Immunohistochemical expression of erythropoietin and erythropoietin receptor in breast carcinoma

Immunohistochemical Expression of Erythropoietinand Erythropoietin Receptor in Breast Carcinoma

Geza Acs, M.D., Ph.D.1

Paul J. Zhang, M.D.1

Timothy R. Rebbeck, Ph.D.2

Peter Acs, M.D., Ph.D.3

Ajay Verma, M.D., Ph.D.4

1 Department of Pathology and Laboratory Medi-cine, University of Pennsylvania Medical Center,Philadelphia, Pennsylvania.

2 Center for Clinical Epidemiology and Biostatisticsand Cancer Center, University of Pennsylvania,Philadelphia, Pennsylvania.

3 Department of Medicine, Huron Hospital, Cleve-land Clinic Health System, Cleveland, Ohio.

4 Department of Neurology, Uniformed ServicesUniversity of the Health Sciences, Bethesda, Mary-land.

Supported in part by a McCabe Award to Geza Acsand National Institutes of Health grant 37814 toAjay Verma

Address for reprints: Geza Acs, M.D., Ph.D., De-partment of Pathology and Laboratory Medicine,University of Pennsylvania Medical Center, 6Founders Pavilion, 3400 Spruce Street, Philadel-phia, PA 19104; Fax: (215) 349-5910; E-mail:[email protected]

Received January 25, 2002; revision received April4, 2002; accepted April 8, 2002.

BACKGROUND. Erythropoietin (Epo), induced by hypoxia, controls the survival,

proliferation, and differentiation of Epo receptor (EpoR)-bearing erythroid progen-

itors and plays a role in the protection of neurons from hypoxic damage. Hypoxia

in malignant disease is associated with invasion, metastasis, resistance to therapy,

and selection for cells with diminished apoptotic potential. The authors recently

demonstrated the basal and hypoxia-stimulated expression of Epo and EpoR in

human breast carcinoma cell lines and in breast carcinomas, suggesting a role for

autocrine Epo signaling in the hypoxic adaptations of mammary neoplasms.

METHODS. The authors characterized the expression of Epo and EpoR by immu-

nohistochemistry in 184 invasive mammary carcinomas and 158 in situ mammary

carcinomas and benign mammary epithelium. They analyzed the correlation of

Epo and EpoR immunostaining with clinicopathologic tumor features and the

patients’ smoking history.

RESULTS. Benign mammary epithelial cells showed weak-to-moderate expression

of Epo and EpoR. EpoR immunostaining was increased in carcinomas compared

with benign epithelium both in nonsmokers and smokers, and Epo immunostain-

ing was increased in carcinomas compared with benign epithelium in nonsmokers

but not in smokers. Prominent Epo staining was seen in tumor cells adjacent to

necrotic areas and at the infiltrating edge of tumors. EpoR staining, but not Epo

staining, was significantly greater in tumors that showed high histologic grade,

tumor necrosis, lymphovascular invasion, lymph node metastases, and loss of

hormone receptor expression.

CONCLUSIONS. The current findings suggest that increased EpoR expression may

play an important role in breast carcinogenesis. The induction of autocrine or

paracrine Epo signaling may represent a novel mechanism by which hypoxia can

promote breast carcinoma. Cancer 2002;95:969 – 81.

© 2002 American Cancer Society.

DOI 10.1002/cncr.10787

KEYWORDS: breast neoplasms, breast carcinoma, erythropoietin, erythropoietinreceptor, smoking, autocrine signaling.

Erythropoietin (Epo), a glycoprotein hormone stimulator of eryth-ropoiesis,1–3 promotes the proliferation and differentiation of ery-

throid precursor cells while also inhibiting their apoptosis.1–5 Epoexerts its actions through its specific receptor (EpoR), a member ofthe cytokine receptor superfamily.1,6 – 8 Major signal-transductionpathways activated by Epo include the Jak/signal transducer andactivator of transduction (STAT) and Ras/mitogen-activated proteinkinase pathways, which are involved in the inhibition of apoptosis andthe stimulation of cell proliferation in response to this hormone.4,7–12

During adult life, Epo normally is produced by the kidney andliver,1,3,5 and its gene expression is modulated primarily by tissue

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© 2002 American Cancer Society

hypoxia.13 However, other sites of Epo productionhave been reported recently, including bone marrowmacrophages,14 brain astrocytes,15,16 and trophoblastcells of the human placenta.17 Considerable amountsof Epo also are present in human milk,18 the source ofwhich is the lactating breast glands, as shown re-cently.19

In recent years, it has become clear that EpoR isexpressed by a variety of nonhematopoietic cell types,including endothelial cells,20 neurons,21 and tropho-blast cells.22 Although the specific function(s) of EpoRin these sites is not yet understood, there is increasingevidence that EpoR expressed in these tissues is func-tional, suggesting a wider biologic role for Epo signal-ing that is not related to erythropoiesis.23–25 Specifi-cally, the brain appears to have a paracrine Epo/EpoRsystem that is independent of the erythropoietic sys-tem: Neurons express EpoR,15,21,26 and astrocytes pro-duce Epo.15,16 Brain Epo signaling appears to protectneurons from ischemic damage by inhibiting theirapoptosis.26,27 EpoR mRNA also is expressed in endo-thelial cells, and Epo stimulates the proliferation andmigration of human endothelial cells28,29 and angio-genesis.30 Another paracrine Epo/EpoR system in theuterus may play an important role in uterine angio-genesis through EpoR that is expressed in endometrialvascular endothelial cells.30

Elevated Epo levels have long been recognized inpatients with renal carcinomas, Wilms tumors, hepa-tomas, and cerebellar hemangioblastomas, all arisingin anatomic sites in which Epo normally is expressedat low levels.13 In addition, symptomatic Epo produc-tion has been reported in a patient with breast carci-noma.31 Moreover, it was found that ectopic Epo ex-pression in erythroleukemia cells mediated theirautonomous growth.32 EpoR expression also was re-ported in patients with renal cell carcinoma, and apotential paracrine or autocrine role for Epo signalingfor promoting the growth of renal carcinomas hasbeen suggested.33

We recently described human breast carcinomacell lines and human breast carcinomas that ex-pressed Epo and EpoR mRNA and protein and showedthat their expression was enhanced by hypoxia.34 Fur-thermore, we demonstrated that Epo signaling is ac-tive biologically and stimulates tyrosine phosphoryla-tion, DNA synthesis, and proliferation in breastcarcinoma cells. We also showed that the vasculatureof solid tumors, including breast carcinomas, ex-presses EpoR. These results suggested a role for auto-crine-paracrine Epo signaling in the hypoxic survivaland neovascularization of tumors.

In the current study, we analyzed the expressionof Epo and EpoR by immunohistochemistry in a large

series of patients with primary breast carcinomas andin benign mammary epithelium. We analyzed the cor-relation between Epo and EpoR expression and vari-ous clinicopathologic tumor features. Because themost common reason for elevated serum Epo levels inhumans is hypoxia caused by cigarette smoking,35 andbecause recent studies have suggested that smokingmay be associated with an increased risk of breastcarcinoma,36 –38 we also analyzed the correlation be-tween Epo and EpoR immunostaining and the per-sonal smoking history of patients.

MATERIALS AND METHODSWe selected 184 breast biopsy specimens from theSurgical Pathology files of the University of Pennsyl-vania Medical Center that included 158 in situ mam-mary carcinomas and 184 invasive mammary carcino-mas. Hematoxylin and eosin-stained slides werereviewed independently by two pathologists (G.A. andP.J.Z.) to confirm the diagnosis, including histologictype and tumor grade, based on established crite-ria.39 – 41 Ductal carcinomas in situ (DCIS) were gradedas described by Scott et al.,42 using primarily nucleargrade. All invasive carcinomas were graded using themodified, combined histologic grading system de-scribed by Elston and Ellis.43 Tumors also were eval-uated to determine the presence or absence of tumornecrosis and lymphovascular invasion. Informationregarding tumor staging, including tumor size, axillarylymph node involvement, and other therapeutic andprognostic biologic markers, such as estrogen andprogesterone receptor status and HER2/neu overex-pression, were retrieved from the pathology reports.Smoking history was collected from a standardizedquestionnaire on 102 individuals who participated inboth the current study and in a separate study ofbreast carcinoma risk factors.

Immunohistochemical assays were performed onformalin fixed, paraffin embedded sections. Five-mi-crometer-thick sections were cut and deparaffinizedin xylene and rehydrated in graded alcohols. Slideswere steamed in 0.01 mol/L sodium citrate buffer, pH6.0, for 20 minutes. Endogenous peroxidase activitywas blocked by 3% hydrogen peroxide in methanol for20 minutes. Slides were incubated with the polyclonalantibody against Epo (rabbit polyclonal, H-162; 1:200dilution; Santa Cruz Biotechnologies Inc., Santa Cruz,CA) and EpoR (rabbit polyclonal, C-20; 1:200 dilution;Santa Cruz Biotechnologies Inc.) overnight at 4 °C.Slides were then washed five times with Tris-bufferedsaline containing Tween 20 (TBST), pH 7.6 (DAKOCorp., Carpinteria, CA), and incubated for 30 minutesat room temperature with horseradish peroxidase-la-beled dextran polymer coupled to antirabbit antibody

970 CANCER September 1, 2002 / Volume 95 / Number 5

(EnVision � System HRP; rabbit; DAKO Corp.). Slideswere then washed three times with TBST, developedwith diaminobenzidine for 10 minutes, and counter-stained with hematoxylin. Slides of fetal liver44 andplacenta17,22 were used as positive controls. A negativecontrol was done in each case by omission of theprimary antibody. The specificity of the antibodieswere confirmed previously.19,21,34,45,46 In addition, thespecificity of the immunoreactivity also was evaluatedwith an antibody absorption test: The primary anti-body was preincubated with blocking peptide forEpoR (Santa Cruz Biotechnologies Inc.) or human re-combinant Epo (rhEpo; 10:1 peptide:antibody ratio;R&D Systems, Minneapolis, MN), which resulted inthe complete abolition of immunohistochemicalstaining. The specificity of the immunostaining reac-tion is supported further by other experiments using amouse monoclonal anti-Epo (clone 9C21D11; R&DSystems) and a rabbit polyclonal anti-EpoR antibody(Upstate Biotechnology, Lake Placid, NY),47 which re-sulted in an immunostaining pattern similar to thatobtained with the antibodies used in the current study(our unpublished observation).

Immunohistochemical stains for Epo and EpoRwere interpreted semiquantitatively by assessing theintensity of staining according to a four-tiered scale.Cytoplasmic and/or membrane immunoreactivity wasconsidered positive. First, the total percentages of pos-itive tumor cells and benign ductal and lobular epi-thelial cells were assessed. Then, the percentages ofweakly, moderately, and strongly staining cells weredetermined, so that the sum of these categoriesequated with the overall percentage of positivity. Astaining score was then calculated as follows: score(from a maximum of 300) � sum of 1 � percentage ofweak staining cells, 2 � percentage of moderatelystaining cells, and 3 � percentage of strong stainingcells. In addition, a differential tumor score also wascalculated by subtracting the staining score of theadjacent benign epithelium from that of the in situand invasive carcinomas. Immunohistochemicalstains were evaluated independently by two patholo-gists (G.A. and P.J.Z.). Slight differences in interpreta-tion were resolved by simultaneous viewing.

The Wilcoxon signed rank test was used for thecomparison of median Epo and EpoR expression lev-els in carcinomas and in adjacent benign mammaryepithelial cells. The correlation between the levels ofEpo and EpoR staining in invasive and in situ compo-nents of carcinomas was estimated using the Spear-man rank correlation test. We examined the correla-tion between median Epo and EpoR expression levelsand tumor type, tumor size, combined tumor grade,lymph node status, estrogen and progesterone recep-

tor status, and HER2/neu overexpression. For the sta-tistical analysis, the Mann–Whitney rank sum test andthe Kruskal–Wallis one-way analysis of variance byranks was used followed by a Dunn multiple-compar-ison test, when appropriate. Statistical significancewas determined if the two-sided P value of a test was� 0.05.

RESULTSExpression of EpoRIn normal ductal and lobular epithelial cells adjacentto tumor, weak, granular, cytoplasmic EpoR immuno-staining was seen in 176 of 184 biopsy specimens(95.6%). Compared with Epo (see below), no increasedEpoR staining was seen in lobules that showed secre-tory changes (not shown). Benign epithelial lesions,including usual hyperplasia, sclerosing adenosis, andpapillomas, showed weak EpoR staining similar to thatseen in normal epithelial cells (Fig. 1A). Diffuse, mod-erate-to-strong cytoplasmic and membrane EpoR im-munostaining was seen in 121 of 122 DCIS tumors(99.2%), in 36 of 36 lobular carcinomas in situ (LCIS)tumors (100%), and in 183 of 184 invasive carcinomas(99.5%) (Fig. 1B–D). Although EpoR staining usuallywas uniform throughout the tumor, increased expres-sion was seen in tumor cells adjacent to necrotic areas(Fig. 2A,B). Necrotic tissue itself showed no stainingfor EpoR. EpoR immunostaining was similar in the insitu and invasive components of the tumors (correla-tion coefficient [r] � 0.795; P � 0.0001; Spearman rankcorrelation test). In addition, strong EpoR expressionwas found in the tumor vasculature (not shown).

Compared with benign epithelial cells, EpoR im-munostaining was increased significantly in DCIS (P� 0.0001, Wilcoxon signed rank test; rs � 0.467; P� 0.0001), LCIS (P � 0.0001; Wilcoxon signed ranktest; rs � 0.483; P � 0.005), and invasive carcinomas (P� 0.0001; Wilcoxon signed rank test; rs � 0.355; P� 0.0001) (Tables 1, 2; Figs. 1B–D, 3). EpoR immuno-staining in DCIS and invasive carcinomas was signif-icantly greater compared with EpoR immunostainingin benign epithelial cells both in nonsmokers (DCIS: n� 30 tumors; P � 0.0001; rs � 0.331; P � 0.05; invasivecarcinoma: n � 41 tumors; P � 0.0001; rs � 0.420; P� 0.005; Wilcoxon signed rank test) and in smokers(DCIS: n � 35 tumors; P � 0.0001; rs � 0.608; P� 0.0001; invasive carcinomas: n � 61 tumors; P� 0.0001; rs � 0.494; P � 0.0001; Wilcoxon signed ranktest). There was no difference in the level of EpoRimmunostaining of benign epithelial cells in non-smokers (median score, 100; mean � standard error ofthe mean [SEM], 85.3 � 9.9; n � 41 tumors) comparedwith smokers (median score, 100; mean � SEM, 95.6� 6.6; n � 61 tumors; P � 0.05; Mann–Whitney test).

Epo and Epo Receptor in Breast Carcinoma/Acs et al. 971

We found no significant difference in EpoR immu-nostaining based on the EpoR staining scores betweeneither DCIS and LCIS or invasive ductal and lobularcarcinomas and carcinomas with mixed ductal andlobular features (P � 0.05; Kruskal–Wallis test) (Table2). Conversely, based on the differential tumor scores,a highly significant difference in EpoR expression wasseen between ductal carcinomas and lobular carcino-mas, with mixed tumors showing intermediate levelsof EpoR staining (P � 0.0001; Kruskal–Wallis test) (Ta-ble 2). A similar difference was found in EpoR stainingbetween DCIS and LCIS as well (P � 0.0066; Mann–Whitney test) (Table 1).

We found no correlation between EpoR immuno-staining and tumor size (P � 0.05; Mann–Whitneytest). In DCIS, a significant correlation was seen be-tween EpoR staining and tumor grade and the pres-ence of tumor necrosis (Table 1). Similarly, in invasivecarcinomas, significantly increased EpoR immuno-staining was seen in carcinomas with high histologicgrade, in carcinomas that showed tumor necrosis andlymphovascular invasion, and in tumors that wereassociated with lymph node metastases (Table 2).EpoR staining also was significantly greater in breasttumors with negative hormone receptor expressioncompared with hormone receptor positive tumors(Table 2). No correlation was found between EpoRexpression and HER2/neu overexpression by the tu-mors (Table 2).

Expression of EpoIn benign mammary epithelial cells, we found weak-to-moderate, granular, cytoplasmic staining for Epo in169 of 184 specimens (91.8%). Epo staining was mostpronounced at the apical part of epithelial cells in thelobules (Fig. 4A). We found prominent, strong Epoimmunostaining in lobules that showed secretorychanges (Fig. 4B). In benign epithelial lesions (hyper-plasia without atypia, sclerosing adenosis, and papil-loma), Epo staining was similar to the Epo stainingseen in normal epithelial cells (not shown). Epo im-munostaining was found in 112 of 122 DCIS tumors(91.8%), in 34 of 36 LCIS tumors (94.4%) (Table 3), andin 174 of 184 invasive breast carcinomas (94.6%) (Ta-ble 4). Epo staining was usually weak to moderate andheterogeneous; however, strong, prominent stainingwas present in viable tumor cells adjacent to necroticareas (Fig. 5A,B) and at the infiltrating edge of carci-nomas (Fig. 5C). Necrotic tissue showed no stainingfor Epo. Epo expression was similar in the in situ andinvasive components of the tumors (r � 0.844; P� 0.0001; Spearman rank correlation test).

Compared with benign epithelial cells, Epo im-munostaining was increased significantly in DCIS (P� 0.0001; Wilcoxon signed rank test; rs � 0.683; P� 0.0001), LCIS (P � 0.005; Wilcoxon signed rank test;rs � 0.477; P � 0.005), and invasive carcinomas (P� 0.0001; Wilcoxon signed rank test; rs � 0.731; P� 0.0001) (Fig. 5D). It is interesting to note that, when

FIGURE 1. Expression of erythropoietin

receptor (EpoR) in benign mammary epi-

thelial cells and invasive breast carcinoma.

(A) Weak immunostaining for EpoR is seen

in usual ductal hyperplasia without atypia.

(B) Strong cytoplasmic and membrane im-

munostaining for EpoR is seen in invasive

ductal carcinoma. Note that the benign

duct in the center shows only weak stain-

ing for EpoR. (C) Strong EpoR immuno-

staining is seen in invasive ductal carci-

noma. Only weak staining is present in the

benign duct in the center. (D) Moderate-

to-strong immunostaining for EpoR is seen

in invasive lobular carcinoma. The benign

duct in the center shows only weak EpoR

staining; immunohistochemical staining

for EpoR with hematoxylin counterstain).

Original magnification �200 (A); �400

(B–D).


smokers (n � 61 patients) and nonsmokers (n � 41patients) were analyzed separately, we found signifi-cantly increased Epo immunostaining in invasive car-cinomas compared with benign epithelial cells only innonsmokers (P � 0.0018; Wilcoxon signed rank test; rs� 0.816; P �0.0001) and not in smokers (P � 0.05;Wilcoxon signed rank test; rs � 0.703; P � 0.0001) (Fig.6). Similarly, whereas Epo immunostaining was in-creased significantly in DCIS compared with benignepithelial cells in nonsmokers (P � 0.001; n � 30specimens; Wilcoxon signed rank test; rs � 0.733; P� 0.0001), the difference was only marginally signifi-cant in smokers (P � 0.0444; n � 35 specimens; Wil-coxon signed rank test; rs � 0.687; P � 0.0001). Al-though Epo immunostaining was increased in benignepithelial cells in smokers (median score, 100; mean� SEM, 90.2 � 7.3; n � 61 patients) compared withnonsmokers (median score, 60; mean � SEM, 79.0� 10.1; n � 41 patients), this did not reach statisticalsignificance (P � 0.05; Mann–Whitney test).

Epo immunostaining was similar in invasive ductaland lobular carcinomas and in carcinomas with mixedductal and lobular features (P � 0.05; Kruskal–Wallistest) (Table 4). Similarly, no difference was found in Epoimmunostaining between DCIS and LCIS (P � 0.05;Mann–Whitney test). We found no correlation betweenEpo immunostaining and tumor size, tumor grade, pres-ence of necrosis, lymphovascular invasion, lymph nodestatus, hormone receptor status, or HER2/neu overex-pression (P � 0.05; Kruskal–Wallis or Mann–Whitneytest) (Table 4). Similarly, no correlation was found be-tween Epo immunostaining and grade of DCIS or pres-ence of comedo-type necrosis (P � 0.05; Kruskal–Wallisor Mann–Whitney test) (Table 3).

FIGURE 2. Increased erythropoietin receptor (EpoR) expression in necrotic

tumor areas. (A) Moderate immunostaining for EpoR is seen in ductal carci-

noma in situ. Note the increased EpoR staining in the tumor cells adjacent to

the central necrotic area. (B) Strong EpoR immunostaining is seen in cells of

invasive ductal carcinoma adjacent to necrotic area (asterisk; immunohisto-

chemical staining for EpoR with hematoxylin counterstain). Original magnifi-

cation, � 400 (A, B).

TABLE 1Erythropoietin Receptor Expression in In Situ Mammary Carcinomas

In situ carcinoma No.

EpoR staining score EpoR DTSa

Median Mean � SEM P valueb Median Mean � SEM P valueb

TypeDCIS 122 200 175.7 � 6.8 � 0.05 100 95.1 � 6.3 0.0066LCIS 36 115 164.5 � 10.3 40 58.4 � 10.0 —

DCIS gradeLow 22 95 149.0 � 16.9 0.0041 60 63.5 � 13.1 0.0111Intermediate 51 105 164.4 � 10.3 — 100 89.6 � 9.8 —High 49 160 204.1 � 9.3 — 100 120.8 � 9.8 —

NecrosisAbsent 59 115 162.4 � 9.1 0.03 100 89.5 � 7.9 � 0.05Present 63 140 194.8 � 9.1 — 100 112.3 � 9.1 —

EpoR: erythropoietin receptor; DTS: differential tumor score; SEM: standard error of the mean; DCIS: ductal carcinoma in situ; LCIS: lobular carcinoma in situ.a EpoR DTS: EpoR staining score of tumor minus EpoR staining score of corresponding benign epithelium.b Mann–Whitney or Kruskal–Wallis test.


It is noteworthy that, when the analysis was doneusing the differential tumor scores for Epo, we found asignificant correlation between increased Epo immuno-staining in the tumors and the presence of lymph nodemetastases (P � 0.0397; Mann–Whitney test) (Table 4).When Epo immunostaining in DCIS and invasive carci-nomas was compared with the adjacent benign epithe-lium in the same specimens, we found significantlygreater Epo staining in intermediate grade tumors (P� 0.01) and high-grade tumors (P � 0.0001) only, andnot in low-grade carcinomas (P � 0.05; Wilcoxon signedrank test).

DISCUSSIONWe characterized the expression of Epo and EpoR byimmunohistochemistry in a series of 158 in situ and184 invasive breast carcinomas and adjacent benign

mammary epithelium. We found weak-to-moderate,granular, cytoplasmic Epo and EpoR immunostainingin benign mammary epithelial cells in 91.8% and95.6% of the specimens, respectively. This pattern ofimmunostaining is similar to that described previ-ously in breast tissues19 and other tissues.17,22,48,49 Innormal ducts and lobules, Epo staining was mostprominent in the luminal aspect of lobular epithelialcells. We found strong Epo immunostaining in lobulesthat showed secretory changes; however, EpoR stain-ing was not increased in such secretory lobules. Thesefindings are similar to those of Juul et al.,19 who re-cently also reported strong Epo immunoreactivity inmammary epithelial cells from lactating breast tissue,with less reactivity noted during gestation and in non-lactating breast. Those authors also found weak EpoRimmunoreactivity in mammary epithelial cells regard-

TABLE 2Erythropoietin Receptor Expression in Invasive Mammary Carcinomas

IMC No.

EpoR staining score EpoR DTSa


Total 184 190 172.5 � 5.3 N/A 100 93.0 � 5.3 N/AType

Ductal 126 185 176.7 � 6.5 � 0.05 100 106.0 � 5.9 � 0.0001Mixed 27 200 166.3 � 15.2 — 100 86.6 � 17.4 —Lobular 31 200 161.0 � 10.3 — 40 46.0 � 9.4

Grade1 44 155 149.3 � 10.6 0.026 60 63.9 � 10.9 .00032 89 200 177.0 � 7.4 — 100 96.4 � 6.93 51 195 183.6 � 10.0 — 120 117.1 � 8.3 —

SizeT1 91 180 159.2 � 8.2 � 0.05 90 87.0 � 7.6 � 0.05T2–T4 93 195 178.0 � 6.8 — 100 95.6 � 6.5 —

Tumor necrosisAbsent 141 180 159.2 � 8.2 0.027 90 87.0 � 7.6 0.0123Present 43 200 193.3 � 10.3 — 110 113.9 � 9.2 —

LVIAbsent 119 170 161.3 � 6.9 0.0069 80 79.9 � 6.5 0.0061Present 65 200 189.8 � 7.5 — 110 107.1 � 7.4 —

Lymph node statusNegative 95 170 160.8 � 7.8 0.0075 85 80.4 � 7.6 � 0.05Positive 89 200 187.5 � 7.0 — 100 101.6 � 7.0 —

ER statusNegative 75 200 185.7 � 7.9 0.0375 100 108.3 � 7.2 0.0115Positive 109 180 161.3 � 6.9 — 90 80.4 � 6.7 —

PR statusNegative 77 200 185.9 � 7.6 0.0404 100 101.4 � 7.3 � 0.05Positive 107 170 161.1 � 7.1 — 100 89.2 � 6.8 —

HER2/neu statusNegative 116 180 175.3 � 6.8 � 0.05 100 93.2 � 6.8 � 0.05Positive 68 190 164.6 � 8.6 — 100 93.4 � 7.6 —

EpoR: erythropoietin receptor; DTS: differential tumor score; IMC: invasive mammary carcinoma; SEM: standard error of the mean; N/A: not available; LVI:

lymphovascular invasion; ER: estrogen receptor; PR: progesterone receptor.a EpoR DTS: EpoR staining score of tumor minus EpoR staining score of corresponding benign epithelium.b Mann–Whitney or Kruskal–Wallis test.


less of lactational state. These findings and our resultssupport the concept that Epo present in breast milk issynthesized in the lactating mammary gland epithe-lium. Although the physiologic role of Epo in breast isnot yet clear, and no data are available currently tosuggest a possible role for Epo in lactation, the pres-ence of EpoR suggests a specific role for Epo signalingin the breast.19

We found significantly increased Epo immuno-staining in both in situ and invasive carcinomas com-pared with benign ductal and lobular epithelial cells innonsmokers, but not in smokers. The lack of a differ-ence in Epo staining for smokers appeared to be dueto an increased baseline level of Epo expression insmokers in benign epithelium. However, this differ-ence did not reach statistical significance in our series.Conversely, EpoR immunostaining was increased sig-nificantly in carcinomas compared with benign epi-thelial cells both in nonsmokers and in smokers. Thebaseline level of EpoR expression in benign epithelialcells was similar for the two groups.

Epo immunostaining was heterogeneous in carci-nomas, with most prominent expression in tumorcells adjacent to necrotic areas and at the infiltratingedge of the tumors, sites thought to be the most hy-poxic parts of tumors.50 –52 In contrast, EpoR expres-sion was usually uniform and homogenous, althoughgreater expression was present in tumor cells adjacentto necrotic areas, similar to the expression levels forEpo. These findings support our recent demonstrationof hypoxic up-regulation of both Epo and EpoR inbreast carcinoma cell lines.34

We found no difference in Epo or EpoR immuno-staining between ductal and lobular carcinomas and

carcinomas with mixed features. However, comparedwith benign epithelium present in the specimens,EpoR staining was significantly greater in ductal car-cinomas compared with tumors of the lobular type. Itis interesting to note that tumors with mixed featuresdemonstrated EpoR immunostaining levels that wereintermediate between the levels seen in ductal carci-nomas and lobular carcinomas. Epo immunostainingdid not show a correlation with any of the clinicohis-topathologic features of tumors examined, includingtumor size, grade, lymphovascular invasion, lymphnode status, hormone receptor status, and HER2/neuexpression. In previous studies, these clinicohis-topathologic features also were not correlated withintratumoral hypoxia.53 It is noteworthy that Epo im-munostaining did not show a statistical correlationwith the presence of tumor necrosis despite the prom-inent Epo staining observed near necrotic areas. Thisfinding likely is due to the fact that necrotic regionsusually represented small areas of tumors, and theprominent immunostaining that was restricted tothese areas did not increase the overall staining scoresignificantly.

In contrast, we found significantly increased EpoRimmunostaining in tumors that showed high com-bined histologic grade, tumor necrosis, lymphovascu-lar invasion, lymph node metastases, and loss of hor-mone receptor expression, all features that areconsidered to be associated with an adverse progno-sis.54 No correlation was seen between EpoR expres-sion, tumor size, HER2/neu expression.

Until recently, the action of Epo was thought to berestricted to erythropoiesis.3 There is now evidencethat certain nonerythroid cells express EpoR and re-spond to Epo in vitro and in vivo. The major mecha-nism by which Epo promotes erythropoiesis is by pre-venting the programmed cell death of erythroidprecursors through up-regulation of the Bcl-2 andBcl-XL pathways mediated by STAT-5.55,56 There isalso increasing evidence that EpoR expressed in neu-ronal tissues is functional21 and that Epo may havemechanisms of action in the central nervous systemsimilar to those described in erythroid cells, such asdecreasing apoptosis in neurons during normal braindevelopment, or exerting a neuroprotective effect un-der adverse conditions, such as hypoxia.21,27

EpoR mRNA also is expressed in endothelial cells,and Epo stimulates the proliferation and migration ofhuman endothelial cells28,29 and stimulates angiogen-esis.30 Recent studies indicate that Epo signaling inendothelial cells is mediated through tyrosine phos-phorylation of proteins, including phosphorylation oftranscription factor STAT-5, which is similar to that inerythroid cells.57 It was suggested recently that a para-

FIGURE 3. Expression of erythropoietin receptor (EpoR) in benign mammary

epithelial cells and invasive mammary carcinoma (IMC). Bars indicate the

median values of EpoR immunostaining scores. Asterisks, P � 0.0001 (Wil-

coxon signed rank test).


crine mechanism of Epo signaling may play a role inuterine angiogenesis.30 We have found that the vascu-lature of solid tumors, including breast carcinomas,also expresses EpoR, and a paracrine mechanism ofEpo signaling also may play a role in the vasculariza-tion of these tumors.34 Our current finding of EpoRexpression in tumor vasculature is consistent with thishypothesis.

An autocrine role for Epo in which the hormonecontributes to the survival, proliferation, and differen-tiation of trophoblast cells22 may be analogous to itsrole in erythroleukemia,58 hepatocellular carcinoma,59

and renal cell carcinoma cells.33 We recently showedthat Epo signaling stimulates the proliferation of hu-man breast carcinoma cell lines, suggesting that anautocrine mechanism of Epo signaling also may play arole in the proliferation and hypoxic survival of breasttumors.34 Our current results also support these find-ings.

It has been demonstrated that Epo expression inthe uterus and oviduct is regulated by estrogen inaddition to hypoxia.30 It is noteworthy that, althoughthe importance of an increased estrogen effect inbreast carcinogenesis is well established,60 we foundno correlation of Epo expression with the hormonereceptor status of tumors. Conversely, we did findincreased EpoR expression by tumors with negativehormone receptor status, a feature that is associatedwith poor differentiation and an adverse prognosis.

Regions of low oxygen and necrosis are commonfeatures of solid tumors.51,53 Hypoxic conditions invitro as well as in vivo result in elevated levels ofhypoxia-inducible factor 1 (HIF-1), a transcription fac-tor that, in turn, stimulates the expression of a number

FIGURE 4. Expression of erythropoietin (Epo) in benign mammary epithelium.

(A) Epo immunostaining is seen in benign lobular epithelial cells. Note the more

prominent staining at the apical part of the cells. (B) Prominent immunostaining

for Epo is seen in a lobule that shows secretory change; immunohistochemical

staining for Epo with hematoxylin counterstain). Original magnification �400

(A); �200 (B).

TABLE 3Erythropoietin Expression in In Situ Mammary Carcinomas

In situ carcinoma No.

Epo staining score Epo DTSa


TypeDCIS 122 90 90.3 � 6.4 � 0.05 5 26.4 � 4.9 � 0.05LCIS 36 100 97.9 � 9.5 — 0 25.7 � 8.4 —

DCIS gradeLow 22 60 79.6 � 15.7 � 0.05 7.5 18.8 � 11.1 � 0.05Intermediate 51 80 91.5 � 10.2 — 0 24.3 � 8.2 —High 49 100 96.0 � 9.9 — 17.5 33.4 � 8.0 —

NecrosisAbsent 59 95 91.3 � 9.4 � 0.05 5 27.1 � 8.2 � 0.05Present 63 90 92.8 � 8.9 — 10 27.4 � 6.4 —

Epo: erythropoietin; DTS: differential tumor score; SEM: standard error of the mean; DCIS: ductal carcinoma in situ; LCIS: lobular carcinoma in situ.a Epo DTS: Epo staining score of tumor minus Epo staining score of corresponding benign epithelium.b Mann–Whitney or Kruskal–Wallis test.


of genes important for tumor cell survival.61,62 Theoverexpression of HIF-1 protein was found in 53% ofall primary malignant tumors, including 29% of pri-mary breast tumors and 69% of breast carcinoma me-tastases.63 In colonic adenocarcinoma, tumor cells atthe leading edge of infiltrating carcinoma manifestedthe most intense HIF-1 expression63; this pattern ofstaining is similar to the expression of Epo we found inbreast carcinomas, supporting the hypothesis that in-creased Epo expression in the tumors is stimulated bytissue hypoxia.

HIF-1 regulates the hypoxia-driven expression ofseveral other genes in addition to Epo, including vas-cular endothelial growth factor, glycolytic enzymes,and glucose transporters.13,64 Thus, it is unlikely thatEpo signaling is the only determinant of hypoxic tu-mor cell survival. It should be noted, however, that,

although Epo gene transcription is regulated by HIF-1,Epo mRNA stability, and (hence) translation, is alsoregulated by RNA-stabilizing mechanisms.65 Thus, el-evated Epo protein expression also may result fromdistinct biochemical alterations in tumor cells.

It is believed that hypoxia mediates the selectionof neoplastic cells with diminished apoptotic potentialby providing a growth advantage to cells with geneticalterations that impair the process of apoptosis.66 Thishypoxia-mediated clonal selection of tumor cells withdiminished apoptotic potential has been suggested asan important biologic mechanism of tumor progression.The presence of hypoxia also may be involved in thedevelopment of a more aggressive phenotype and maycontribute to metastasis67–71 and treatment resis-tance.72,73 The potent antiapoptotic actions of Epo sig-naling may contribute to such effects of hypoxia.55,74

TABLE 4Erythropoietin Expression in Invasive Mammary Carcinomas

IMC No.

Epo staining score Epo DTSa


Total 184 80 80.8 � 4.9 N/A 0 14.5 � 3.6 N/AType

Ductal 126 70 78.3 � 6.3 � 0.05 0 13.5 � 4.5 � 0.05Mixed 27 80 74.4 � 11.9 — 0 11.8 � 10.2 —Lobular 31 100 96.5 � 9.9 — 0 20.7 � 7.5 —

Grade1 44 90 88.2 � 10.5 � 0.05 0 7.3 � 5.9 � 0.052 89 80 81.7 � 6.6 — 2 13.7 � 3.8 —3 51 70 73.5 � 10.3 — 0 20.3 � 8.9 —

SizeT1 91 35 62.4 � 12.3 � 0.05 0 6.2 � 4.1 � 0.05T2–T4 93 70 77.6 � 6.9 — 0 19.3 � 5.6 —

Tumor necrosisAbsent 141 80 81.7 � 5.6 � 0.05 0 10.6 � 3.3 � 0.05Present 43 70 77.9 � 10.7 — 5 27.2 � 10.1 —

LVIAbsent 119 80 80.7 � 6.3 � 0.05 0 11.1 � 4.5 � 0.05Present 65 80 80.7 � 8.2 — 0 17.0 � 4.7 —

Lymph node statusNegative 95 75 80.8 � 7.6 � 0.05 0 10.4 � 4.6 0.0397Positive 89 90 84.1 � 6.9 — 10 22.6 � 5.6 —

ER statusNegative 75 80 84.4 � 7.9 � 0.05 0 22.8 � 7.4 � 0.05Positive 109 70 77.4 � 6.6 — 0 10.7 � 3.2 —

PR statusNegative 77 80 80.6 � 7.7 � 0.05 0 13.6 � 5.0 � 0.05Positive 107 70 80.4 � 6.7 — 0 16.9 � 5.3 —

HER2/neu statusNegative 116 80 87.8 � 5.9 � .05 0 12.6 � 4.5 � 0.05Positive 68 75 68.6 � 8.8 1 16.8 � 5.2 —

Epo: erythropoietin; DTS: differential tumor score; IMC: invasive mammary carcinoma; SEM: standard error of the mean; LVI: lymphovascular invasion; ER: estrogen

receptor; PR: progesterone receptor.a Epo DTS: Epo staining score of tumor minus Epo staining score of corresponding benign epithelium.b Mann–Whitney or Kruskal–Wallis test.


Recent studies have suggested that both activesmoking and passive smoking may be associated withan increased risk of breast carcinoma.36 –38 Passivesmokers (exposed to 2 hours of smoke for 25 years)had 3.2 times the risk of breast carcinoma comparedwith women with no such exposure, and active smok-ers (� 20 cigarettes per day) had 4.6 times the risk ofbreast carcinoma. Our findings of increased Epo im-munostaining in the benign mammary epithelium ofsmokers are consistent with the observation of in-creased levels of Epo in the breast milk of smokerscompared with nonsmokers.18 Cigarette smoking is acommon cause of tissue hypoxia, and it is believedthat it induces elevated circulating Epo levels in hu-mans.35 Smokers, indeed, have higher serum Epo lev-els and hematocrits.35 Although it is believed that themajor source of elevated circulating Epo in smokers isdue to systemic hypoxia and is likely to come from thekidneys, the increased Epo immunostaining in benignmammary epithelium of smokers and in breast carci-nomas suggests that increased expression of Epoprobably is related to tissue and tumor hypoxia. EpoR-bearing cells, of course, can respond to locally or sys-

temically elevated Epo levels. These findings raise theintriguing possibility that the high expression of EpoRin breast carcinoma cells may represent a mechanismthat links smoking, tissue hypoxia, elevated Epo levels,and carcinoma. Clearly, further studies are needed toinvestigate this possibility.

Hypoxia also can induce EpoR expression,34 andwe found increased EpoR staining near necrotic areasof tumors. However, hypoxic regions usually are dis-tributed heterogeneously within tumors.53 Althoughthe heterogeneous pattern of Epo immunostaining isconsistent with this phenomenon, the markedly in-creased expression of EpoR throughout breast carci-noma tissue suggests that it may not be attributedsolely to hypoxia. Furthermore, HIF-1 is not known asa regulator of EpoR gene expression. Thus, the bio-chemical mechanisms underlying elevated EpoR ex-pression in breast carcinoma and its hypoxic enhance-ment remain unknown. Elevated EpoR expressionmay increase the sensitivity of the neoplastic cells toavailable Epo. Elevated systemic Epo levels, thus, mayindicate potentially adverse outcomes for patientswith breast carcinoma, as proposed for patients with

FIGURE 5. The expression of erythropoietin (Epo) in invasive mammary carcinoma. (A) Moderate immunostaining for Epo is seen in ductal carcinoma in situ. Note

the strong Epo staining in viable tumor cells adjacent to the central necrotic area. (B) Strong immunostaining for Epo is seen in cells of invasive ductal carcinoma

adjacent to a necrotic area (asterisk). Note that tumor cells away from the necrotic area show only weak Epo expression (lower part of photomicrograph. (C) Weak

Epo immunostaining is seen in invasive ductal carcinoma with strong expression of the hormone in cells at the infiltrating edge of the tumor. (D) Prominent Epo

immunostaining is seen in cells of invasive lobular carcinoma. The carcinoma cells infiltrate around a benign duct, which shows only weak staining for Epo;

immunohistochemical staining for Epo with hematoxylin counterstain). Original magnification �400 (A, B); �200 (C); �40 (D).


renal carcinoma.33 The routine use of rhEpo in pa-tients with malignancies for the treatment of anemiaresulting from chemotherapy, therefore, may need tobe re-evaluated for its potential adverse effects. Fur-ther studies are needed to examine these hypotheses.

In summary, we have demonstrated that benignmammary epithelial cells and cells of in situ and in-vasive breast carcinomas express both Epo and EpoR.Increased immunostaining of EpoR in breast carcino-mas showed a positive correlation with clinicohis-topathologic features suggestive of an adverse prog-nosis. Our findings suggest that increased Eposignaling may represent a novel mechanism by whichhypoxia promotes a malignant phenotype of breastcarcinoma.

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Immunohistochemical expression of erythropoietin and erythropoietin receptor in breast carcinoma