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Address correspondence to Gary L. Francis MD PhD,Department of Pediatrics, Uniformed Services University ofthe Health Sciences, 4301 Jones Bridge Road, Bethesda, MD20814, USA; tel 301 295 9716; fax 301 295 3898; email:[email protected]. The opinions or assertions containedherein are the private views of the authors and are not to beconstrued as official or to reflect the opinions of the UniformedServices University of the Health Sciences, Walter Reed ArmyMedical Center, the Department of the Army, or the Depart-ment of Defense.

Erythropoietin and the Erythropoietin Receptor Are Expressed byPapillary Thyroid Carcinoma fromChildren and Adolescents.Expression of Erythropoietin Receptor Might Be a Favorable Prognostic Indicator

Thomas G. Eccles,1,3 Aneeta Patel,1 Ajay Verma,2 Diarmuid Nicholson,4 Yvonne Lukes,4R. Michael Tuttle,5 and Gary L. Francis1,3,4

Departments of Pediatrics1 and Neurology,2 Uniformed Services University of the Health Sciences, Bethesda,Maryland; Departments of Pediatrics3 and Clinical Investigation,4 Walter Reed Army Medical Center,Washington, District of Columbia; and Department of Endocrinology,5 Memorial Sloan Kettering CancerCenter, New York, New York

Abstract. Erythropoietin (EPO) and the EPO receptor (EPO-R) have been implicated in solid tumors ofthe brain, breast, kidney and female genital tract. Based on their expression by a variety of tumors, wehypothesized that EPO and/or EPO-R might be expressed by thyroid cancers. To test this, we determinedEPO and EPO-R expression by immunohistochemistry in 17 papillary thyroid carcinomas (PTC) fromchildren and adolescents. Only a minority of PTC (4/17, 24%) expressed EPO, and there were no significantdifferences between the PTC that did or did not express EPO. In contrast, EPO-R was detected in themajority of PTC (11/17, 65%). The average tumor size (1.5 ± 0.8 cm), MACIS score (3.6 ± 0.2) and risk ofrecurrence (0/11) for the EPO-R(+) PTC were significantly less than those for PTC that failed to expressEPO-R (average tumor size = 3.6 ± 2.4 cm, p = 0.021; average MACIS score = 4.3 ± 0.7, p = 0.004;recurrence = 3/6, p = 0.029). We conclude that the majority of PTC from children and adolescents expressEPO-R, a finding associated with favorable prognostic indicators and a lower risk of recurrence. (received 24

June 2003; accepted 2 July 2003)

Key Words: thyroid, cancer, erythropoietin, erythropoietin receptor

Introduction

Erythropoietin (EPO) is a 34 kD glycoproteinhormone whose principal function is to regulate redblood cell production [1]. In response to hypoxia,EPO is secreted by the kidney and bound to theerythropoietin receptor (EPO-R) on red cellprogenitors where it promotes differentiation andhemoglobin synthesis [1,2].

The EPO-R belongs to the growth hormonereceptor super family along with granulocyte-colonystimulating factor (GCSF), granulocyte macro-phage-colony stimulating factor (GMCSF), andseveral of the interleukins (IL) [3]. EPO induceshomodimerization of EPO-R, activating receptor-associated Janus kinase 2 (JAK 2), and serving as adocking site for one of the signal transducers andactivators of transcription (STAT 5) [4]. EPO-Ractivation appears to inhibit apoptosis rather thanto affect the commitment of progenitor cells to theerythroid lineage [5].

Of major interest are newly discovered functionsfor EPO that extend beyond the formation of redblood cells. EPO protects a number of tissues fromapoptosis, especially cells of the central nervoussystem [6]. In vitro, EPO protects neurons fromglutamate-induced toxicity, and in vivo, EPO

0091-7370/03/0400/0411 $3.00. © 2003 by the Association of Clinical Scientists, Inc.

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protects neurons against ischemia-induced celldeath. In response to tissue hypoxia, the transcrip-tion factor, hypoxia-induced factor-1 (HIF-1), up-regulates the expression and stabilizes the mRNAsencoding EPO, fibroblast growth factor-2 (FGF-2),and vascular endothelial growth factor (VEGF),suggesting that EPO expression might be ageneralized response to tissue hypoxia [8,9].

EPO and EPO-R have been implicated in anumber of malignant conditions, only a few of whichinvolve cells of erythroid lineage [10,11]. Novel butimportant roles for EPO and EPO-R have beenidentified in solid tumors of the brain, breast, kidneyand female genital tract [12-16]. Several observationssuggest these effects may be related to tissue hypoxia[12-14]. Hypoxia is present in virtually all solidneoplasms, and is associated with invasion,metastasis, resistance to therapy, and selection of cellswith diminished apoptotic potential [17-24]. Basaland hypoxia-stimulated expression of EPO andEPO-R have been demonstrated in human breastcancers and cell lines, suggesting roles for EPOsignaling in the adaptation of these neoplasms tohypoxia [12-14].

EPO-R immunostaining is increased in breastcancers compared to normal tissues, and is mostintense in regions directly adjacent to the infiltratingedge of growing tumors. In addition, EPO-Rexpression is significantly greater in breast tumorswith higher grade histology, tumor necrosis, lympho-vascular invasion, lymph node metastases, and lossof hormone receptor expression [12-14]. In vitro,EPO stimulates tyrosine phosphorylation and prolif-eration of breast cancer cells, both of which areblocked by a neutralizing anti-EPO antibody [13].

Malignant tumors of the ovary and uterus alsoexpress EPO and EPO-R [16,25,26]. Treatmentwith anti-EPO antibody reduced the size of ovarianand uterine cancer xenografts in nude mice[16,25,26]. Immunohistochemical examination ofthe treated tumors revealed apoptotic death ofmalignant and endothelial cells [16].

Based on the expression of EPO and EPO-Rby a variety of solid tumors, we hypothesized thatEPO and/or EPO-R might be expressed by thyroidcancers. To our knowledge, no previous study hasexamined this possibility, nor has any study

examined the potential impact of EPO and EPO-Rexpression on the clinical behavior of thyroidcancers. Our data indicate that EPO and EPO-Rare expressed by papillary thyroid cancers (PTCs)from children and adolescents. The data show thatPTCs that express EPO-R are smaller, have lowerMACIS (metastasis-age-completeness-of-resection-invasion-size) scores, and a lower risk of recurrence.

Materials and Methods

Human experimentation. This study protocol wasapproved the Human Use Committee of theDepartment of Clinical Investigation, Walter ReedArmy Medical Center, Washington, DC.

Patients. The automated centralized tumor registryof the Department of Defense (ACTUR) wassearched to identify all patients with PTC who were<21 yr old at the time of diagnosis. A computerizeddatabase containing demographic features, tumorcharacteristics, surgical and adjunctive treatments,and clinical outcomes was generated from these dataand used for this and previous publications [27-38].

The extent of disease at diagnosis was classifiedby the method of DeGroot et al [39], and the metas-tasis-age-completeness-of-resection-invasion-sizescore (MACIS) [40]. According to DeGroot et al[39], Class 1 disease was confined to the thyroidgland; Class 2 involved the regional lymph nodes;Class 3 either extended beyond the capsule or wasinadequately resected; and Class 4 had distantmetastasis. Because all patients were <39 years ofage, MACIS scores were calculated as 3.1 + (size x0.3) + 1 (if incomplete resection) + 1 (if directinvasion) + 3 (if distant metastasis) [40].

As in previous studies, recurrence was definedas the appearance of new disease (identified by radio-active iodine scan or biopsy) in any patient who hadbeen free of disease (no disease palpable or identifiedby radioactive iodine scan) for a period of 4 mofollowing initial therapy [41]. Serum thyroglobulin(Tg) values were determined on contemporarypatients (normal range: 3 - 40 ng/ml, University ofSouthern California Clinical Laboratories, LosAngeles, CA). Levels <2 ng/ml were considered toindicate freedom from disease [42]. The clinical

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details for these and additional patients have beenpreviously published [41].

Formalin-fixed, paraffin-embedded archivedtumor blocks from 17 patients with PTC wereavailable for study. Sufficient material was alsoavailable to examine EPO and EPO-R staining onregions of presumably “normal” thyroid tissue thatwere immediately adjacent to a few of the PTC.Adjacent “normal” tissue was identified in 4 samplesthat were stained for EPO and 6 samples that werestained for EPO-R. These are included in the textas presumably “normal” thyroid, but there were notruly normal thyroid glands available for this study.

Immunohistochemistry. Tumor blocks weresectioned and stained with hematoxylin and eosinto confirm the diagnosis [43]. The sections immed-iately adjacent (5 µm) were deparaffinized (xylene)and rehydrated through a series of graded alcoholsolutions. Endogenous peroxidase was inactivated(3% H2O2, 30 min) and antigen was retrieved incitrate buffer (pH 6.0, 30 min, 100°C, steamer).For EPO determination, tissue sections weresequentially incubated with primary monoclonalanti-EPO antibody (H-162, 1:50, Santa CruzBiotechnology, Santa Cruz, CA), followed bypreformed avidin-biotinylated horse radishperoxidase complex, diaminobenzidine (DAB), andhematoxylin counterstain using the Ventana Nexesautomated immunostainer (Ventana DABDetection Kit, Ventana Medical Systems, Tucson,AZ). Sections of normal human kidney were usedas the positive control, and phosphate-buffered salinewas substituted for the primary and secondaryantibodies and used as the negative controls.

The intensity of EPO staining was based on theintensity of chromogen developed throughout themajority of each tumor. Staining intensity wasdetermined by two blinded, independent examinersand graded as follows: 0 = absent, 1 = minimal, 2 =moderate, and 3 = intense. The inter-observeragreement was >95%, and the few discordant slideswere graded by a third examiner. The two scores inagreement were then used as the final intensity grade.The same procedures were used to evaluate EPO-Rexpression; the primary antibody was directedagainst EPO-R (C-20, 1:50, Santa Cruz Biotech-nology, Santa Cruz, CA).

Amplification by polymerase chain reaction.Expression of EPO and EPO-R was confirmed byreverse transcription and polymerase chain reaction(RT-PCR) amplification of specific regions of theEPO and EPO-R messenger ribonucleic acids(mRNAs). These were then sequenced using dye-deoxy terminators and the ABI-Prism 377 SequenceDetection System (Applied Biosystems, Inc., FosterCity, CA.). One µg of cDNA was reverse transcribedusing the Impromptu RT kit (Promega, Inc.,Madison, Wisconsin) at 42°C for 1 hr, followed by15 min at 70°C. PCR was performed in optimizedPCR buffer (25 µl) containing enhancer and stabil-izer, along with DNTPs (Maxim Biotech), equalamounts of sense and antisense primers (10 pmolof each) and Platinum Taq polymerase (0.625 units,Invitrogen, Carlsbad, CA).

A 2-step PCR cycling regimen was used for EPOamplification. The parameters used included aninitial denaturation step at 96°C for 5 min, followedby an annealing step at 63°C for 4 min, 30 ampli-fication cycles at 94°C for 1 min, 63°C for 2 min,and a final extension at 72°C for 7 min. To increasedetection sensitivity, 1 µl of this PCR product wastransferred to a fresh PCR mixture for 7 additionalcycles. A consistent amplicon was detected in allamplifications by 2 cycles.

A 3-step cycling regimen was used for EPO-Rand GAPDH. The 3-step cycling parameters forEPO-R included an initial denaturation step at 96°Cfor 1 min, followed by 32 cycles of 1 min at 96°C, 1min at 58°C, 1 min at 72°C, and a final extensionat 72°C for 10 min. The cycling parameters forGAPDH were identical, except that the annealingtemperature was 60°C, and 30 amplification cycleswere used.

To test the integrity of the RT-PCR process, anexternal positive control (Maxim Biotech, SanFrancisco, CA), an RT negative control, andexpression of the internal housekeeping gene,glyceraldehyde-3-PO4 dehydrogenase (GAPDH)were performed for each RT reaction. The PCRproducts were resolved by electrophoresis on 2%agarose gels and visualized by ethidium bromidestaining. The primers for EPO were designed usingPrimer Express (V2.0 Applied Biosystems, FosterCity, CA) and the primers for EPO-R and GAPDH

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were obtained from Maxim Biotech (San Francisco,CA). The sequences are as follows:EPO:

Sense: 5’-CCCTGTTGGTCAACTCTTCC;Antisense: 5’-GTGTACAGCTTCAGCTTTCC;Product = 233 bp.

EPO-R:Sense: 5’-CCCAGGTCGGCTCCCTTTGT;Antisense:5’-GTCCTCCAACCGCTCGGTGA;Product = 149 bp.

GAPDH:Sense: 5’-ATCCCTCCAAAATCAAGTGG;Antisense: 5’-CAGAGATGATGACCCTTTTGG;Product = 126 bp.The sequence of the EPO amplimer was then

confirmed using dyedeoxy terminators and the ABIPrism 377 sequence detection instrument (AppliedBiosystems, Inc., Foster City, CA). Amplified cDNAwas extracted from the electrophoretic bandcorresponding to EPO using an Ultrafree™ DNAcentrifugal filter device (Millipore, Bedford, MA).The gel containing this band was placed in the GelNebulizer sample cup and centrifuged (5,000 x g,10 min). DNA was then re-amplified by PCR underthe following conditions: 50 mM KCl; 20 mM Tris-HCl, pH 8.0; 2 mM MgCl2; 0.2 mM dNTP; 0.4units TAQ polymerase; 0.2 µM forward and reverseprimers; one cycle at 95°C for 5 min, 35 cycles at95°C for 20 sec, one cycle at 60°C for 20 sec, and afinal cycle at 72°C for 20 sec. Excess primers wereremoved (Microcon column, Amicon, Beverly, MA)and the PCR products were diluted (500 µl) andcentrifuged (500 x g, 8 min). Samples wererecovered, dried, suspended (10 µl H2O), andsequenced (BigDye™ Terminator v 3.0 CycleSequencing Kit, Applied Biosystems Inc., FosterCity, CA). Excess terminator dyes were removed(CentriSep Spin Column, Princeton Separations,Aldelphia, NJ), and the fluorescent fragments wereseparated and analyzed (Model 377, AppliedBiosystems, Foster City, CA). The BLAST data base(National Library of Medicine, National Institutesof Health, Bethesda, MD) was then queried forsequence similarity.

Data analysis and statistical comparisons.Statistical analyses were performed using SPSS forWindows 95 (Version 7.5, SPSS Inc., Chicago, IL).The proportion of PTCs and surrounding “normal”

tissues that showed specific EPO and EPO-Rimmunostaining was compared using Fisher’s exacttest. The proportion of PTCs that showed aggressiveclinical behavior (invasion, metastasis, persistence,or recurrence) was then compared between EPO (+)and EPO (-) groups, and between EPO-R (+) andEPO-R (-) groups using Fisher’s exact test. Theaverage MACIS score, tumor size, and time torecurrence were compared between EPO (+) andEPO (-) PTC, as well as between EPO-R (+) andEPO-R (-) PTC by ANOVA.

Results

The demographic features, tumor characteristics,details of treatment and outcome for the 17 patientsin this study are shown in Tables 1 and 2. Themajority were female (n = 15, 88%) with either Class1 (n = 8, 47%), Class 2 (n = 8, 47%), or Class 3 (n= 1, 6%) disease. Their ages ranged from 6 – 21years (mean = 16.5 ± 4.5 yr), tumor sizes rangedfrom 0.7 – 7.5 cm in diameter (mean = 2.26 ± 1.8cm), and the MACIS scores ranged from 3.31 – 5.35(mean = 3.84 ± 0.6). All were treated with total (n =13, 76%) or subtotal (n = 3, 18%) thyroidectomy(details of one patient were insufficient to assign theextent of surgery) and the majority (60%, 9/15 forwhom details were available) received radioactiveiodine ablation. The patients were followed forperiods ranging from 0 – 118 mo (mean = 59 ± 42mo) after the initial therapy. Only 3 patientsdeveloped recurrent disease (17 - 67 mo, mean = 37± 26 mo). Demographic features, extent of disease,and type of therapy are similar to those of the largercohort previously studied by our group [41].

Typical immunostaining for EPO and EPO-Rare shown in Fig. 1. Specific staining was seen inthe cytoplasm of the PTC, but was absent from thesurrounding presumably “normal” thyroid tissue in4 samples stained for EPO and 6 samples stainedfor EPO-R. Although EPO was only detected inPTC and not in adjacent “normal” thyroid, thedifference was not significant [4/17 PTC were EPO(+), 0/4 adjacent “normal” samples were EPO (+),p = 0.55]. However, EPO-R expression was morefrequently detected in PTC than in presumably“normal” adjacent thyroid tissue [11/17 PTC were


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EPO-R (+) compared to 0/6 adjacent”“normal”thyroid samples, p = 0.014].

Expression of EPO and EPO-R mRNA wasconfirmed by RT-PCR and is shown in Fig. 2. RNAwith sufficient integrity to amplify the internalhousekeeping gene, GAPDH, was successfullyextracted from two EPO (+) PTC, two EPO (-)PTC, three EPO-R (+) PTC, and two EPO-R (-)PTC. These tissue samples were then used todetermine the expression of EPO and EPO-R. Asshown, EPO and EPO-R were detected by RT-PCRin all samples that stained for EPO and EPO-R.Conversely, all samples that failed to stain for EPOand EPO-R also failed to express EPO and EPO-Rby RT-PCR. Neither EPO nor EPO-R was detectedin any of the RT negative controls. Sequence analysisconfirmed that the EPO signal corresponds to thatof human EPO with a probability >99.9%.

Four of 17 (24%) PTC expressed EPO byimmunostaining (Tables 1 and 2). EPO staining was

intense (grade 3) in two (2/17, 12%), moderate(grade 2) in one (1/17, 6%), and minimal (grade 1)in the fourth (1/17, 6%). All of these PTC werefrom young women with Class 1 or Class 2 disease.The majority (3/4, 75%) of these tumors weremultifocal (a feature associated with a higher risk ofrecurrence), but none recurred.

The majority of PTC (n = 13, 76%) failed toexpress EPO by immunostaining (Tables 1 and 2).Most of these PTC were from young women (11/13, 85%) with Class 1 (n = 6) or Class 2 (n = 6)disease. The majority of EPO (-) tumors wereunifocal (n = 7, a feature associated with a low riskof recurrence) but 3 recurred (23%). There were nosignificant differences in demographic features,extent of disease at diagnosis, treatment, or risk ofrecurrence between the EPO (+) and EPO (-)groups.

EPO-R was detected by immunostaining in themajority of PTC (11/17, 65%) and was either

Table 1. Demographics, treatment and outcome for patients stratified according to EPO-R expression.

Gender Age Tumor Focal Class1 MACIS2 Extent RAI4 Recur Time Follow EPO EPO-R(yr) size (uni-/ of (Y/N) (Y/N) to up

(cm) multi-) surgery3 recur (mo)(mo)

EPO-R Positive PTCM 20 1.2 Uni 1 3.46 ST N N 118 0 2F 21 1.2 Multi 1 3.46 T Y N 107 0 3F 11 1.5 Uni 1 3.55 ST N N 18 0 3F 21 1.8 Uni 1 3.64 T N N 68 3 2F 21 2.3 Multi 1 3.79 T UNK N 18 2 3F 6 2.6 Uni 1 3.88 T N N 0 0 2F 18 2.9 Uni 1 3.97 T Y N 100 0 3F 15 0.7 Multi 2 3.31 T Y N 85 1 2F 10 0.8 Uni 2 3.34 T Y N 15 0 2F 20 1.0 Uni 2 3.40 T Y N 101 0 2F 21 1.0 Multi 2 3.40 T UNK N 22 0 2

EPO-R Negative PTCF 17 2.4 Uni 1 3.82 ST Y N 50 0 0M 19 1.0 Multi 2 3.4 UNK N N 104 0 0F 14 3.0 Multi 2 4.0 T Y Y 67 86 0 0F 13 5.5 Multi 2 4.75 T N N 0 3 0F 15 7.5 Multi 2 5.35 T Y Y 27 27 0 0F 19 2.0 Multi 3 4.7 T Y Y 17 86 0 0

1 Class according to DeGroot et al [39].2 MACIS according to Hay et al [40].3 Initial surgical procedure plus completion operation (if performed). T = total, ST = subtotal thyroidectomy, UNK = unknown.4 RAI refers to first (if any) radioactive iodine and includes “remnant ablation.”


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Fig. 1. (legend on opposite page)


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moderate (n = 7, 64%) or intense (n = 4, 36%)(Tables 1 and 2). Most of these patients were youngwomen (10/11, 91%) with unifocal tumors (7/11,64%) and either Class 1 (n = 7, 64%) or Class 2 (n= 4, 36%) disease. They were all treated with total(n = 9) or subtotal thyroidectomy (n = 2) and themajority (5/9 for whom details were available)received radioactive iodine. No patient developedrecurrent disease. There were suggestions that EPO-

R (+) tumors were more likely to be unifocal [7/11,64% vs 1/5, 20% for EPO-R (-) tumors, p = 0.08]and confined to the gland at diagnosis [7/11, 64%vs 1/6, 17% for EPO-R (-) tumors, p = 0.09] butthe differences only approached statisticalsignificance (Table 2).

Six PTC (6/17, 35%) failed to express EPO-Rby immunostaining (Tables 1 and 2). The majorityof these patients were also young women (5/6, 83%)with multifocal tumors (5/6, 83%) that were Class2 at diagnosis (n = 4, 67%). Surgical details wereavailable for 5 of these patients, all of whomunderwent total (4/5, 80%) or subtotal (1/5, 20%)thyroidectomy. Four (4/6, 67%) received radioactiveiodine ablation. Over time, three patients (3/6, 50%)

Fig. 1. EPO and EPO-R immunostaining of PTC from childrenand adolescents. Sections were stained by immunoperoxidasefor EPO and EPO-R. The presence and intensity of stainingwere graded from absent (grade 0) to intense (grade 3) for bothEPO (panels A – D) and EPO-R (panels E – H). Allphotomicrographs are shown at 100x magnification.

Table 2. PTC stratified according to EPO and EPO-R expression.

All PTC EPO (+) EPO (-) EPO (+) vs EPO-R (+) EPO-R (-) EPO-R (+) vs(n = 17) (n = 4) (n = 13) EPO (-) (p) (n = 11) (n = 6) EPO-R (-) (p)

Age (yr) 16.5 ± 4.5 18 ± 3.5 16.1 ± 4.8 ns 16.7 ± 5.4 16.2 ± 2.6 nsGender (F/M) 15/2 4/0 11/2 ns 10/1 5/1 nsTumor size (cm) 2.26 ± 1.8 3.1 ± 3.0 2.0 ± 1.3 ns 1.5 ± 0.8 3.6 ± 2.4 0.021Focal (uni-/multi-) 8/9 1/3 7/6 ns 7/4 1/5 0.08

Class1 ns 0.091 8 2 6 7 12 8 2 6 4 43 1 0 1 0 14 0 0 0 0 0

MACIS2 3.84 ± 0.6 4.0 ± 0.9 3.8 ± 0.5 ns 3.6 ± 0.2 4.3 ± 0.7 0.004

Extent of surgery3 nsTotal 13 4 10 9 4Subtotal 3 0 3 2 1Lobectomy 0 0 0 0 0Unknown 1 0 0 0 1

RAI ablation4 nsYes 9 1 8 5 4No 6 2 4 2 0Unknown 2 1 1 4 2

Recur 3 0 3 ns 0 3 0.029

Follow up (mo) 59 ± 42 50 ± 32 62 ± 45 ns 59 ± 45 58.8 ± 40.2 nsTime to recur (mo) 37 ± 26 None 37 ± 26 ns None 37 ± 26 ns

1 Class according to DeGroot et al [39].2 MACIS according to Hay et al [40].3 Surgery refers to initial procedure plus completion operation (if performed).4 RAI refers to first (if any) radioactive iodine and includes “remnant ablation.”


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Fig. 2. (legend on opposite page)


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developed recurrent disease. Of interest, the averagetumor size (1.5 ± 0.8 cm), MACIS score (3.6 ± 0.2)and risk of recurrence (0/11) for the EPO-R (+) PTCwere significantly less than those for PTC that failedto express EPO-R (average tumor size = 3.6 ± 2.4cm, p = 0.021; average MACIS score = 4.3 ± 0.7, p= 0.004; recurrence = 3/6, p = 0.029).

Discussion

Erythropoietin (EPO) primarily functions toregulate red blood cell production. However, recentstudies have shown that EPO and the EPO-R haveimportant roles in solid tumors of the brain, breast,kidney and female genital tract [12-16]. Severalobservations suggest these effects might be relatedto tissue hypoxia [12-14]. Based on the expressionof EPO and EPO-R by a variety of solid tumors, wehypothesized that EPO and/or EPO-R might beexpressed by thyroid cancers. To our knowledge, noprevious study has examined this possibility.

Our data show that the majority of PTC fromchildren and adolescents express the EPO-R (11/17, 65%) and a minority express EPO (4/17, 24%).Only one PTC expressed EPO and not EPO-R. Theexpression of EPO and EPO-R were confirmed bytwo independent methods: immunohistochemicalstaining and RT-PCR with sequence analysis.Although sufficient RNA could only be extractedfrom a small number of samples (4 for EPO and 5for EPO-R), the results of immunostaining and RT-PCR were concordant in all cases. To our knowledge,these data are the first to identify EPO and EPO-Rexpression by thyroid cancers.

We did not detect EPO or EPO-R expressionin any sample of presumably “normal” thyroid tissueadjacent to the PTC in this study. However, due tothe small number of samples, we could only identify

4 surrounding areas of “normal” thyroid that hadbeen stained for EPO. All 4 were negative, but thisdid not differ from the proportion of PTC that failedto express EPO [4/4 “normal” and 13/17 PTC wereEPO (-), p = 0.55]. We were able to identify asufficient number of surrounding “normal” sectionsstained for EPO-R to determine that EPO-Rexpression was more common in PTC (11/17) thanin surrounding “normal” thyroid (0/6, p = 0.014).These data suggest that the expression of EPO-Rand possibly EPO are features of PTC and notnormal thyroid.

The impact of EPO expression on the clinicalbehavior of individual PTC was then examined.There were no significant differences in tumor sizeor the extent of disease at diagnosis between the PTCthat did and did not expressed EPO. However,recurrence only developed in PTC that failed toexpress EPO (n = 3). The number of cases in eachgroup was too small to achieve statistical significance.The patients with PTC were then stratified into twogroups based on EPO-R expression. Whencompared to PTC that expressed EPO-R, PTC thatfailed to express EPO-R had larger tumor size (3.6± 2.4 vs 1.5 ± 0.8 cm, p = 0.021) and MACIS scores(4.3 ± 0.7 vs 3.6 ± 0.2, p = 0.004). Previous studieshave shown that larger tumor size and MACIS scoreare associated with a greater risk of tumor recurrence[40,41]. In agreement with these observations, thePTC that failed to express EPO-R had a greater riskof recurrence (3/6, 50% vs 0/11, p = 0.029).

Previous studies of EPO and EPO-R expressionin breast cancer showed that EPO-R immuno-staining was increased in carcinomas compared tonormal tissues, and most intense in areas directlyadjacent to necrotic foci or at the infiltrating tumoredge [12-14]. EPO-R but not EPO staining, wasalso greater in tumors showing higher grade histo-logy, tumor necrosis, lymphovascular invasion,lymph node metastases, and loss of hormone recep-tor expression [12,14]. In agreement with thesestudies, we only found EPO and EPO-R expressionin PTC, and not in surrounding “normal” thyroid.

However, in contrast, our data suggest thatexpression of EPO-R is associated with favorablerisk factors (smaller tumor size, lower MACIS scores,and lower recurrence risk). The reason for these

Fig. 2. EPO and EPO-R amplification by RT-PCR. TotalRNA was reverse transcribed and specific EPO (→), EPO-R(⇒), and GAPDH (solid triangle) sequences were amplified.Lanes 1 - 6 contain PTC in which EPO was identified byimmunohistochemistry. Lanes 7 - 12 contain PTC that failedto stain for EPO. Lanes 13 – 21 contain PTC in which EPO-R was detected by immunohistochemistry. Lanes 22 – 27contain PTC that failed to stain for EPO-R. RT negativecontrols and GAPDH amplification are shown for each samplealong with the results of EPO and EPO-R expression


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differences is not clear. It is possible that EPO-Rmay have different functions in thyroid and breastcancer cells, but it is also possible that the differencescould relate to the selection of patients in our study.Our interest is in thyroid carcinoma of childhood,and our tissue samples were obtained only frompatients with well-differentiated PTC who wereunder 21 yr of age. For these patients, the prognosisis highly favorable [41]. It is possible that the impactof EPO-R expression might have been different hadwe examined poorly differentiated or anaplasticthyroid cancers.

Our data are the first to show that EPO andEPO-R are expressed by thyroid cancers, but arelimited by several factors. First, the number oftumors is small (n = 17), and second, all these tumorsare highly differentiated PTC from children andyoung adults with a favorable prognosis [41].

Study of a larger number of more diversepatients is clearly indicated. In addition, the clinicaldata in our study are derived from a retrospectivedatabase [41]. Although treatments received by thepatients were similar, they were not randomized andwe have no information by which to determine whyany individual patient was selected to receive aspecific treatment. A randomized, prospectiveanalysis of EPO and EPO-R expression wouldeliminate this potential bias.

Finally, the patients reported in this studyreceived their care over the preceeding 20 yr.Sensitive thyroglobulin (Tg) assays were not availableduring this entire period and for that reason, serumTg values were not included in our definitions ofrecurrence and freedom from disease. Nevertheless,serum Tg levels were <2 ng/ml in all thecontemporary patients who were defined as free fromdisease (n = 4). It is still possible, however, that serumTg measurements might have resulted inreclassification of some of the historical subjects andmight have altered the outcomes of this study.

In summary, our data are the first to show thatPTC from children and adolescents express EPOand EPO-R. PTC that fail to express EPO-R arelarger, have higher MACIS scores, and have a higherrisk of recurrence.

Acknowledgement

This study was funded by an intramural researchgrant (WU# 02-65011d) from the Department ofClinical Investigation, Walter Reed Army MedicalCenter, Washington, DC.

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