ACLS
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Eccles, T. G.
Right arrow Articles by Francis, G. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Eccles, T. G.
Right arrow Articles by Francis, G. L.
Annals of Clinical & Laboratory Science 33:411-422 (2003)
© 2003 Association of Clinical Scientists

Erythropoietin and the Erythropoietin Receptor Are Expressed by Papillary Thyroid Carcinoma fromChildren and Adolescents.

Expression of Erythropoietin Receptor Might Be a Favorable Prognostic Indicator

Thomas G. Eccles1,3, Aneeta Patel1, Ajay Verma2, Diarmuid Nicholson4, Yvonne Lukes4, R. Michael Tuttle5 and Gary L. Francis1,3,4
1 Departments of Pediatrics and 2 Neurology, Uniformed Services University of the Health Sciences, Bethesda, Maryland;
3 Departments of Pediatrics and 4 Clinical Investigation, Walter Reed Army Medical Center, Washington, District of Columbia; and
5 Department of Endocrinology, Memorial Sloan Kettering Cancer Center, New York, New York

Address correspondence to Gary L. Francis MD PhD, Department of Pediatrics, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814, USA; tel 301 295 9716; fax 301 295 3898; email: gfrancis{at}usuhs.mil. The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or to reflect the opinions of the Uniformed Services University of the Health Sciences, Walter Reed Army Medical Center, the Department of the Army, or the Department of Defense.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgement
 References
 
Erythropoietin (EPO) and the EPO receptor (EPO-R) have been implicated in solid tumors of the brain, breast, kidney and female genital tract. Based on their expression by a variety of tumors, we hypothesized that EPO and/or EPO-R might be expressed by thyroid cancers. To test this, we determined EPO and EPO-R expression by immunohistochemistry in 17 papillary thyroid carcinomas (PTC) from children and adolescents. Only a minority of PTC (4/17, 24%) expressed EPO, and there were no significant differences between the PTC that did or did not express EPO. In contrast, EPO-R was detected in the majority of PTC (11/17, 65%). The average tumor size (1.5 ± 0.8 cm), MACIS score (3.6 ± 0.2) and risk of recurrence (0/11) for the EPO-R(+) PTC were significantly less than those for PTC that failed to express EPO-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 express EPO-R, a finding associated with favorable prognostic indicators and a lower risk of recurrence.

(received 24 June 2003; accepted 2 July 2003)

Keywords: thyroid, cancer, erythropoietin, erythropoietin receptor


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Acknowledgement
 References
 
Erythropoietin (EPO) is a 34 kD glycoprotein hormone whose principal function is to regulate red blood cell production [1]. In response to hypoxia, EPO is secreted by the kidney and bound to the erythropoietin receptor (EPO-R) on red cell progenitors where it promotes differentiation and hemoglobin synthesis [1,2].

The EPO-R belongs to the growth hormone receptor super family along with granulocyte-colony stimulating factor (GCSF), granulocyte macrophage-colony stimulating factor (GMCSF), and several of the interleukins (IL) [3]. EPO induces homodimerization of EPO-R, activating receptor-associated Janus kinase 2 (JAK 2), and serving as a docking site for one of the signal transducers and activators of transcription (STAT 5) [4]. EPO-R activation appears to inhibit apoptosis rather than to affect the commitment of progenitor cells to the erythroid lineage [5].

Of major interest are newly discovered functions for EPO that extend beyond the formation of red blood cells. EPO protects a number of tissues from apoptosis, especially cells of the central nervous system [6]. In vitro, EPO protects neurons from glutamate-induced toxicity, and in vivo, EPO protects neurons against ischemia-induced cell death. In response to tissue hypoxia, the transcription factor, hypoxia-induced factor-1 (HIF-1), up-regulates the expression and stabilizes the mRNAs encoding EPO, fibroblast growth factor-2 (FGF-2), and vascular endothelial growth factor (VEGF), suggesting that EPO expression might be a generalized response to tissue hypoxia [8,9].

EPO and EPO-R have been implicated in a number of malignant conditions, only a few of which involve cells of erythroid lineage [10,11]. Novel but important roles for EPO and EPO-R have been identified in solid tumors of the brain, breast, kidney and female genital tract [12–16]. Several observations suggest these effects may be related to tissue hypoxia [12–14]. Hypoxia is present in virtually all solid neoplasms, and is associated with invasion, metastasis, resistance to therapy, and selection of cells with diminished apoptotic potential [17–24]. Basal and hypoxia-stimulated expression of EPO and EPO-R have been demonstrated in human breast cancers and cell lines, suggesting roles for EPO signaling in the adaptation of these neoplasms to hypoxia [12–14].

EPO-R immunostaining is increased in breast cancers compared to normal tissues, and is most intense in regions directly adjacent to the infiltrating edge of growing tumors. In addition, EPO-R expression is significantly greater in breast tumors with higher grade histology, tumor necrosis, lymphovascular invasion, lymph node metastases, and loss of hormone receptor expression [12–14]. In vitro, EPO stimulates tyrosine phosphorylation and proliferation of breast cancer cells, both of which are blocked by a neutralizing anti-EPO antibody [13].

Malignant tumors of the ovary and uterus also express EPO and EPO-R [16,25,26]. Treatment with anti-EPO antibody reduced the size of ovarian and uterine cancer xenografts in nude mice [16,25,26]. Immunohistochemical examination of the treated tumors revealed apoptotic death of malignant and endothelial cells [16].

Based on the expression of EPO and EPO-R by a variety of solid tumors, we hypothesized that EPO and/or EPO-R might be expressed by thyroid cancers. To our knowledge, no previous study has examined this possibility, nor has any study examined the potential impact of EPO and EPO-R expression on the clinical behavior of thyroid cancers. Our data indicate that EPO and EPO-R are expressed by papillary thyroid cancers (PTCs) from children and adolescents. The data show that PTCs that express EPO-R are smaller, have lower MACIS (metastasis-age-completeness-of-resection-invasion-size) scores, and a lower risk of recurrence.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Acknowledgement
 References
 
Human experimentation.  This study protocol was approved the Human Use Committee of the Department of Clinical Investigation, Walter Reed Army Medical Center, Washington, DC.

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

The extent of disease at diagnosis was classified by the method of DeGroot et al [39], and the metastasis-age-completeness-of-resection-invasion-size score (MACIS) [40]. According to DeGroot et al [39], Class 1 disease was confined to the thyroid gland; Class 2 involved the regional lymph nodes; Class 3 either extended beyond the capsule or was inadequately resected; and Class 4 had distant metastasis. Because all patients were <39 years of age, MACIS scores were calculated as 3.1 + (size x 0.3) + 1 (if incomplete resection) + 1 (if direct invasion) + 3 (if distant metastasis) [40].

As in previous studies, recurrence was defined as the appearance of new disease (identified by radioactive iodine scan or biopsy) in any patient who had been free of disease (no disease palpable or identified by radioactive iodine scan) for a period of 4 mo following initial therapy [41]. Serum thyroglobulin (Tg) values were determined on contemporary patients (normal range: 3 – 40 ng/ml, University of Southern California Clinical Laboratories, Los Angeles, CA). Levels <2 ng/ml were considered to indicate freedom from disease [42]. The clinical details for these and additional patients have been previously published [41].

Formalin-fixed, paraffin-embedded archived tumor blocks from 17 patients with PTC were available for study. Sufficient material was also available to examine EPO and EPO-R staining on regions of presumably "normal" thyroid tissue that were immediately adjacent to a few of the PTC. Adjacent "normal" tissue was identified in 4 samples that were stained for EPO and 6 samples that were stained for EPO-R. These are included in the text as presumably "normal" thyroid, but there were no truly normal thyroid glands available for this study.

Immunohistochemistry.  Tumor blocks were sectioned and stained with hematoxylin and eosin to confirm the diagnosis [43]. The sections immediately adjacent (5 µm) were deparaffinized (xylene) and rehydrated through a series of graded alcohol solutions. Endogenous peroxidase was inactivated (3% H2O2, 30 min) and antigen was retrieved in citrate buffer (pH 6.0, 30 min, 100°C, steamer). For EPO determination, tissue sections were sequentially incubated with primary monoclonal anti-EPO antibody (H-162, 1:50, Santa Cruz Biotechnology, Santa Cruz, CA), followed by preformed avidin-biotinylated horse radish peroxidase complex, diaminobenzidine (DAB), and hematoxylin counterstain using the Ventana Nexes automated immunostainer (Ventana DAB Detection Kit, Ventana Medical Systems, Tucson, AZ). Sections of normal human kidney were used as the positive control, and phosphate-buffered saline was substituted for the primary and secondary antibodies and used as the negative controls.

The intensity of EPO staining was based on the intensity of chromogen developed throughout the majority of each tumor. Staining intensity was determined by two blinded, independent examiners and graded as follows: 0 = absent, 1 = minimal, 2 = moderate, and 3 = intense. The inter-observer agreement was >95%, and the few discordant slides were graded by a third examiner. The two scores in agreement were then used as the final intensity grade. The same procedures were used to evaluate EPO-R expression; the primary antibody was directed against EPO-R (C-20, 1:50, Santa Cruz Biotechnology, Santa Cruz, CA).

Amplification by polymerase chain reaction.  Expression of EPO and EPO-R was confirmed by reverse transcription and polymerase chain reaction (RT-PCR) amplification of specific regions of the EPO and EPO-R messenger ribonucleic acids (mRNAs). These were then sequenced using dyedeoxy terminators and the ABI-Prism 377 Sequence Detection System (Applied Biosystems, Inc., Foster City, CA.). One µg of cDNA was reverse transcribed using the Impromptu RT kit (Promega, Inc., Madison, Wisconsin) at 42°C for 1 hr, followed by 15 min at 70°C. PCR was performed in optimized PCR buffer (25 µl) containing enhancer and stabilizer, along with DNTPs (Maxim Biotech), equal amounts of sense and antisense primers (10 pmol of each) and Platinum Taq polymerase (0.625 units, Invitrogen, Carlsbad, CA).

A 2-step PCR cycling regimen was used for EPO amplification. The parameters used included an initial denaturation step at 96°C for 5 min, followed by an annealing step at 63°C for 4 min, 30 amplification cycles at 94°C for 1 min, 63°C for 2 min, and a final extension at 72°C for 7 min. To increase detection sensitivity, 1 µl of this PCR product was transferred to a fresh PCR mixture for 7 additional cycles. A consistent amplicon was detected in all amplifications by 2 cycles.

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

To test the integrity of the RT-PCR process, an external positive control (Maxim Biotech, San Francisco, CA), an RT negative control, and expression of the internal housekeeping gene, glyceraldehyde-3-PO4 dehydrogenase (GAPDH) were performed for each RT reaction. The PCR products were resolved by electrophoresis on 2% agarose gels and visualized by ethidium bromide staining. The primers for EPO were designed using Primer Express (V2.0 Applied Biosystems, Foster City, CA) and the primers for EPO-R and GAPDH 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 ABI Prism 377 sequence detection instrument (Applied Biosystems, Inc., Foster City, CA). Amplified cDNA was extracted from the electrophoretic band corresponding to EPO using an UltrafreeTM DNA centrifugal filter device (Millipore, Bedford, MA). The gel containing this band was placed in the Gel Nebulizer sample cup and centrifuged (5,000 x g, 10 min). DNA was then re-amplified by PCR under the following conditions: 50 mM KCl; 20 mM Tris-HCl, pH 8.0; 2 mM MgCl2; 0.2 mM dNTP; 0.4 units TAQ polymerase; 0.2 µM forward and reverse primers; one cycle at 95°C for 5 min, 35 cycles at 95°C for 20 sec, one cycle at 60°C for 20 sec, and a final cycle at 72°C for 20 sec. Excess primers were removed (Microcon column, Amicon, Beverly, MA) and the PCR products were diluted (500 µl) and centrifuged (500 x g, 8 min). Samples were recovered, dried, suspended (10 µl H2O), and sequenced (BigDyeTM Terminator v 3.0 Cycle Sequencing Kit, Applied Biosystems Inc., Foster City, CA). Excess terminator dyes were removed (CentriSep Spin Column, Princeton Separations, Aldelphia, NJ), and the fluorescent fragments were separated and analyzed (Model 377, Applied Biosystems, Foster City, CA). The BLAST data base (National Library of Medicine, National Institutes of Health, Bethesda, MD) was then queried for sequence similarity.

Data analysis and statistical comparisons.  Statistical analyses were performed using SPSS for Windows 95 (Version 7.5, SPSS Inc., Chicago, IL). The proportion of PTCs and surrounding "normal" tissues that showed specific EPO and EPO-R immunostaining was compared using Fisher’s exact test. The proportion of PTCs that showed aggressive clinical behavior (invasion, metastasis, persistence, or recurrence) was then compared between EPO (+) and EPO (-) groups, and between EPO-R (+) and EPO-R (-) groups using Fisher’s exact test. The average MACIS score, tumor size, and time to recurrence were compared between EPO (+) and EPO (-) PTC, as well as between EPO-R (+) and EPO-R (-) PTC by ANOVA.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Acknowledgement
 References
 
The demographic features, tumor characteristics, details of treatment and outcome for the 17 patients in this study are shown in Tables 1Go and 2Go. The majority were female (n = 15, 88%) with either Class 1 (n = 8, 47%), Class 2 (n = 8, 47%), or Class 3 (n = 1, 6%) disease. Their ages ranged from 6 – 21 years (mean = 16.5 ± 4.5 yr), tumor sizes ranged from 0.7 – 7.5 cm in diameter (mean = 2.26 ± 1.8 cm), 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 the extent of surgery) and the majority (60%, 9/15 for whom details were available) received radioactive iodine ablation. The patients were followed for periods ranging from 0 – 118 mo (mean = 59 ± 42 mo) after the initial therapy. Only 3 patients developed recurrent disease (17 – 67 mo, mean = 37 ± 26 mo). Demographic features, extent of disease, and type of therapy are similar to those of the larger cohort previously studied by our group [41].


View this table:
[in this window]
[in a new window]
  		Table 1. Demographics, treatment and outcome for patients stratified according to EPO-R expression.
 

View this table:
[in this window]
[in a new window]
  		Table 2. PTC stratified according to EPO and EPO-R expression.
 
Typical immunostaining for EPO and EPO-R are shown in Fig. 1Go. Specific staining was seen in the cytoplasm of the PTC, but was absent from the surrounding presumably "normal" thyroid tissue in 4 samples stained for EPO and 6 samples stained for EPO-R. Although EPO was only detected in PTC and not in adjacent "normal" thyroid, the difference was not significant [4/17 PTC were EPO (+), 0/4 adjacent "normal" samples were EPO (+), p = 0.55]. However, EPO-R expression was more frequently detected in PTC than in presumably "normal" adjacent thyroid tissue [11/17 PTC were EPO-R (+) compared to 0/6 adjacent""normal" thyroid samples, p = 0.014].



View larger version (150K):
[in this window]
[in a new window]
  		Fig. 1. EPO and EPO-R immunostaining of PTC from children and adolescents. Sections were stained by immunoperoxidase for EPO and EPO-R. The presence and intensity of staining were graded from absent (grade 0) to intense (grade 3) for both EPO (panels A – D) and EPO-R (panels E – H). All photomicrographs are shown at 100x magnification.

 
Expression of EPO and EPO-R mRNA was confirmed by RT-PCR and is shown in Fig. 2Go. RNA with sufficient integrity to amplify the internal housekeeping gene, GAPDH, was successfully extracted from two EPO (+) PTC, two EPO (-) PTC, three EPO-R (+) PTC, and two EPO-R (-) PTC. These tissue samples were then used to determine the expression of EPO and EPO-R. As shown, EPO and EPO-R were detected by RT-PCR in all samples that stained for EPO and EPO-R. Conversely, all samples that failed to stain for EPO and EPO-R also failed to express EPO and EPO-R by RT-PCR. Neither EPO nor EPO-R was detected in any of the RT negative controls. Sequence analysis confirmed that the EPO signal corresponds to that of human EPO with a probability >99.9%.



View larger version (66K):
[in this window]
[in a new window]
  		Fig. 2. EPO and EPO-R amplification by RT-PCR. Total RNA was reverse transcribed and specific EPO (->), EPO-R ({Rightarrow}), and GAPDH (solid triangle) sequences were amplified. Lanes 1 – 6 contain PTC in which EPO was identified by immunohistochemistry. Lanes 7 – 12 contain PTC that failed to stain for EPO. Lanes 13 – 21 contain PTC in which EPO-R was detected by immunohistochemistry. Lanes 22 – 27 contain PTC that failed to stain for EPO-R. RT negative controls and GAPDH amplification are shown for each sample along with the results of EPO and EPO-R expression

 
Four of 17 (24%) PTC expressed EPO by immunostaining (Tables 1Go and 2Go). 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 were from young women with Class 1 or Class 2 disease. The majority (3/4, 75%) of these tumors were multifocal (a feature associated with a higher risk of recurrence), but none recurred.

The majority of PTC (n = 13, 76%) failed to express EPO by immunostaining (Tables 1Go and 2Go). 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 were unifocal (n = 7, a feature associated with a low risk of recurrence) but 3 recurred (23%). There were no significant differences in demographic features, extent of disease at diagnosis, treatment, or risk of recurrence between the EPO (+) and EPO (-) groups.

EPO-R was detected by immunostaining in the majority of PTC (11/17, 65%) and was either moderate (n = 7, 64%) or intense (n = 4, 36%) (Tables 1Go and 2Go). Most of these patients were young women (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 the majority (5/9 for whom details were available) received radioactive iodine. No patient developed recurrent 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] but the differences only approached statistical significance (Table 2Go).

Six PTC (6/17, 35%) failed to express EPO-R by immunostaining (Tables 1Go and 2Go). The majority of these patients were also young women (5/6, 83%) with multifocal tumors (5/6, 83%) that were Class 2 at diagnosis (n = 4, 67%). Surgical details were available for 5 of these patients, all of whom underwent total (4/5, 80%) or subtotal (1/5, 20%) thyroidectomy. Four (4/6, 67%) received radioactive iodine ablation. Over time, three patients (3/6, 50%) developed recurrent disease. Of interest, the average tumor size (1.5 ± 0.8 cm), MACIS score (3.6 ± 0.2) and risk of recurrence (0/11) for the EPO-R (+) PTC were significantly less than those for PTC that failed to express EPO-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).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Acknowledgement
 References
 
Erythropoietin (EPO) primarily functions to regulate red blood cell production. However, recent studies have shown that EPO and the EPO-R have important roles in solid tumors of the brain, breast, kidney and female genital tract [12–16]. Several observations suggest these effects might be related to tissue hypoxia [12–14]. Based on the expression of EPO and EPO-R by a variety of solid tumors, we hypothesized that EPO and/or EPO-R might be expressed by thyroid cancers. To our knowledge, no previous study has examined this possibility.

Our data show that the majority of PTC from children 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. The expression of EPO and EPO-R were confirmed by two independent methods: immunohistochemical staining and RT-PCR with sequence analysis. Although sufficient RNA could only be extracted from a small number of samples (4 for EPO and 5 for 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-R expression by thyroid cancers.

We did not detect EPO or EPO-R expression in any sample of presumably "normal" thyroid tissue adjacent to the PTC in this study. However, due to the small number of samples, we could only identify 4 surrounding areas of "normal" thyroid that had been stained for EPO. All 4 were negative, but this did not differ from the proportion of PTC that failed to express EPO [4/4 "normal" and 13/17 PTC were EPO (-), p = 0.55]. We were able to identify a sufficient number of surrounding "normal" sections stained for EPO-R to determine that EPO-R expression was more common in PTC (11/17) than in surrounding "normal" thyroid (0/6, p = 0.014). These data suggest that the expression of EPO-R and possibly EPO are features of PTC and not normal thyroid.

The impact of EPO expression on the clinical behavior of individual PTC was then examined. There were no significant differences in tumor size or the extent of disease at diagnosis between the PTC that did and did not expressed EPO. However, recurrence only developed in PTC that failed to express EPO (n = 3). The number of cases in each group was too small to achieve statistical significance. The patients with PTC were then stratified into two groups based on EPO-R expression. When compared to PTC that expressed EPO-R, PTC that failed 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 studies have shown that larger tumor size and MACIS score are associated with a greater risk of tumor recurrence [40,41]. In agreement with these observations, the PTC that failed to express EPO-R had a greater risk of recurrence (3/6, 50% vs 0/11, p = 0.029).

Previous studies of EPO and EPO-R expression in breast cancer showed that EPO-R immunostaining was increased in carcinomas compared to normal tissues, and most intense in areas directly adjacent to necrotic foci or at the infiltrating tumor edge [12–14]. EPO-R but not EPO staining, was also greater in tumors showing higher grade histology, tumor necrosis, lymphovascular invasion, lymph node metastases, and loss of hormone receptor expression [12,14]. In agreement with these studies, we only found EPO and EPO-R expression in PTC, and not in surrounding "normal" thyroid.

However, in contrast, our data suggest that expression of EPO-R is associated with favorable risk factors (smaller tumor size, lower MACIS scores, and lower recurrence risk). The reason for these differences is not clear. It is possible that EPO-R may have different functions in thyroid and breast cancer cells, but it is also possible that the differences could relate to the selection of patients in our study. Our interest is in thyroid carcinoma of childhood, and our tissue samples were obtained only from patients with well-differentiated PTC who were under 21 yr of age. For these patients, the prognosis is highly favorable [41]. It is possible that the impact of EPO-R expression might have been different had we examined poorly differentiated or anaplastic thyroid cancers.

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

Study of a larger number of more diverse patients is clearly indicated. In addition, the clinical data in our study are derived from a retrospective database [41]. Although treatments received by the patients were similar, they were not randomized and we have no information by which to determine why any individual patient was selected to receive a specific treatment. A randomized, prospective analysis of EPO and EPO-R expression would eliminate this potential bias.

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

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


   Acknowledgement
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgement
References
 
This study was funded by an intramural research grant (WU# 02-65011d) from the Department of Clinical Investigation, Walter Reed Army Medical Center, Washington, DC.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgement
 References
 

  1. Romanowski RR, Sytkowski AJ. The molecular structure of human erythropoietin. Hematol Oncol Clin North Am 1994;8:885–894.[Medline]
  2. Semenza GL, Traystman MD, Gearhart JD, Antonarakis SE. Polycythemia in transgenic mice expressing the human erythropoietin gene. Proc Natl Acad Sci USA 1989;86: 2301–2305.[Abstract/Free Full Text]
  3. Mulcahy L. The erythropoietin receptor. Semin Oncol 2001;28:19–23.[Medline]
  4. Fuste B, Serradell M, Escolar G, Cases A, Mazzara R, Castillo R, Ordinas A, Diaz-Ricart M. Erythropoietin triggers a signaling pathway in endothelial cells and increases the thrombogenicity of their extracellular matrices in vitro. Thromb Haemost 2002;88:678–685.[Medline]
  5. Uddin S, Kottegoda S, Stigger D, Platanias LC, Wickrema A. Activation of the Akt/FKHRL1 pathway mediates the antiapoptotic effects of erythropoietin in primary human erythroid progenitors. Biochem Biophys Res Commun 2000;275:16–19.[Medline]
  6. Sakanaka M, Wen TC, Matsuda S, Masuda S, Morishita E, Nagao M, Sasaki R. In vivo evidence that erythropoietin protects neurons from ischemic damage. Proc Natl Acad Sci USA 1998;95:4635–4640.[Abstract/Free Full Text]
  7. Siren AL, Fratelli M, Brines M, Goemans C, Casagrande S, Lewczuk P, Keenan S, Gleiter C, Pasquali C, Capobianco A, Mennini T, Heumann R, Cerami A, Ehrenreich H, Ghezzi P. Erythropoietin prevents neuronal apoptosis after cerebral ischemia and metabolic stress. Proc Natl Acad Sci USA 2001;98:4044–4049.[Abstract/Free Full Text]
  8. Rosenberger C, Mandriota S, Jurgensen JS, Wiesener MS, Horstrup JH, Frei U, Ratcliffe PJ, Maxwell PH, Bachmann S, Eckardt KU. Expression of hypoxia-inducible factor-1alpha and -2alpha in hypoxic and ischemic rat kidneys. J Am Soc Nephrol 2002;13:1721–1732.[Abstract/Free Full Text]
  9. Grimm C, Wenzel A, Groszer M, Mayser H, Seeliger M, Samardzija M, Bauer C, Gassmann M, Reme CE. HIF-1-induced erythropoietin in the hypoxic retina protects against light-induced retinal degeneration. Nat Med 2002;8:718–724.[Medline]
  10. Longmore GD, Lodish HF. An activating mutation in the murine erythropoietin receptor induces erythroleukemia in mice: a cytokine receptor superfamily oncogene. Cell 1991;67:1089–1102.[Medline]
  11. Winkelmann JC, Ward J, Mayeux P, Lacombe C, Schimmenti L, Jenkins RB. A translocated erythropoietin receptor gene in a human erythroleukemia cell line (TF-1) expresses an abnormal transcript and a truncated protein. Blood 1995;85:179–185.[Abstract/Free Full Text]
  12. Acs G, Zhang PJ, Rebbeck TR, Acs P, Verma A. Immunohistochemical expression of erythropoietin and erythropoietin receptor in breast carcinoma. Cancer 2002; 95:969–981.[Medline]
  13. Arcasoy MO, Amin K, Karayal AF, Chou SC, Raleigh JA, Varia MA, Haroon ZA. Functional significance of erythropoietin receptor expression in breast cancer. Lab Invest 2002;82:911–918.[Medline]
  14. Acs G, Acs P, Beckwith SM, Pitts RL, Clements E, Wong K, Verma A. Erythropoietin and erythropoietin receptor expression in human cancer. Cancer Res 2001;61:3561–3565.[Abstract/Free Full Text]
  15. Westenfelder C, Baranowski RL. Erythropoietin stimulates proliferation of human renal carcinoma cells. Kidney Int 2000;58:647–657.[Medline]
  16. Yasuda Y, Musha T, Tanaka H, Fujita Y, Fujita H, Utsumi H, Matsuo T, Masuda S, Nagao M, Sasaki R, Nakamura Y. Inhibition of erythropoietin signalling destroys xenografts of ovarian and uterine cancers in nude mice. Br J Cancer 2001;84:836–843.[Medline]
  17. Kim CY, Tsai MH, Osmanian C, Graeber TG, Lee JE, Giffard RG, DiPaolo JA, Peehl DM, Giaccia AJ. Selection of human cervical epithelial cells that possess reduced apoptotic potential to low-oxygen conditions. Cancer Res 1997;57:4200–4204.[Abstract/Free Full Text]
  18. Yu JL, Rak JW, Carmeliet P, Nagy A, Kerbel RS, Coomber BL. Heterogeneous vascular dependence of tumor cell populations. Am J Pathol 2001;158:1325–1334.[Abstract/Free Full Text]
  19. Rak JW, St Croix BD, Kerbel RS. Consequences of angiogenesis for tumor progression, metastasis and cancer therapy. Anticancer Drugs 1995;6:3–18.[Medline]
  20. Folkman J. Tumor angiogenesis: therapeutic implications. N Engl J Med 1971;285:1182–6.
  21. Folkman J. Tumor angiogenesis. Adv Cancer Res 1985;43: 175–203.[Medline]
  22. Folkman J, Hanahan D. Switch to the angiogenic phenotype during tumorigenesis. Princess Takamatsu Symp 1991;22:339–347.[Medline]
  23. Folkman J. The role of angiogenesis in tumor growth. Semin Cancer Biol 1992;3:65–71.[Medline]
  24. Dachs GU, Tozer GM. Hypoxia modulated gene expression: angiogenesis, metastasis and therapeutic exploitation. Eur J Cancer 2000;36:1649–1660.
  25. Spurlin GW, Van Nagell JR, Jr., Parker JC, Jr., Roddick JW, Jr. Uterine myomas and erythrocytosis. Obstet Gynecol 1972;40:646–651.[Medline]
  26. Shimosato Y, Kameya T, Hirohashi S. Growth, morphology, and function of xenotransplanted human tumors. Pathol Annu 1979;14:215–257.
  27. Fenton C, Anderson J, Lukes Y, Dinauer CA, Tuttle RM, Francis GL. Ras mutations are uncommon in sporadic thyroid cancer in children and young adults. J Endocrinol Invest 1999;22:781–789.[Medline]
  28. Fenton CL, Patel A, Tuttle RM, Francis GL. Autoantibodies to p53 in sera of patients with auto-immune thyroid disease. Ann Clin Lab Sci 2000;30:179–183.[Abstract]
  29. Fenton C, Patel A, Dinauer C, Robie DK, Tuttle RM, Francis GL. The expression of vascular endothelial growth factor and the type 1 vascular endothelial growth factor receptor correlate with the size of papillary thyroid carcinoma in children and young adults. Thyroid 2000; 10:349–357.[Medline]
  30. Fenton CL, Lukes Y, Nicholson D, Dinauer CA, Francis GL, Tuttle RM. The ret/PTC mutations are common in sporadic papillary thyroid carcinoma of children and young adults. J Clin Endocrinol Metab 2000;85:1170–1175.[Abstract/Free Full Text]
  31. Fenton CL, Patel A, Burch HB, Tuttle RM, Francis GL. Nuclear localization of thyroid transcription factor-1 correlates with serum thyrotropin activity and may be increased in differentiated thyroid carcinomas with aggressive clinical course. Ann Clin Lab Sci 2001;31:245–252.[Abstract/Free Full Text]
  32. Gupta S, Patel A, Folstad A, Fenton C, Dinauer CA, Tuttle RM, Conran R, Francis GL. Infiltration of differentiated thyroid carcinoma by proliferating lymphocytes is associated with improved disease-free survival for children and young adults. J Clin Endocrinol Metab 2001;86: 1346–1354.[Abstract/Free Full Text]
  33. Patel A, Fenton C, Ramirez R, Dinauer CA, Tuttle RM, Nikiforov YE, Francis GL. Tyrosine kinase expression is increased in papillary thyroid carcinoma of children and young adults. Front Biosci 2000;5:A1–9.[Medline]
  34. Patel A, Jhiang S, Dogra S, Terrell R, Powers PA, Fenton C, Dinauer CA, Tuttle RM, Francis GL. Differentiated thyroid carcinoma that express sodium-iodide symporter have a lower risk of recurrence for children and adolescents. Pediatr Res 2002;52:737–744.[Medline]
  35. Patel A, Fenton C, Terrell R, Powers PA, Dinauer C, Tuttle RM, Francis GL. Nitrotyrosine, inducible nitric oxide synthase (iNOS), and endothelial nitric oxide synthase (eNOS) are increased in thyroid tumors from children and adolescents. J Endocrinol Invest 2002;25:675–683.[Medline]
  36. Patel A, Straight AM, Mann H, Duffy E, Fenton C, Dinauer C, Tuttle RM, Francis GL. Matrix metalloproteinase (MMP) expression by differentiated thyroid carcinoma of children and adolescents. J Endocrinol Invest 2002;25:403–408.[Medline]
  37. Shah R, Banks K, Patel A, Dogra S, Terrell R, Powers PA, Fenton C, Dinauer CA, Tuttle RM, Francis GL. Intense expression of the b7–2 antigen presentation coactivator is an unfavorable prognostic indicator for differentiated thyroid carcinoma of children and adolescents. J Clin Endocrinol Metab 2002;87:4391–4397.[Abstract/Free Full Text]
  38. Straight A, Patel A, Fenton C, Dianuer C, Tuttle RM, Francis G. Thyroid carcinomas that express telomerase follow a more aggressive clinical course for children and adolescents. J Endocrinol Invest 2002;25:302–308.[Medline]
  39. DeGroot LJ, Kaplan EL, McCormick M, Straus FH. Natural history, treatment, and course of papillary thyroid carcinoma. J Clin Endocrinol Metab 1990;71:414–424.[Abstract/Free Full Text]
  40. Hay ID, Bergstralh EJ, Goellner JR, Ebersold JR, Grant CS. Predicting outcome in papillary thyroid carcinoma: development of a reliable prognostic scoring system in a cohort of 1779 patients surgically treated at one institution during 1940 through 1989.Surgery 1993;114:1050–1057; discussion 1057–1058.[Medline]
  41. Welch Dinauer CA, Tuttle RM, Robie DK, McClellan DR, Svec RL, Adair C, Francis GL. Clinical features associated with metastasis and recurrence of differentiated thyroid cancer in children, adolescents and young adults. Clin Endocrinol 1998;49:619–628.[Medline]
  42. Mazzaferri EL, Kloos RT. Is diagnostic iodine-131 scanning with recombinant human TSH useful in the follow-up of differentiated thyroid cancer after thyroid ablation? J Clin Endocrinol Metab 2002;87:1490–1498.[Abstract/Free Full Text]
  43. Hedinger C, Williams ED, Sobin LH. The WHO histological classification of thyroid tumors: a commentary on the second edition. Cancer 1989;63:908–911.[Medline]



This article has been cited by other articles:


Home page
 Eur J EndocrinolHome page
P Iglesias and J J Diez
Thyroid dysfunction and kidney disease
Eur. J. Endocrinol., April 1, 2009; 160(4): 503 - 515.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
S. Elliott, L. Busse, M. B. Bass, H. Lu, I. Sarosi, A. M. Sinclair, C. Spahr, M. Um, G. Van, and C. G. Begley
Anti-Epo receptor antibodies do not predict Epo receptor expression
Blood, March 1, 2006; 107(5): 1892 - 1895.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
M. E. Hardee, M. O. Arcasoy, K. L. Blackwell, J. P. Kirkpatrick, and M. W. Dewhirst
Erythropoietin Biology in Cancer
Clin. Cancer Res., January 15, 2006; 12(2): 332 - 339.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Eccles, T. G.
Right arrow Articles by Francis, G. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Eccles, T. G.
Right arrow Articles by Francis, G. L.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS