expression of erythropoietin and erythropoietin receptor in breast carcinoma. Cancer 95:969-981, 2002[CrossRef][Medline] 24. Elliott S, Busse L, Bass MB, et al: Anti-Epo receptor antibodies do not predict Epo receptor expression. Blood 107:1892-1895, 2006 25. Acs G, Zhang PJ, McGrath CM, et al: Hypoxia-inducible erythropoietin signaling in squamous dysplasia and squamous cell carcinoma of the uterine cervix and its potential role in cervical carcinogenesis and tumor progression. Am J Pathol 162:1789-1806, 2003 26. Ribatti D, Marzullo A, Nico B, et al: Erythropoietin as an angiogenic factor in gastric carcinoma. Histopathology 42:246-250, 2003[CrossRef][Medline] Submitted February 21, 2006; accepted June 30, 2006. Related Editorial
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ABSTRACT
INTRODUCTION
14 g/dL in women; 
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