Expression of c-erbB-3 and c-erbB-4 proteins in papillary thyroid carcinomas.

c-erbB-3 and c-erbB-4 protein expression was analyzed using immunohistochemistry in 138 fresh-frozen thyroid tissue samples from 106 patients, including 56 cases of papillary thyroid carcinoma. Increased expression of c-erbB-3 and c-erbB-4 proteins was observed in papillary carcinomas compared to nonneoplastic thyroid tissue. No amplifications of the c-erbB-3 and c-erbB-4 genes were detected. Coexisting overexpression of epidermal growth factor receptor, c-erbB-2, c-erbB-3, and c-erbB-4 was demonstrated in 36 (64%) of 56 papillary thyroid carcinomas. These findings suggest a common regulatory mechanism for the type I (epidermal growth factor receptor-related) receptors in papillary thyroid carcinomas and provide numerous possibilities for functional receptor interactions.

Expression and alternative splicing of c-ret RNA in papillary thyroid carcinomas.

Somatic rearrangements of the ret receptor tyrosine kinase have been consistently reported in papillary thyroid carcinomas (PTC). It is unclear whether the expression of wild-type c-ret may also be implicated in thyroid tumorigenesis. We studied ret mRNA expression in PTC from Norwegian patients. Using RT-PCR, wild-type ret mRNA was detected in all of 22 PTC and in a PTC cell line. c-ret mRNA was clearly overexpressed in PTC as compared to non-neoplastic thyroid tissue. Hybridization using ret exon DNA dot blot arrays and complex cDNA probes confirmed expression of ret RNA in thyroid biopsies. In accordance with the RNA data, Western immunoblotting showed evidence of wild-type Ret protein in PTC. Rearrangements generating the ret/PTC oncogenes co-existed with c-ret mRNA in PTC. Multiple alternative ret splicing variants were detected in PTC. Four novel ret splicing events were found in the region encoding the extracellular domain. The open reading frames of these transcripts were all in-frame with the Ret tyrosine kinase domain. In the central ret mRNA region encoding the cysteine-rich, transmembrane, and main tyrosine kinase domains, no evidence of alternative splicing was detected. Two alternative splice events were detected in the ret mRNA encoding the C-terminal part of Ret protein harboring tyrosine residues important for Ret signaling, excluding exon 19, or retaining intron 19, respectively. Ribonuclease protection assays confirmed the presence of ret alternative splicing events in thyroid biopsies. We conclude that in addition to ret/PTC rearrangements, wild-type c-ret mRNA and alternatively spliced ret transcripts are present in PTC. Transcriptional up-regulation and post-transcriptional mechanisms of c-ret RNA processing may contribute to differences in expression of Ret protein observed in PTC compared to non-neoplastic thyroid tissue.

Evidence against involvement of APC mutation in papillary thyroid carcinoma.

Papillary thyroid carcinoma (PTC) is one of several tumours associated with familial adenomatous polyposis (FAP), an inherited tumour syndrome which appears to result from germ-line mutation of the APC tumour suppressor gene. Here we investigate the possibility that somatic mutation of APC might play a role in sporadic PTC. 16 cases of PTC together with matched normal tissue were examined by single-strand conformation polymorphism (SSCP) analysis, concentrating on the mutation cluster region (MCR) of the APC gene (codons 1286-1513). No evidence of mutation was observed in any sample. We conclude that APC mutation, at least in the MCR, is not a significant causal mechanism in sporadic PTC.

Enhanced expression of EGF receptor and low frequency of ras mutations in X-ray-induced rat thyroid tumours.

Radiation is recognized as a carcinogenic factor for the thyroid gland. In this experimental study, oncogene expression was investigated in radiation-induced rat thyroid tumours. Forty 3-month-old Wistar rats received X-ray-irradiation to the neck region; 40 animals were untreated controls. After 14 months, thyroid tumours had developed in 25 of the 29 irradiated animals still alive; 76% of these tumours were considered malignant. No tumours developed in controls. Mutations of codons 12-13 and 59-63 of H-, K- and N-ras were analysed by PCR-SSCP (single-strand conformation polymorphism analysis) and sequencing of DNA from thyroid tissue. SSCP indicated a ras mutation frequency of 8%, but only one K-ras codon 12 (Gly-Cys) mutation was confirmed by sequencing. Protooncogene expression was analysed by mRNA slot blot hybridization analysis and immunohistochemistry. K-ras mRNA expression and EGF receptor mRNA and protein expression were significantly increased in the irradiated animals compared with controls, and in tumours versus nontumour tissue. This study of radiation-induced rat thyroid tumours demonstrates that ras expression may be subject to changes apart from activating mutations. Increased expression of EGF receptor in the tumours parallels the situation in human thyroid cancer.

Difference in patterns of Met expression in papillary thyroid carcinomas and nonneoplastic thyroid tissue.

Met protein is a tyrosine kinase receptor for hepatocyte growth factor (HGF). c-Met has morphogenic, mitogenic, and motogenic properties and is overexpressed in many solid tumors. We studied c-met mRNA and protein expression in papillary thyroid carcinomas and nonneoplastic thyroid tissue. The c-met mRNA was detected in all biopsies by reverse transcriptase-polymerase chain reaction and by hybridization of complex cDNA probes to a c-met-specific DNA fragment in a dot blot array. Immunohistochemistry on fresh frozen biopsies showed Met protein localized along the basal cell membrane of normal thyrocytes in 32 of 35 nonneoplastic thyroid tissue specimens, sometimes associated with weak cytoplasmic reactivity but without apical cell membrane staining. In papillary carcinomas an increased Met protein expression was seen, comprising a cytoplasmic (33 of 49) and apical cell membrane (24 of 49) immunoreactivity, whereas only 1 of 49 biopsies showed basal cell membrane staining. A 145-kDa Met-specific band was detected by Western immunoblotting on protein extracts from papillary carcinomas. The tight junction protein zona occludens-1 (ZO-1), studied by immunohistochemistry, was weakly expressed along the apical cell membrane in 10 nonneoplastic biopsies. In contrast, increased and cytoplasmic/apical membranous ZO-1 immunostaining was seen in 11 of 15 papillary carcinomas. Nuclear ZO-1 staining was present in a few papillary carcinomas with partial dedifferentiation. The concomitant overexpression and subcellular redistribution of Met and ZO-1 proteins indicate a change in cell polarity in papillary carcinomas compared to nonneoplastic thyroid tissue. These observations may reflect an important feature of the tumorigenesis of papillary thyroid carcinomas. No significant association was found between semiquantitative immunohistochemical assessment of Met protein and clinical parameters in papillary carcinoma patients.

Demonstration of a TGF-alpha-EGF-receptor autocrine loop and c-myc protein over-expression in papillary thyroid carcinomas.

Autocrine growth stimulation has been identified in several types of human cancer. In the present study we wanted to establish whether autocrine stimulation of the epidermal-growth-factor receptor (EGF-r) by its ligand, transforming growth factor alpha (TGF-alpha) occurs in thyroid neoplasia. We examined 190 fresh, frozen thyroid tissue samples from 70 patients by immunohistochemistry with antibodies to EGF-r, TGF-alpha, c-erbB-2 and c-myc. EGF-r expression was detected in 17 out of 19 papillary carcinomas, TGF-alpha expression in 10, and c-erbB-2 expression in 15. No papillary carcinoma expressed TGF-alpha without also expressing EGF-r. Concomitant expression of EGF-r, TGF-alpha and c-erbB-2 was seen in 7 papillary carcinomas. No EGF-r, TGF-alpha or c-erbB-2 immunopositivity was found in normal-appearing thyroid tissue (25 cases), whereas a few of the non-neoplastic lesions (colloid goitres and diffuse hyperplasias) expressed either EGF-r or TGF-alpha. c-myc expression was detectable in all tissue samples, and expression was invariably nuclear. Increased expression was observed in 10 out of 19 papillary carcinomas, and 8 of these also co-expressed EGF-r and TGF-alpha. In situ hybridization confirmed the presence of TGF-alpha mRNA in tumour epithelium of TGF-alpha-immunopositive samples. The concomitant expression of EGF-r, TGF-alpha and TGF-alpha mRNA gives evidence for a TGF-alpha-EGF-r autocrine loop in thyroid papillary carcinomas. The increased c-myc expression may reflect the proliferative advantage of these tumours.

Expression of heregulins and associations with the ErbB family of tyrosine kinase receptors in papillary thyroid carcinomas.

Heregulin (Hrg) growth factors are natural ligands for ErbB3 and ErbB4. Because these receptors are involved in papillary thyroid carcinomas, we studied expression of Hrgs in fresh-frozen thyroid tissue and analyzed for possible coexpressions among the 4 members of the ErbB family of growth factor receptors and Hrgs in papillary carcinomas. Immunohistochemistry for the Hrg precursor isoform (134 biopsies from 101 patients) showed nuclear immunostaining in 83% of papillary carcinomas but not in normal thyroid tissue. Cytoplasmic immunopositivity for the Hrg precursor isoform was moderate or strong in 78% of papillary carcinoma specimens and weak in 13% of normal thyroid tissue samples. Western blot for the Hrg precursor isoform showed the expected protein band of approximately 70 kDa in papillary carcinomas, but not in non-neoplastic thyroid biopsies. Whereas weak cytoplasmic immunostaining for the mature Hrg alpha, beta1, and beta3, was present in 48, 38, and 51% of papillary carcinomas, respectively, normal thyroid tissue samples were negative. Hrg mRNA was present in both tumor and nontumor tissue, with evidence of increased mRNA expression in 5 of 12 papillary carcinomas. RT-PCR of hrg mRNA, with subsequent DNA sequencing, confirmed the presence of hrg alpha, beta1, beta2, and beta3 mRNA in papillary carcinomas. In 55 papillary carcinomas, increased cytoplasmic immunostaining of the ErbB2 and ErbB3 receptors was significantly associated with each other and with cytoplasmic epidermal growth factor receptor (EGFR) immunoreactivity, indicating a common regulatory mechanism. Cytoplasmic staining for Hrg beta3 was significantly associated with ErbB3 immunostaining, indicating this receptor as the cognate one. The overexpression and nuclear localization of the Hrg precursor isoform were not associated with the expression of ErbB-receptors. This may reflect an unknown mechanism of action, possibly independent of the ErbB receptor system.

Expression of c-erbB-2 protein in papillary thyroid carcinomas.

c-erbB-2 protein expression was investigated immunohistochemically in frozen thyroid tissue specimens from 42 patients using a polyclonal sheep antibody. c-erbB-2 immunoreactivity was detected in 12 out of 17 papillary carcinomas, while no c-erbB-2 protein immunostaining was seen in cases of follicular adenoma (five cases), follicular carcinoma (five cases) or medullary carcinoma (one case), nor in cases of non-neoplastic tissue, including normal thyroid tissue from tumour-bearing glands. RNA was extracted from 51 thyroid tissue samples from 34 of the above patients, and c-erbB-2 mRNA was analysed by slot-blot hybridisation. c-erbB-2 mRNA was detectable in all samples, but papillary carcinomas and lymph node metastases showed significantly higher levels of c-erbB-2 mRNA compared to non-neoplastic tissue. The present demonstration of positive c-erbB-2 immunostaining in papillary thyroid carcinomas is contradictory to previous findings on formalin-fixed, paraffin-embedded material, and emphasises the importance of tissue quality for c-erbB-2 protein detection.

[Diagnostic molecular biology in solid tumors--thyroid gland]. / Diagnostisk molekylaerbiologi ved solide svulster--thyreoidea.

Thyroid cancer is not a common disease. It includes tumour types of great diversity in clinical course and molecular basis. Mutations of TSH-receptor, rearrangements of ret proto-oncogene, and altered expression of other tyrosine kinase growth factor receptors are characteristics of the follicular neoplasias and papillary carcinomas, while undifferentiated tumours harbour p53 mutations. Knowledge acquired to date has led to an increased understanding of thyroid growth and tumour development, but it has had no significant impact on diagnostic and treatment measures. On the other hand, the C-cell derived medullary carcinomas include familial cases where identification of germ-line ret mutations provides the basis for prophylactic thyroidectomy in affected individuals.

Cytoplasmic localization of EGF receptor in papillary thyroid carcinomas: association with the 150-kDa receptor form.

We have previously reported that papillary thyroid carcinomas show an increased expression of EGFR mRNA and protein, compared to non-tumorous thyroid tissue. EGFR immunoreactivity was localized to the cytoplasm as well as to the membrane in papillary carcinomas. To further study EGFR protein expression in human thyroid tissue, we performed immunohistochemistry and Western blots of 64 different thyroid tissue samples from 36 patients, including 23 patients with papillary carcinomas. Two receptor forms were identified in human thyroid tissue, a 170-kDa and a 150-kDa form. The 150-kDa receptor form was more pronounced in papillary carcinomas, while the 170-kDa receptor was the dominant form in non-malignant thyroid tissues. Predominance of the 150-kDa EGFR in the tumour samples was associated with strong cytoplasmic EGFR staining. EGFR gene structure, protein synthesis and maturation were found to be normal. Immunoprecipitation and Western-blot analysis of EGFR from the human thyroid SGHTL-34 cells after increased ligand concentration showed a decreased amount of the mature 170-kDa receptor and a relative increase in the 150-kDa receptor. We have previously demonstrated the presence of a TGF-alpha-EGFR autocrine loop in papillary thyroid carcinomas, and this may explain increased receptor turnover and accumulation of a cytoplasmic degradation product.