Epidermal growth factor receptor gene mutations in papillary thyroid carcinoma

Authors


Abstract

Recent studies have indicated that somatic mutations in the epidermal growth factor receptor (EGFR) gene have been identified in a subset of patients with nonsmall-cell lung cancer (NSCLC) and are associated with sensitivity to the EGFR-tyrosine-kinase inhibitors. These mutations have been reported to be almost exclusively found in a pulmonary adenocarcinoma subgroup of NSCLC, with a low frequency in other solid tumors. We describe a patient with advanced-stage papillary thyroid carcinoma (PTC) whose disease had been diagnosed as pulmonary adenocarcinoma at first, and who had a marked response to the EGFR-tyrosine-kinase inhibitor, gefitinib. An in-frame deletion in exon 19 that eliminated 4 amino acids at positions 746 through 750, which is one of the common drug-sensitive mutations in pulmonary adenocarcinoma, and a serine-to-proline substitution at codon 752, were found in a tumor specimen of the patient. We subsequently searched for mutations in the EGFR tyrosine kinase domain in primary tumors from 23 patients with PTC, and drug-sensitive mutations commonly observed in pulmonary adenocarcinoma were found in 7 of these patients. Our observation of a high frequency of the EGFR-activating mutations in PTC suggests that the EGFR mutation may be an important event in the development of PTC. EGFR gene amplification, also considered to be a predictor of response to EGFR-tyrosine-kinase inhibitors, was evaluated by fluorescence in situ hybridization (FISH); however, only 1 FISH-positive tumor was detected. Our data suggest that EGFR-tyrosine-kinase inhibitors may deserve consideration in the treatment of a subset of patients with PTC, just as with pulmonary adenocarcinoma. © 2008 Wiley-Liss, Inc.

Thyroid cancer is the most common malignancy of the endocrine system, and more than 30,000 patients are diagnosed with thyroid cancer annually in the United States.1 A major histological subtype of thyroid malignancy is papillary thyroid carcinoma (PTC), which is associated with a good prognosis. However, ∼10–30% of patients thought to be disease-free after initial treatment will develop recurrence and/or metastases. Moreover, distant metastasis as the initial presenting diagnosis is not uncommon, with a frequency of 1–9%.2 As is the case for other malignancies, treatment of PTC with distant metastases is usually not curative. Standard treatment options for distant metastasis consist of radioactive iodine therapy, thyroid-stimulating hormone suppression, external beam radiotherapy and palliative resection. Given that the 10-year survival rate is <20% once distant metastases occur,3 more effective treatment options are needed.

Understanding of the signal-transduction pathways that facilitate neoplastic transformation and progression has led to the development of molecular-targeted anticancer agents. Epidermal growth factor receptor (EGFR) is a tyrosine kinase of the ErbB family that regulates signaling pathways for cellular proliferation and survival. Targeting EGFR as a means for anticancer therapy has been proposed on the basis of its ubiquitous expression in human solid tumors.4 Inhibition of kinase activation with small molecule drugs has proved to be an effective approach in the treatment of malignant tumors,5, 6 and EGFR-tyrosine-kinase inhibitors are approved for the therapy of nonsmall-cell lung cancer (NSCLC). Recent molecular studies of patients with marked responsiveness to EGFR-tyrosine-kinase inhibitors have revealed that the majority have tumors exclusively of adenocarcinoma histology and harboring activating mutations within the EGFR kinase domain that cause altered functions of the receptor.7, 8 We describe a patient with metastatic PTC whose disease had been initially diagnosed with a double cancer (pulmonary adenocarcinoma and PTC), and who had a marked response to the EGFR-tyrosine-kinase inhibitor, gefitinib. Analyses of the tumor specimens revealed that a subgroup of Japanese patients with PTC harbor the same EGFR-activating mutations observed in pulmonary adenocarcinoma. We also analyzed the EGFR gene copy number, recently reported to correlate with an improved response and survival with EGFR-tyrosine-kinase inhibitor treatment.9–11 The association among these biomarkers and BRAF mutations, which is prevalent and is considered by many to be a poor prognostic factor in PTC, is presented in this report.12–14

Case reports

The patient was a 65-year-old Japanese woman without a smoking history. She had been in good health until 4 months before admission, when she had a persistent headache. Evaluation at another facility revealed 2 solitary masses in the brain, multiple calcified nodular lesions in the thyroid gland and randomly-distributed multiple small nodules in both lungs, measuring <4 mm in diameter. Serum thyrogloblin concentration was not elevated. Pathologic examination of the specimens obtained by fine needle aspiration biopsy from the thyroid gland revealed a class V adenocarcinoma. Cytologic results of endobronchial washings were also consistent with adenocarcinoma, and she was admitted to Kyoto University Hospital for treatment.

Two lesions in the brain were diagnosed as symptomatic metastatic thyroid carcinomas, and she underwent stereotactic radiosurgery. She subsequently had a total thyroidectomy for the purpose of maximizing the effect of radioactive iodine therapy. Although the surgically-obtained tumor specimen revealed poorly differentiated PTC (Fig. 1a), treatment with radioactive iodine therapy was rejected by the patient.

Figure 1.

Results of histopathologic and genetic analyses of the case and radiographic images of the lung. High-power magnification of a tumor specimen of the thyroid (a) shows medium-sized atypical cells with irregular nuclei arranged in grand-like structure with dense fibrous tissue (hematoxylin and eosin stain). Malignant cells in the resected neck lymph node (b) also have a character of papillary growth pattern, surrounded by an abundant fibrous parenchyma (hematoxylin and eosin stain). (c) shows weakly positive immunoperoxidase staining for thyroglobulin of the neck lymph node. In (d), alignment of the EGFR kinase domain in DNA from the thyroid tumor showed the in-frame deletion mutation (2 peaks) and heterozygous missense mutations (black arrow) at nucleotide 2,254, resulting in amino acid substitutions. A chest radiograph revealed left upper lobe mass and diffuse small pulmonary nodules; both were considered to be derived from the thyroid carcinoma (e). 6 months after receiving gefitinib, the response was maintained (f).

A follow-up CT of the chest obtained 5 months after the thyroidectomy revealed a solitary pulmonary nodule in the left upper lobe, which was 25 mm in diameter. The lesion showed pleural indentation and spiculation and had different characteristics from other unchanged small nodules in both lungs. Nine months later, this dominant pulmonary lesion increased in size, from 25 to 40 mm, in contrast to the other small nodules, and an enlarged cervical lymph node was newly palpable. CT-guided biopsy of the solitary pulmonary mass was unsuccessful. She underwent lymphadenectomy, and pathologic examination of the resected node showed poorly differentiated adenocarcinoma; the primary site was not determined (Figs. 1b and 1c).

Considering the radiographic appearance and the aggressiveness during the follow-up period, the patient seemed to have experienced sequelae of 2 epithelial malignancies (PTC and pulmonary adenocarcinoma). Primary PTC had been diagnosed at presentation, and subsequently, pulmonary adenocarcinoma in the left lung progressed with at least cervical lymph node involvement. The source of the metastatic lesions in the brain and both lungs could not be determined. Therapeutic intervention was recommended for possible pulmonary adenocarcinoma because of its fast growth rate. After discussion of further management, she sought EGFR-activating mutation testing of the tumor for reference.

On the basis of the results of molecular analysis (Fig. 1d), the patient received the oral EGFR-tyrosine-kinase inhibitor, gefitinib. Both the rapidly growing solitary lesion and the multiple stable smaller pulmonary lesions showed partial response, as assessed by response evaluation criteria in solid tumors15 (Figs. 1e and 1f). The intracranial lesions were stable in size after stereotactic radiosurgery. She has remained on medication gefitinib and has been without evidence of disease progression for more than 12 months.

Material and methods

EGFR mutational analysis

The study was approved by the Ethics Committee at the Graduate School of Medicine of Kyoto University. Written informed consent was obtained from each patient. Formalin-fixed, paraffin-embedded tissue blocks were used for DNA analysis.

With respect to the EGFR mutational analyses of the tumor specimens, the dense calcifications commonly observed in PTC often make the thin-slice tissue sections necessary for microdissection technically difficult. To detect EGFR mutations in a large background of wild-type EGFR genes derived from normal cells, we therefore adopted the peptic nucleic acid-locked nucleic acid (PNA-LNA) polymerase chain reaction (PCR) clamp method, according to protocols described elsewhere.16 Briefly, PNA clamp primers inhibit amplification of the wild-type sequence, and LNA probes are used to specifically detect mutant sequences in the presence of wild-type sequences. A synergic effect of these primers causes specific PCR amplification of mutant sequences. Specific PNA-LNA probe sets to each mutation were developed to cover >95% of the EGFR mutations previously reported in Japan.17 PCR products from both conventional PCR and the PNA-LNA PCR were purified and directly sequenced on an ABI PRISM 3100 automated capillary sequencer in both the sense and antisense directions.

Fluorescence in situ hybridization analysis

EGFR gene copy numbers were investigated by fluorescence in situ hybridization (FISH), using the LSI EGFR SpectrumOrange/CEP 7 SpectrumGreen probe (Vysis, Tokyo, Japan). Reagents and methods used in the EGFR FISH analysis have been described elsewhere.18 We analyzed at least 60 individual tumor-cell nuclei to assess copy numbers of the EGFR and chromosome 7 probes as controls.

The patients were classified into 6 categories according to EGFR copy numbers per cell and the frequency of tumor cells with a specific number of copies of the EGFR gene and the chromosome 7 centromere, as described by Cappuzzo et al.11 Samples with high polysomy or high gene copy numbers were classified as FISH-positive.

BRAF mutational analysis

Because the thymine-to-adenine missense mutation at nucleotide 1,799 in exon 15 of the BRAF gene that leads to the substitution of a valine for a glutamic acid at the number 600 residue (Val600Glu) represents the vast majority of reported BRAF-activating mutations in thyroid carcinoma, this hotspot was chosen for BRAF mutational analysis. The template DNA extracted from the specimens manually microdissected with a needle was amplified by PCR. The purified PCR products were sequenced on an automated sequencer. Primers amplified a 231 base pair fragment of BRAF gene exon 15 (forward: 5′-AAACTCTTCATAATG CTTGCTCTG; reverse: 5′-GGCCAAAAATTTAATCAGTGGA) at an annealing temperature of 56° C13.

Results

Results of EGFR-activating mutation

Both the resected cervical lymph node and thyroid tumor specimens were subjected to analysis. We searched for somatic mutations of the EGFR kinase domain in the cervical lymph node and found an in-frame deletion in exon 19 that eliminated 4 amino acids at positions 746 to 750, which is one of the common drug-sensitive mutation regions in pulmonary adenocarcinoma (Fig. 1d). A serine-to-proline substitution at codon 752 in exon 19 was also observed. The same EGFR mutation was detected in the thyroid tumor, suggesting that both specimens originated from the same epithelial malignancy. The resected PTC was expected to be the primary lesion; however, differential diagnosis of the primary pulmonary adenocarcinoma with widespread metastasis to the brain, lung, lymph node and thyroid gland could not be excluded.

Results of further analysis for cases with papillary thyroid carcinoma

To obtain a more comprehensive view of EGFR-activating mutations and other associated biomarkers, we further analyzed 22 PTCs. All patients were native-Japanese of Asian ethnicity and without other malignancies, a family history of thyroid cancer or a history of radiation exposure. To analyze certificated primary thyroid cancer, tumor specimens of patients with no evidence of distant metastasis at the time of surgery were selected. There was a female predominance (F:M = 17:6), reflecting the epidemiologic nature of the disease. The mean age of the patients at diagnosis was 61.9 years (range, 23 to 77 years). Fifteen patients had conventional PTC. There were 6 patients with poorly differentiated PTC and 2 with follicular variant PTC. Surgical specimens of 6 patients with stage I, 2 stage II, 14 stage III and 1 stage IV (the presented case) were evaluated.

EGFR-activating mutations commonly encountered in pulmonary adenocarcinoma were detected in 7 of the 23 patients (30.4%; Table I). Three tumors had small in-frame deletions, including codons 746–750 in exon 19. Four tumors contained the Leu858Arg missense mutation in exon 21. There was no correlation between these EGFR mutation status and clinicopathologic parameters: The frequency of EGFR mutation was 2 of 6 (33%) in men and 5 of 17 (29%) in women; 4 of 15 (27%) in conventional PTC and 3 of 8 (38%) in other histologies; 3 of 8 (38%) in stage I/II tumors and 4 of 15 (27%) in stage III/IV tumors. (p values not significant; Fisher's exact test).

Table I. Observed Somatic Mutations in Exons 19 and 21 of EGFR in Patients With Papillary Thyroid Carcinoma
Patient No.GenderAgeStage1HistologyDNA alterationProtein productBRAF (Val600Glu) mutation
  • 1

    American Joint Committee on Cancer, TNM classification 6th edition, 2002.

1 (presented case)F66IVPoorly dif. PTCDeletion of 15 nucleotides (2,235–2,249) and substitution of C for T at nucleotide 2,254In-frame deletion (746–750) and amino acid substitution (S752P)Yes
2F74IIIConventional PTCDeletion of 15 nucleotides (2,236–2,250)In-frame deletion (746–750)No
3M77IIIConventional PTCDeletion of 15 nucleotides (2,235–2,249)In-frame deletion (746–750)Yes
4F43IConventional PTCSubstitution of G for T at nucleotide 2,573Amino acid substitution (L858R)No
5F76IIPoorly dif. PTCSubstitution of G for T at nucleotide 2,573Amino acid substitution (L858R)Yes
6F76IIFollicular variant PTCSubstitution of G for T at nucleotide 2,573Amino acid substitution (L858R)No
7M73IIIConventional PTCSubstitution of G for T at nucleotide 2,573Amino acid substitution (L858R)No

Among 23 patients analyzed, 19 patients (82.6%) had tissue available for EGFR FISH analysis. We could not obtain appropriate tumor tissue slides from 3 patients because of dense calcifications and from 1 patient because of a technical error. One patient's tumor had high EGFR gene copy numbers (gene amplification; Fig. 2a). The remaining 18 samples of patients were classified as FISH-negative (Fig. 2b). Disomy for the EGFR gene was present in 6 of 19 patients (31.6%), low trisomy in 10 patients (52.6%) and low polysomy in 2 patients (10.5%).

Figure 2.

EGFR gene copy number analysis by fluorescence in situ hybridization (FISH) and BRAF mutational analysis. FISH with EGFR (red signals) and chromosome 7 (green signals) probes in PTC sections showed clustered gene amplification in tumor classified as FISH-positive (a). A FISH-negative (b) tumor represented with 2 gene copies. Sequence of wild-type and mutant BRAF are shown in (c). The thymine-to-adenine transition introduces a substitution of valine-to-glutamic acid at codon 600 (Val600Glu, black arrow).

BRAF mutation status was assessed in all tumor samples. A BRAF mutation (Val600Glu) was detected in 12 of 23 cases (52.2%, Fig. 2c). We compared associations between EGFR mutation status and BRAF mutation status in each tumor; however, there was no significant association between these mutations (p > 0.5; Fisher's exact test).

Discussion

Our investigation has revealed that the EGFR-activating mutations commonly found in pulmonary adenocarcinoma are also identified in a proportion of PTC specimens from Japanese patients. Many types of somatic mutations in the EGFR gene have been reported in NSCLC, but few are known to be sensitive to an EGFR-tyrosine-kinase inhibitor. The most common of these drug-sensitive mutations are in-frame deletions in exon 19 that include 4 amino acids (leucine-arginine-glutamate-alanine: codon 747 to 750) in the tyrosine kinase domain and a thymine-to-guanine transversion that results in an arginine-for-leucine substitution at amino acid 858 in exon 21, together representing ∼90% of EGFR mutations.19, 20 The precise mechanism by which mutant-bearing tumors are more sensitive to an EGFR-tyrosine-kinase inhibitor remains to be evaluated. Various groups have indicated that the EGFR mutant (deletion in exon 19 or point mutation in exon 21) cells exhibit ligand-independent activation and prolonged tyrosine-kinase activity by ligand stimulation.7, 8, 21 In cell lines bearing mutations, signal transducers and activators of transcription (STAT) 3 and 5 and Akt, which are all included in prosurvival pathways, are selectively activated. Suppression of these survival signals leads to rapid apoptosis, because these cells are completely dependent on mutant EGFR pathway for maintenance of their malignant phenotype.22 Although biologic aggressiveness differs between pulmonary adenocarcinoma and PTC, parental cells of these tumors have the same developmental properties. The formation of both thyroid gland and lung originates from budding of the embryonic foregut and requires thyroid transcription factor 1 for organ maturation.23, 24 The EGFR mutation may be one of the main genetic alterations for oncogenic transformation to PTC.

Previous studies have failed to identify the EGFR-activating mutations in thyroid cancer.25, 26 There may be 2 reasons. One is that our study was focused on a specific population, i.e., Asian patients with PTC. As for NSCLC, the EGFR-activating mutations are observed in a particular subset of patients; specifically, mutations are more common in never-smokers, women, Asians and patients with adenocarcinoma. Lack of a smoking history, the most common carcinogen of NSCLC, implies the possibility that other genetic and environmental factors contribute to the development of EGFR mutations. Interestingly, one cohort study of thyroid cancer revealed that risk was independently and positively related to the female gender and Asian race, as well as radiation of the neck, a family history of thyroid disease and a history of a goiter.27 Comprehensive studies should augment our understanding of the epidemiology of these 2 types of epidermal malignancies with the same EGFR mutational profile. Another reason is a problem of sample heterogeneity and methodology. Several authors have reported that NSCLC, including adenocarcinoma, is often heterogeneous at the molecular level, with a varying mutational status within the same tumor.28 The heterogeneity of the EGFR mutational status requires careful assay interpretation. Moreover, nontumor cells, such as stromal, vascular and inflammatory cells inevitably exist in the sample. During conventional PCR, DNA derived from both normal cells and tumor cells may be equally amplified and sequencing results may reflect the ratio of tumor cells recovered for PCR amplification. We adopted the PNA-LNA PCR clamp method, one of the highly sensitive detection methods for specific EGFR mutations to overcome these issues. On further analysis of mutational status in different histologic subtypes of thyroid cancer and patients with various ethnicities, the means of molecular assay is a matter of fundamental importance.

In this study, we found that the increase in copy number is rare, i.e., 1 of 19 analyzed samples. Although there is no consistent literature on EGFR gene copy number in PTC, the frequency seems to be low compared with that of NSCLC (30 to 45%).9–11 With respect to anaplastic thyroid carcinoma, however, one small study on Korean patients revealed that increased EGFR gene copy number was observed in 14 of 23 analyzed samples, suggesting that EGFR gene gain may correlate with biological dedifferentiation process in primary thyroid carcinoma.26

BRAF belongs to the family of serine-threonine kinase RAF proteins, which are effectors of the MAPK pathway. RAS binding and subsequent recruitment to the cell membrane activates MEK, which in turn promotes activation and intracellular translocation of ERK. Somatic mutation within the kinase domain of BRAF was reported to facilitate constitutive activation of BRAF kinase and has been found in various human solid tumors.12 In multiple studies, a high prevalence of the BRAF mutation (∼45%) was documented in sporadic PTC.13, 14 In this study, the prevalence of 53.2% is compatible to these reports and EGFR-activating mutations were found to be independent to the BRAF mutation. Provided that in pulmonary adenocarcinoma cell lines mutant EGFR can activates the protein kinase C/Akt and the STAT 3/5 signal cascade,22 independent occurrence of these mutations implies that multiple and different alterations of cellular differentiation, proliferation, and/or the survival signal cascade are involved in the development of human PTC.

Chromosomal rearrangement involving the RET receptor tyrosine kinase gene is another characteristic feature of PTC and RNA-based methods are mandatory for its detection.29–31 Paraffin-embedded samples, with expected profound degeneration of RNA, were only available in this study and the association between RET rearrangement and EGFR mutation status needs to be studied further.

In summary, we have described a patient with PTC whose tumor harbored the EGFR-activating mutation and showed a marked response to an EGFR-tyrosine-kinase inhibitor. A high frequency of the EGFR-activating mutations in PTC of Japanese patients suggests an important role in tumorigenesis and may offer a new strategy in the treatment of these tumors.

Acknowledgements

The authors acknowledge assistance of the Mitsubishi Chemical Medience Cooperation and SRL, Inc. for tumor DNA analyses with the PNA-LNA PCR clamp method and FISH analyses. They thank Dr. Toshiaki Manabe and Dr. Hirokazu Kotani for help with performance of histopathologic studies.

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