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Epidermal Growth Factor Receptor as a Therapeutic Target in Human Thyroid Carcinoma: Mutational and Functional Analysis

Department of Medical Oncology (C.S.M., J.N., G.F., N.M.), Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115; Department of Pathology (V.K., E.C.), School of Medicine, Aristotle University of Thessaloniki, Thessaloniki 54006, Greece; Massachusetts Eye and Ear Infirmary (V.P.), Harvard Medical School, Boston, Massachusetts 02114; Department of Pathology (E.S., G.F., S.T.-B.), University of Athens, Athens 11527, Greece; and Laboratory of Environmental Mutagenesis and Carcinogenesis (G.V.), Institute of Biology, National Centre for Scientific Research"Demokritos", GR-15310, Athens, Greece

Address all correspondence and requests for reprints to: Constantine S. Mitsiades, M.D., Ph.D., Department of Medical Oncology, Dana-Farber Cancer Institute, Mayer Building, Room M555, 44 Binney Street, Boston, Massachusetts 02115. E-mail: Constantine_Mitsiades{at}dfci.harvard.edu.

	Abstract

Top Abstract Introduction Patients and Methods Results Discussion References

 
Context: The epidermal growth factor receptor (EGFR), a transmembrane tyrosine kinase (TK) receptor that mediates proliferation and survival signaling, is expressed in a wide variety of normal and neoplastic tissues. EGFR inhibitors have produced objective responses in patients with non-small-cell lung carcinomas harboring activating EGFR TK domain somatic mutations.

Objective and Methods: Because the EGFR pathway has been reported to be important for the pathophysiology of thyroid carcinoma, we investigated the expression and mutational status of EGFR in 14 thyroid carcinoma cell lines as well as its functional role by evaluating their in vitro sensitivity to AEE788, a new dual-family EGFR/ErbB2 and vascular endothelial growth factor receptor TK inhibitor. We also evaluated the mutational status, mRNA and protein expression, as well as phosphorylation status of EGFR in a panel of thyroid carcinoma specimens.

Results: EGFR expression and phosphorylation in the thyroid carcinoma cell lines and tissue specimens were present but not stronger than in noncancerous thyroid tissue. EGFR TK domain mutations were detected in two of 62 histological specimens (3.2%) but not in cell lines. All thyroid carcinoma cell lines were significantly less sensitive (IC₅₀ at least 25-fold higher) in vitro to AEE788 than a primary culture of EGFR-mutant lung carcinoma cells.

Conclusions: Thyroid carcinoma cells overall are poorly responsive to clinically relevant concentrations of AEE788 in vitro. The presence of EGFR-activating TK domain mutations may identify a small minority of thyroid cancer patients that may benefit from EGFR inhibitors, but additional preclinical evidence of efficacy is needed.

	Introduction

Top Abstract Introduction Patients and Methods Results Discussion References

 
APPROXIMATELY 25,700 PATIENTS are diagnosed annually with thyroid cancer in the United States (1), leading to 1500 deaths annually and suggesting that novel, effective therapies are needed. The epidermal growth factor receptor (EGFR) is frequently overexpressed in breast, lung, colon, ovarian, and brain tumors, suggesting that it may be a valid target for novel anticancer therapies (2). Gefitinib (Iressa; AstraZeneca Pharmaceuticals, Wilmington, DE), erlotinib (Tarceva; Genentech, San Francisco, CA), and AEE788 (Novartis Pharma, Basel, Switzerland) disrupt EGFR kinase activity. Gefitinib and erlotinib have resulted in objective responses in patients with non-small-cell lung cancers (NSCLC). However, the response rate to any EGFR inhibitor (EGFRI) is strongly associated with the presence of somatic activating mutations within the EGFR tyrosine kinase (TK) domain (3, 4, 5, 6), which activate Akt and signal transducer and activator of transcription-5 and promote cell survival (7). Overall, fewer than 10% of NSCLCs in the United States carry such mutations (although the incidence may be 30% in Japan) (3, 4).

Both EGF and EGFR have been detected in thyroid carcinomas, and this pathway has been proposed to be important for thyroid carcinoma proliferation and metastasis (8). In the present study, we found that EGFR TK mutations are rare, and EGFR is usually not overexpressed or overactivated in thyroid carcinomas, even in those with TK mutations. Overall, our study did not demonstrate a pivotal role for the EGFR pathway in thyroid carcinomas.

	Patients and Methods

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Human tissues

Formalin-fixed and paraffin-embedded thyroid carcinoma specimens from 62 patients followed at the Evgenidion Hospital, Athens, Greece [14 males and 48 females; 43 papillary, two follicular, eight anaplastic, and nine medullary; age, 16–74 yr (mean ± SD, 46.4 ± 15.6 yr); 25 stage I, four stage II, 14 stage III, and 19 stage IV patients] were studied in accordance with the Declaration of Helsinki principles and Institutional Review Board policies.

Cell lines

The papillary thyroid carcinoma (PTC) cell lines BHP-2, BHP-5, BHP-7, BHP-10, BHP-14, BHP-17, BHP-18, and BHP-19 were generous gifts of Dr. Jerome M. Hershman (West Los Angeles Veterans Affairs Medical Center, Los Angeles, CA). The SW579 (papillary) and TT (medullary) cell lines were from American Type Culture Collection (Manassas, VA). The NPA (papillary), FRO (anaplastic), ARO (anaplastic), and WRO (follicular) cell lines were a generous gift of Dr. James A. Fagin (University of Cincinnati School of Medicine, Cincinnati, OH) (9).

A primary culture of NSCLC cells, known to carry an AlaGlu substitution at codon 750 and a 746–748 deletion (kindly provided by Dr. Bruce E. Johnson, Dana-Farber Cancer Institute), served as positive control.

Reagents

AEE788 is a potent, combined inhibitor of both EGF and vascular endothelial growth factor receptor (VEGFR) TK family members and was provided by Dr. Peter Traxler (Novartis Pharma). The chemical structure and biochemical characteristics of AEE788 are described in Ref. 10 and published as supplemental data on The Endocrine Society’s Journals Online web site at http://jcem.endojournals.org.

Relative quantification of EGFR mRNA expression was performed with real-time relative quantification of the cDNA amplification products using TaqMan-FAM-MGB assays and automated analysis in an Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA). More details on this method as well as on direct EGFR TK domain DNA sequencing are described in the supplemental data. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric survival assay was performed as previously described (11).

Immunohistochemistry (IHC)

For IHC, we used the EGFR PharmDX kit (Dako, Carpinteria, CA), the anti-nuclear factor-B (anti-NF-B) p65 subunit MAB3026 (Chemicon, Temecula, CA), and the following antibodies from Cell Signaling (Beverly, MA): anti-phospho-EGFR Tyr992, anti-phospho-EGFR Tyr1068, anti-phospho-EGFR Tyr1173, and anti-phospho-Akt Ser473.

Complete description of the IHC protocol and evaluation of immunostaining is available in the supplemental data. Tissue sections were evaluated by two expert pathologists (V.K. and S.T.-B.).

Statistical analysis

Statistical significance was examined by a two-way ANOVA, followed by Duncan’s post hoc test, and by Pearson’s ² test and likelihood ratio for qualitative IHC results. In all analyses, P < 0.05 was considered statistically significant.

	Results

Top Abstract Introduction Patients and Methods Results Discussion References

 
Thyroid carcinoma cell lines are refractory to AEE788 in vitro

All thyroid carcinoma cell lines tested were resistant to AEE788 at pharmacologically relevant concentrations (as high as 1 µM) for 96 h (Fig. 1A). The IC₅₀ values for all thyroid cell lines were at least 2 µM. For comparison, EGFR-mutant NSCLC cells were very sensitive to AEE788 (IC₅₀ = 75 nM).

View larger version (36K): [in this window] [in a new window]   FIG. 1. A, Resistance of thyroid carcinoma cells, including anaplastic cells, to the growth-inhibitory effect of EGFR inhibition at clinically relevant concentrations in vitro. Percent cell viability (mean ± SD) was quantified by metallothionein in a panel of thyroid carcinoma cell lines treated with the EGFR/VEGFR inhibitor AEE788 for 96 h. All thyroid carcinoma cells lines (SW579, NPA, BHP-2, BHP-5, BHP-7, BHP-10, FRO, ARO, WRO, and TT) were resistant to AEE788 treatment within this range of clinically relevant concentrations. A primary culture of NSCLC cells was very sensitive to even sub-micromolar concentrations of AEE788 and served as a positive control. All cells were grown in DMEM (BioWhittaker, Walkersville, MD) with 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% fetal bovine serum (Invitrogen, Carlsbad, CA). Experiments were repeated at least three times, and each experimental condition was repeated at least in quadruplicate wells in each experiment. Data reported are average values ± SD from representative experiments. B, Relative quantification of EGFR mRNA expression in thyroid carcinoma cell lines and primary tumor specimens, compared with NCTT (used as calibrator). After RNA extraction and RT, relative quantification of EGFR expression was assessed with real-time PCR of the cDNA amplification products using a TaqMan-FAM-MGB assay in an Applied Biosystems 7500 Real-Time PCR System supported by the SDS software version 1.3.0 (default protocol, 40 cycles). A TaqMan-FAM-MGB assay for ß-glucuronidase was used as endogenous reference. Relative quantification analysis was performed automatically with threshold set at 0.1. The cutoff for overexpression was set at greater than 2-fold increase. For analysis, threshold was set at 0.1. Data shown are from one representative of three equivalent runs. None of the samples was found to overexpress EGFR (i.e. >2-fold higher than NCTT). FRO cells (derived from an anaplastic carcinoma) and one primary papillary thyroid carcinoma (PTC-03, representing a tumor with favorable histology) expressed higher EGFR levels than NCTT but was still less than 2-fold of NCTT. Cell lines derived from papillary carcinomas (NPA and all BHP lines as well as SW579 cells from a poorly differentiated papillary tumor with squamous differentiation), one medullary carcinoma (TT), and one of the two anaplastic carcinoma cell lines (ARO) expressed relatively lower EGFR mRNA levels than NCTT. EGFR mRNA levels in all primary anaplastic and in 11 of 12 papillary carcinomas were lower than in NCTT. Among primary tumors, medullary carcinomas expressed the lowest EGFR mRNA levels. No signal was obtained from the no-template (negative) control.

Only one of 11 cell lines (FRO) and one of 20 primary carcinomas (a PTC with favorable histology) expressed EGFR mRNA levels higher than noncancerous thyroid tissue (NCTT) (Fig. 1B), but even these samples did not reach the cutoff of more than 2-fold increase to be defined as overexpression.

EGFR expression and phosphorylation patterns in thyroid carcinomas

Detailed presentation of the IHC results is provided in the supplemental data. Briefly, EGFR immunostaining was focally observed in NCTT and PTCs. No difference was observed in EGFR expression or phosphorylation at Tyr1068 and Tyr1173 between NCTT and PTC (supplemental Fig. 1). Phosphorylation at EGFR Tyr992 was stronger in NCTT than PTCs (P < 0.001).

There was no correlation between unfavorable histology and EGFR expression or phosphorylation at Tyr992 or Tyr1068. However, unfavorable histology PTCs exhibited stronger EGFR phosphorylation at Tyr1173 (P = 0.006).

Medullary and anaplastic carcinomas exhibited only faint immunopositivity for EGFR PharmDX, EGFR phosphorylation at Tyr992, Tyr1173, and (with the exception of two strongly immunopositive medullary carcinomas) Tyr1068.

Akt phosphorylation and NF-B activation patterns in thyroid carcinomas

Immunostaining for pAkt was present in 86% of our PTC panel, with stronger intensity in the invasive than in the central part of the tumor and in unfavorable than in favorable histology PTCs.

Activated NF-B immunostaining was practically absent from NCTT (supplemental Fig. 1). By contrast, activated NF-B was detected in 20 of 28 PTCs (71%) and was more common in unfavorable than in favorable histology PTCs (P = 0.015).

The intensity of immunostaining for activated NF-B was positively associated with EGFR phosphorylation at Tyr992 (P = 0.002), Tyr1173 (P = 0.008), and Tyr1068 (P = 0.038) and was not related to EGFR expression.

Finally, pAkt and activated NF-B immunostaining were highly correlated (P < 0.0001).

EGFR TK domain mutations in thyroid carcinoma

We sequenced exons 18, 19, and 21 (where mutations cluster in NSCLC) of the EGFR gene in our panel of 14 thyroid carcinoma cell lines. No mutations were detected.

Sequencing of 62 thyroid carcinoma specimens revealed two specimens with mutations (3.2%). One was a G2500A base substitution (GTGATG) in codon 834 (exon 21), resulting in a valinemethionine substitution in a stage I PTC from a 24-yr-old female (supplemental Fig. 2). The same tumor specimen exhibited a silent polymorphism at codon 838 (G2514A). The second mutation was a A2038G base substitution (AGTGGT) in codon 695 (exon 18), resulting in a serineglycine substitution in a stage III PTC from a 56-yr-old female (supplemental Fig. 2).

Thyroid carcinoma specimens with EGFR TK domain mutations do not exhibit evidence of EGFR pathway overactivation

EGFR TK mutations in NSCLC cells promote EGF-induced receptor autophosphorylation and Akt activation (7). Moreover, the antiapoptotic transcription factor NF-B is activated by EGF and Akt (11, 12). We thus compared the presence of EGFR, phospho-EGFR, pAkt, and activated NF-B between the two specimens carrying EGFR TK domain mutations and the wild-type specimens in our panel. We detected immunostaining for these EGFR pathway mediators in the EGFR-mutant thyroid carcinoma specimens, but it was not more intense than in noncancerous thyrocytes from the same patients or in carcinoma cells from EGFR wild-type specimens (Fig. 2), suggesting that it is unlikely that the detected mutations lead to constitutive overactivation of the EGFR pathway.

View larger version (126K): [in this window] [in a new window]   FIG. 2. Thyroid carcinoma specimens with EGFR TK domain mutations do not exhibit evidence of EGFR pathway overactivation. Immunohistochemical evaluation of EGFR pathway activation in thyroid carcinoma tissue sections from an EGFR wild-type (wt) papillary carcinoma (left column) and the two EGFR-mutant papillary carcinomas (a stage I PTC from a 24-yr-old female carrying a Val834Met substitution (middle column), and a stage III PTC from a 56-yr-old female carrying a Ser695Gly substitution (right column) for EGFR-DX (A–C), phospho-EGFR Tyr992 (D–F), phospho-EGFR Tyr1068 (G–I), phospho-EGFR Tyr1173 (J-L), phospho-Akt Ser473 (M–O), and activated NF-B p65 (P–R). A–C, EGFR PharmDX, with faint staining in B that can be recognized only at high power, in comparison with A. Overall, the wild-type specimen (A) had strong membranous EGFR PharmDX immunostaining, whereas each mutant specimen (B and C) had, on average, faint immunostaining. D–F, Phospho-EGFR Tyr992, with faint immunostaining in D (wild type), strong immunostaining in E, and absence of staining in F (<10% positive cells). G–I, Phospho-EGFR Tyr1068, with faint staining in G (wild type), more than 50% of cells with faint staining in H, and more than 50% with strong staining in I. J–L, Phospho-EGFR Tyr1173, with strong immunostaining in more than 50% of cells in J (wild type) and K and absence of staining in L. M–O, pAKT, with tumor cells uniformly exhibiting cytoplasmic staining. In M (wild type), the higher presence of cytoplasmic pAKT immunostaining in tumor compared with and nonneoplastic follicular cells can be easily recognized. The two mutant specimens (N and O) exhibit less pronounced pAkt immunostaining. P–R, Activated NF-B, with strong immunostaining in P (wild type) and Q and absence of immunostaining in R. Overall, the two EGFR-mutant papillary carcinomas do not exhibit evidence of higher activation of the EGFR signaling pathway and its downstream mediators compared with wild-type tumors. Magnification, x400 (B, D, and E); x200 (all others).

	Discussion

Top Abstract Introduction Patients and Methods Results Discussion References

EGFRIs such as gefitinib and erlotinib have been approved for the treatment of NSCLC. NSCLCs that responded to EGFRIs harbored somatic mutations in the EGFR TK domain (3, 4), which promote receptor autophosphorylation, Akt and STAT activation, and resistance to conventional chemotherapy (7), highlighting EGFR as a therapeutic target for any malignancies harboring such mutations. All thyroid carcinoma cell lines in our panel were EGFR wild type and, in agreement with the NSCLC data (where EGFR wild-type cells are EGFRI refractory), were refractory to the EGFRI AEE788 in vitro.

Recent studies have suggested that gefitinib and AEE788 have in vitro activity against thyroid carcinomas, including anaplastic ones (13, 14, 15, 16). However, the concentrations of EGFRIs used in those studies (>10 µM, with IC₅₀ values in the range of 12–17 µM) were significantly higher than those (1 µM) used in our current study. Moreover, EGFR-mutant NSCLC cells were not used as controls in those studies, which would have given them an important perspective. We believe that the available evidence is yet insufficient to support the clinical use of EGFRIs as monotherapy for the treatment of aggressive thyroid carcinoma. Combination therapies using EGFRIs and conventional chemotherapy could be considered in an effort to improve efficacy; however, the disappointing results of similar clinical trials in NSCLC (17, 18) again suggest caution.

Overall, our study did not identify a significant correlation between EGFR expression or phosphorylation and thyroid carcinoma histology (with the exception of Tyr1173). However, immunostaining for pAkt and activated NF-B correlated strongly with unfavorable histology. We have previously reported that activated NF-B is present in thyroid carcinomas (21) and may represent a therapeutic target. Although we did detect a correlation between EGFR phosphorylation and strong immunostaining for pAkt and NF-B, it is obvious that pAkt and NF-B represent common downstream mediators of several other autocrine and paracrine signals that play an important role in thyroid cancer cell growth and survival, such as the Ret/Ras/Raf cascade (19), the IGF-I pathway (11), and the fibroblast growth factor pathway (20) and in cell motility and invasiveness, such as the hepatocyte growth factor/Met pathway (20). This suggests that Akt and NF-B may represent better therapeutic targets than EGFR for the treatment of thyroid cancer. Alternatively, a multitargeted inhibitor, such as AEE788, may have in vivo activity via inhibiting angiogenesis pathways, such as the VEGF pathway, which also plays a role in thyroid carcinoma pathophysiology (20), or other receptors (10, 16).

In conclusion, EGFR is usually not overexpressed or overactivated in thyroid carcinomas compared with adjacent nonmalignant thyroid tissue. Although the presence of EGFR-activating TK domain mutations in a minority of thyroid carcinomas may raise hope for clinical efficacy of EGFRIs in a carefully selected patient subset, our study did not demonstrate a pivotal role of the EGFR pathway even in this subgroup, suggesting that additional preclinical evidence of activity is still required before recommending such a treatment.

	Footnotes

 
Disclosure Summary: C.S.M. has received honoraria from Novartis Pharma for participation as consultant in scientific advisory meetings. All other authors have nothing to declare.

First Published Online July 5, 2006

Abbreviations: EGFR, Epidermal growth factor receptor; EGFRI, EGFR inhibitor; IHC, immunohistochemistry; NCTT, noncancerous thyroid tissue; NF-B, nuclear factor-B; NSCLC, non-small-cell lung cancer; PTC, papillary thyroid carcinoma; TK, tyrosine kinase; VEGFR, vascular endothelial growth factor receptor.

Received January 10, 2006.

Accepted June 23, 2006.

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Top Abstract Introduction Patients and Methods Results Discussion References

 

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This Article Abstract Full Text (PDF) Supplemental Data Submit a related Letter to the Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Mitsiades, C. S. Articles by Mitsiades, N. Search for Related Content PubMed PubMed Citation Articles by Mitsiades, C. S. Articles by Mitsiades, N. Related Collections Thyroid Endocrine Oncology