Malignant Thyroid Tissues Research Articles

A phase II study of gefitinib in patients with advanced thyroid cancer

6020 Background: There are few effective treatments for locally advanced or metastatic thyroid cancers that are refractory to radioactive iodine (RaI). The epidermal growth factor receptor (EGFR) is highly expressed on normal and malignant thyroid tissue, and has been associated with a worse prognosis in well-differentiated thyroid cancer. The EGFR tyrosine kinase inhibitor gefitinib is effective in inhibiting the growth of thyroid cancer cells in vitro. This study sought to determine the efficacy and tolerability of gefitinib in patients with advanced thyroid cancer. Methods: In this open-label phase II trial, patients with RaI-refractory, locally advanced or metastatic thyroid cancer received 250mg of daily gefitinib. Anaplastic and medullary thyroid carcinoma were considered RaI refractory based on histology. The primary endpoint was overall response rate. Secondary endpoints were toxicity, progression-free survival (PFS), overall survival (OS), and tumor marker responses. Tumor markers consisted of serum thyroglobulin (Tg) in the differentiated cancers and serum calcitonin and carcinoembryonic antigen in medullary thyroid cancer. Results: A total of 27 patients were enrolled. 59% were male and the median age was 65. Histologic subtypes included papillary (41%), follicular (22%), anaplastic (19%), medullary (15%), and Hürthle cell carcinoma (4%). There were no objective tumor responses among the 25 patients evaluated, at which point the trial was halted. However, 32% of patients had objective reductions in tumor volume that did not meet criteria for PR. 48%, 24%, and 12% of patients had stable disease (SD) after 3, 6, and 12 months of treatment. Median PFS and OS were 3.7 [95% CI; 1.8, 5.7] and 17.5 months [9.2, not yet reached]. Five patients with SD had a decrease in Tg to <90% of baseline level that was maintained for at least 3 months. Gefitinib was well tolerated, with 11% of patients experiencing grade 3 toxicity. Conclusions: Gefitinib therapy did not result in any objective tumor responses. However, reductions in tumor volume, falling Tg levels, and prolonged SD in a subset of patients may indicate biologic activity. The OS of 17.5 months was superior to historical rates in this population. However, the significance of this is difficult to interpret in the absence of a randomized comparison group. No significant financial relationships to disclose.

Stability of mRNA and carcinogenesis: The role of AUF1 in thyroid carcinoma progression

AUF1/ heterogeneous nuclear ribonucleoprotein D (hnRNPD) is an adenylate uridylate-rich elements (ARE) binding protein. AUF1 regulates the mRNA stability of many genes related to growth regulation, including proto-oncogenes, growth factors, cytokines and cell cycle regulatory genes. AUF1 is expressed in kidney, liver, lymphoid tissues and melanocytes, and is involved in apoptosis, tumorigenesis and development by its interactions with ARE-bearing mRNAs. Here we provide evidence for a role of AUF1 during thyroid carcinogenesis. We show that in human thyroid carcinoma cell lines AUF1 affects cell cycle progression and provide evidence for an altered nuclear/ cytoplasmic ratio of AUF1 expression during cell division. Moreover, siRNA knock-down of AUF1 resulted in increased cellular levels of c-Myc oncoprotein, the down-regulation of wild type p53 and elevated expression of cyclin-dependent kinase inhibitors. These findings correlate with a decreased proliferation rate of thyroid carcinoma cells. Subcellular fractionation and immunohistochemistry of thyroid tissues revealed significantly increased cytoplasmic expression of AUF1 in benign and malignant tissues as compared to normal thyroid tissues. Furthermore, logarithmic nuclear/ cytoplasmic ratio of total AUF1 expression in normal, goiter, adenoma and follicular thyroid carcinoma decreased along with tissue malignancy. Thus, we hypothesize that cytoplasmic AUF1 promotes thyroid carcinogenesis by affecting the half-life of mRNA transcripts contributing to thyroid carcinoma progression. This is the first report of the expression analysis of AUF1 in normal, benign and malignant thyroid tissues implicating AUF1 as a potential novel marker for human thyroid carcinoma.

A multi-class predictor based on a probabilistic model: application to gene expression profiling-based diagnosis of thyroid tumors

BackgroundAlthough microscopic diagnosis has been playing the decisive role in cancer diagnostics, there have been cases in which it does not satisfy the clinical need. Differential diagnosis of malignant and benign thyroid tissues is one such case, and supplementary diagnosis such as that by gene expression profile is expected.ResultsWith four thyroid tissue types, i.e., papillary carcinoma, follicular carcinoma, follicular adenoma, and normal thyroid, we performed gene expression profiling with adaptor-tagged competitive PCR, a high-throughput RT-PCR technique. For differential diagnosis, we applied a novel multi-class predictor, introducing probabilistic outputs. Multi-class predictors were constructed using various combinations of binary classifiers. The learning set included 119 samples, and the predictors were evaluated by strict leave-one-out cross validation. Trials included classical combinations, i.e., one-to-one, one-to-the-rest, but the predictor using more combination exhibited the better prediction accuracy. This characteristic was consistent with other gene expression data sets. The performance of the selected predictor was then tested with an independent set consisting of 49 samples. The resulting test prediction accuracy was 85.7%.ConclusionMolecular diagnosis of thyroid tissues is feasible by gene expression profiling, and the current level is promising towards the automatic diagnostic tool to complement the present medical procedures. A multi-class predictor with an exhaustive combination of binary classifiers could achieve a higher prediction accuracy than those with classical combinations and other predictors such as multi-class SVM. The probabilistic outputs of the predictor offer more detailed information for each sample, which enables visualization of each sample in low-dimensional classification spaces. These new concepts should help to improve the multi-class classification including that of cancer tissues.

Diagnosis of Suspicious Thyroid Nodules Using Four Protein Biomarkers

Fine-needle aspiration (FNA) cytology, a standard method for thyroid nodule diagnosis, cannot distinguish between benign follicular thyroid adenoma (FTA) and malignant follicular thyroid carcinoma (FTC). Previously, using expression profiling, we found that a combination of transcript expression levels from DDIT3, ARG2, C1orf24, and ITM1 distinguished between FTA and FTC. The goal of this study was to determine if antibody markers used alone or in combination could accurately distinguish between a wider variety of benign and malignant thyroid lesions in fixed sections and FNA samples. Immunohistochemistry was done on 27 FTA, 25 FTC, and 75 other benign and malignant thyroid tissue sections using custom antibodies for chromosome 1 open reading frame 24 (C1orf24) and integral membrane protein 1 (ITM1) and commercial antibodies for DNA damage-inducible transcript 3 (DDIT3) and arginase II (ARG2). FNA samples were also tested using the same antibodies. RNA expression was measured by quantitative PCR in 33 thyroid lesions. C1orf24 and ITM1 antibodies had an estimated sensitivity of 1.00 for distinguishing FTA from FTC. For the expanded analysis of all lesions studied, ITM1 had an estimated sensitivity of 1.00 for detecting malignancy. Because all four cancer biomarkers did well, producing overlapping confidence intervals, not one best marker was distinguished. Transcript levels also reliably predicted malignancy, but immunohistochemistry had a higher sensitivity. Malignant cells were easily detected in FNA samples using these markers. We improved this diagnostic test by adding C1orf24 and ITM1 custom antibodies and showing use on a wider variety of thyroid pathology. We recommend that testing of all four cancer biomarkers now be advanced to larger trials. Use of one or more of these antibodies should improve diagnostic accuracy of suspicious thyroid nodules from both tissue sections and FNA samples.

Expression of Aurora kinases in human thyroid carcinoma cell lines and tissues

The Aurora kinases are involved in the regulation of cell cycle progression, and alterations in their expression have been shown to associate with cell malignant transformation. In the present study, we demonstrated that human thyrocytes express all 3 Aurora kinases (A, B and C) at both protein and mRNA level and this expression is cell cycle-regulated. An increase in the protein level of the 3 kinases was found, with respect to normal human thyrocytes (HTU5), in the human cell lines derived from follicular (FTC-133), papillary (B-CPAP) and anaplastic (8305C) thyroid carcinomas, but not in cells derived from a follicular adenoma (HTU42). These observations were mirrored in RT-PCR experiments for Aurora-A and B. In contrast, Aurora-C mRNA levels were not significantly different among the different cell types analyzed, suggesting that posttranscriptional mechanism(s) modulate its expression. The expression at the protein level of all 3 Aurora kinases was significantly higher in 3 thyroid papillary carcinomas with respect to normal matched tissues obtained from the same patients. Similar modifications, at the mRNA level, could be observed in 7 papillary carcinoma tissues for Aurora-A and B, but not for Aurora-C. In conclusion, we demonstrated that normal human thyrocytes express all 3 members of the Aurora kinase family, and their expression is amplified in malignant thyroid cell lines and tissues. These results suggest that the Aurora kinases may play a relevant role in malignant thyroid cancers, and may represent a putative therapeutic target for thyroid neoplasms.

Identification of candidates for tumor‐specific alternative splicing in the thyroid

Alternative splicing is the differential processing of exon junctions to produce a new transcript variant from one gene. Some aberrant splicing, however, has been shown to be cancer specific. Identification of these specific splice variations will provide important insight into the molecular mechanism of normal cellular physiology as well as the disease processes. To gain knowledge about whether alternative splicing is linked to thyroid tumorigenesis, we used our prediction database to select targets for analysis. Fifteen putatively new alternative splicing isoforms were selected on the basis of their expression in thyroid libraries and/or their origin in genes previously associated with carcinogenesis. Using a set of 66 normal, benign, and malignant thyroid tissue samples, new splicing events were confirmed by RT-PCR for 13 of 15 genes (a validation rate of 87%). In addition, new alternative splicing isoforms not predicted by the system and not previously described in public databases were identified. Five genes (PTPN18, ABI3BP, PFDN5, SULF2, and ST5) presented new and/or additional unpredicted isoforms differentially expressed between malignant and benign or normal thyroid tissues, confirmed by sequencing. PTPN18, ABI3BP, and PFDN5 revealed a statistically significant differential splicing profile. In addition, real-time PCR analysis revealed that expression of an alternative PFDN5 variant was higher in malignant lesions than in benign lesions or normal tissues.

Allelic Variation in Gene Expression in Thyroid Tissue

Heritable allelic variation in gene expression may contribute to sporadic and familial disease, but is relatively unexplored. Papillary thyroid cancer (PTC) is characterized by strong heritability but predisposing genes have not been detected. Here we tested the hypothesis that inherited variation in allelic expression occurs in thyroid tissue and so might contribute to disease. We studied genes with a role in thyroid function, signaling, and/or tumorigenesis (PRKAR1A, DUOX1, IP3RI, ARGBP2, TPO, TG, and PEG10). We screened thyroid tissues from controls and patients with thyroid disease and lymphoblastoid cell lines from 40 healthy individuals. We demonstrated the robustness of the technique, a fluorescent dideoxy terminator-based method distinguishing the mRNA products of alleles in individuals who are heterozygous for a polymorphism in the transcript. We confirmed that the PEG10 gene transcript was derived only from one allele because of genetic imprinting. Three other genes, DUOX1, TPO, and ARGBP2 showed allelic variation in thyroid tissue and/or lymphoblastoid cells. Benign and malignant thyroid tissue from the same patient always gave concordant results. DUOX1 variation in expression was seen in 3 of 13 patients with PTC and 6 of 27 patients with benign disease. Further studies are needed to determine the significance of these and other allelic variations.

Expression of pendrin in benign and malignant human thyroid tissues

The Pendred syndrome gene (PDS) encodes a transmembrane protein, pendrin, which is expressed in follicular thyroid cells and participates in the apical iodide transport. Pendrin expression has been studied in various thyroid neoplasms by means of immunohistochemistry (IHC), Western blot and RT–quantitative real-time PCR. The expression was related to the functional activity of the thyroid tissue. Follicular cells of normal, nodular goitre and Graves' disease tissues express pendrin at the apical pole of the thyrocytes. In follicular adenomas, pendrin was detected in cell membranes and cytoplasm simultaneously in 10 out of 15 cases. Pendrin protein was detected in 73.3 and 76.7% of the follicular (FTC) and papillary (PTC) thyroid carcinomas, respectively, where pendrin was solely localised inside the cytoplasm. An extensive intracellular immunostaining of pendrin was observed in six out of 11 (54.5%) of positive FTCs and 19 out of 23 (82%) of PTCs. Focal reactivity was detected in one follicular- and three papillary carcinomas, whereas pendrin protein was absent in three of 15 FTC and four of 30 PTC; mRNA of pendrin was detected in 92.4% of thyroid tumours. The relative mRNA expression of pendrin was lower in cancers than in normal thyroid tissues (P<0.001). The pendrin protein level was found to parallel its mRNA expression, which was not, however, related to the tumour size and tumour stage. In conclusion, pendrin is expressed in the majority of differentiated thyroid tumours with high individual variability but its targeting to the apical cell membrane is affected.

Differential expression of alphaB-crystallin and Hsp27-1 in anaplastic thyroid carcinomas because of tumor-specific alphaB-crystallin gene (CRYAB) silencing

Expression of the small heat shock protein alphaB-crystallin in differentiated thyroid tumors has been described recently. In this study, we investigated the molecular mechanisms that affect the expression of alphaB-crystallin in benign goiters (n = 7) and highly malignant anaplastic thyroid carcinomas (ATCs) (n = 3). AlphaB-crystallin expression was compared with that of Hsp27-1. Immunoblot and quantitative real-time (RT) polymerase chain reaction revealed marked downregulation of alphaB-crystallin in all the tested ATCs and the ATC-derived cell line C-643 . In contrast, considerable expression of Hsp27-1 in benign and malignant thyroid tissue was demonstrated. Immunofluorescence analysis revealed no relevant topological differences between benign and malignant thyrocytes in the cytoplasmic staining of both proteins. Consistent and marked downregulation of TFCP2L1 was identified as one of the main mechanisms contributing to CRYAB gene silencing in ATCs. In addition, CRYAB gene promoter methylation seems to occur in distinct ATCs. In silico analysis revealed that the differential expression of alphaB-crystallin and Hsp27-1 results from differences between the alphaB-crystallin and Hsp27-1 promoter fragments (712 bp upstream from the transcriptional start site). Biological activity of the analyzed promoter element is confirmed by its heat shock inducibility. In conclusion, we demonstrate downregulation of alphaB-crystallin expression in highly dedifferentiated ATCs because of a tumor-specific transcription factor pattern. The differential expression of alphaB-crystallin and Hsp27-1 indicates functional differences between both proteins.

Id1 Gene Expression Is Up-Regulated in Hyperplastic and Neoplastic Thyroid Tissue and Regulates Growth and Differentiation in Thyroid Cancer Cells

The Id (inhibitor of DNA binding) proteins are a family of helix-loop-helix (HLH) proteins (Id1, Id2, Id3, and Id4) that lack the basic domain necessary for DNA binding. The Id1 protein enhances cell proliferation and inhibits cellular differentiation in a variety of cell types. We have previously demonstrated that the Id1 gene is up-regulated in papillary and medullary thyroid cancers. In this study we characterized the expression and distribution of the Id1 protein in normal, hyperplastic, and neoplastic human thyroid tissue. We also evaluated the effect of the Id1 gene on thyroid cancer cell growth and markers of thyroid cell differentiation. We used semiquantitative immunohistochemistry to characterize Id1 protein expression in normal, hyperplastic (multinodular goiter and Graves' disease), and neoplastic thyroid tissue from 103 patients. Normal thyroid tissue had the lowest level of Id1 protein expression (P < 0.0001). Anaplastic thyroid cancer had the highest level (vs. benign and malignant thyroid tissues, P < 0.01). Id1 protein expression was higher in malignant thyroid tissue than in hyperplastic thyroid tissue (P < 0.02). We found no significant association between the level of Id1 protein expression and patient age, sex, tumor-node-metastasis stage, tumor size, primary tumor vs. lymph node metastasis, primary tumor vs. recurrent tumors, and extent of tumor differentiation. Inhibiting Id1 mRNA expression in thyroid cancer cell lines using Id1 antisense oligonucleotides resulted in growth inhibition (P < 0.03) and decreased thyroglobulin and sodium-iodine symporter mRNA expression (P < 0.02). In conclusion, Id1 is overexpressed in hyperplastic and neoplastic thyroid tissue and directly regulates the growth of thyroid cancer cells of follicular cell origin, but is not a marker of aggressive phenotype in differentiated thyroid cancer.

PAX8 and peroxisome proliferator-activated receptor gamma 1 gene expression status in benign and malignant thyroid tissues.

Genetic alterations involving the thyroid transcription factor PAX8 and the peroxisome proliferator-activated receptor gamma 1 (PPARgamma1) genes have been described in thyroid neoplasms. We investigated in a series of thyroid samples, including 14 normal, 13 hyperfunctioning tissues, 26 follicular adenomas, 21 follicular and 41 papillary carcinomas, both the frequency of the PAX8-PPARgamma1 rearrangement and the expression of the PAX8 and PPARgamma transcripts. Using RT-PCR followed by sequencing PCR products, PAX8-PPARgamma1 translocation was not detected in benign tissues nor in papillary carcinomas and was detected in 4 (19%) of 21 follicular carcinomas and in one (4%) of 26 follicular adenomas. Specific real-time quantitative RT-PCR (Q RT-PCR) methods detected high levels of PPARgamma transcripts in follicular carcinomas presenting the rearrangement. Interestingly, the level of PPARgamma transcripts was significantly decreased in papillary carcinomas in comparison with those found in benign adenomas and follicular carcinomas. Finally, PAX8 gene expression was decreased in both papillary and follicular thyroid carcinomas, and in these tumors to the same extent in the presence or absence of the rearrangement. These alterations in both PPARgamma and PAX8 gene expression may explain the poorly differentiated histotype of follicular carcinomas harboring the translocation. Immunohistochemistry showed that nuclear PPARgamma staining was weak in normal tissues, adenomas, papillary carcinomas and in some follicular carcinomas, and strong in the follicular carcinomas positive for the PAX8-PPARgamma1 translocation, but also in some follicular tumors in which no translocation could be evidenced. These observations confirm that the PAX8-PPARgamma1 translocation characterizes a subset of thyroid follicular carcinomas but is not a specific marker of carcinoma and that its frequency is lower than that initially reported. Finally, immunohistochemistry is not a reliable method for the specific detection of the translocation, that can be specifically evidenced by Q RT-PCR.

Differential expression of thyroperoxidase mRNA splice variants in human thyroid tumors

The levels of human thyroperoxidase (hTPO) mRNA expression and the rates of hTPO mRNA with alternatively spliced exons 10, 14, and 16 were analyzed in normal, benign, and malignant thyroid tissues (13 normal thyroid tissues, 9 adenomas, 4 papillary carcinomas, 11 follicular variant of papillary carcinomas, 16 minimally invasive follicular carcinomas, 6 widely invasive follicular carcinomas) using a semi-quantitative reverse-transcription polymerase chain reaction procedure. The level of hTPO mRNA decreased in the follicular variant of papillary carcinomas and in minimally invasive follicular carcinomas and was more heterogeneous in the other pathological tissues than in normal tissues. Based on the mean values recorded, the splicing of exons 10 and 16 increased by at least 50% in all the carcinomas, as well as in the benign tissues in the case of exon 10. By contrast, no significant increase was observed in the splicing of exon 14 except in the case of the follicular variant of papillary carcinomas. In conclusion, the results of this study show that the splicing of hTPO increases in benign and malignant thyroid tissues. This event might partly explain the decrease in both the quantity and the level of activity of hTPO observed in thyroid cancer due to the loss of stability of the spliced isoforms. In addition, an increase in the alternative splicing of other mRNAs may contribute to the process of malignancy.

Expression of cyclooxygenase-2 and thromboxane synthase in non-neoplastic and neoplastic thyroid lesions.

Cyclooxygenase-2 (COX-2) and thromboxane synthase (TBXAS) are important enzymes involved in the arachidonic acid pathway and synthesis of prostaglandins. We examined COX-2 and TBXAS immunoreactivity in 150 surgically resected thyroid specimens using immunohistochemistry to determine expression in benign and malignant thyroid lesions and to examine their roles in thyroid tumor progression. Papillary thyroid carcinomas and follicular carcinomas expressed higher levels of COX-2 compared to follicular adenomas and adenomatous nodules. We showed for the first time that TBXAS was expressed in thyroid tissues, with higher levels in papillary carcinomas compared to non-neoplastic and benign thyroid tissues. Western blot was performed on seven thyroid samples. These results indicate that both COX-2 and TBXAS are expressed in benign and malignant thyroid tissues. Although some malignant thyroid tumors showed higher levels of COX-2 expression, COX-2 and TBXAS are probably not useful in the immunohistochemical diagnosis of thyroid malignancies. However, the expression of both COX-2 and TBXAS by thyroid tissue may provide insight into the role of these enzymes in progression from benign to malignant thyroid tumors.

Her2 Expression in Thyroid Tumors

Similarities exist in hormone receptors of breast, prostate, and thyroid tumors. HER2 oncogene expression is known to be present in breast and prostate tumors, but conflicting data have been published about its presence in thyroid tumors. This uncertainty prompted us to examine the incidence of HER2 overexpression in normal and malignant thyroid tissue. Normal and neoplastic thyroid tissue samples from 46 female and 9 male patients were assayed for HER2 expression by immunohistochemical assay. Of the 55 total samples, 36 were from neoplasms and 19 were from benign tissues. Significant HER2 overexpression was not found in any benign or malignant thyroid tissue. Two of 6 thyroid carcinomas from male patients showed 1+ reactivity for HER2 expression on immunohistochemistry assay, but remained negative on fluorescene in sito hybridization confirmatory testing. No significant expression of HER2 was noted in benign or malignant thyroid tissue. These results cast doubt on the value of HER2 as a prognostic factor or possible target for specific antitumor therapy for thyroid cancer.

Expression of Galectin-3 in Normal and Malignant Thyroid Tissue by Quantitative PCR and Immunohistochemistry

Galectin-3 has been extensively studied as an immunohistochemical marker of thyroid malignancy, and a high diagnostic accuracy has been reported even for difficult pathological diagnoses, such as minimal invasive follicular carcinoma. We consequently hypothesized that the quantitative analysis of galectin-3 mRNA rather than the more observer-dependent immunohistological determination might enhance the diagnostic workup of ambiguous thyroid lesions. In the present study, we set out to validate this approach by analyzing concomitantly the expression and production of galectin-3 in benign and malignant thyroid tumors by means of quantitative PCR and immunohistochemistry. Twenty-eight benign and 31 malignant thyroid samples were quantified by real-time PCR for the mRNA levels of galectin-3 and thyroglobulin. Galectin-3 protein expression was examined by immunohistochemistry in 13 benign and 14 malignant thyroid samples. There was a significant increase in galectin-3 at both the mRNA (12/20) and protein levels in papillary cancer (8/8), although the mRNA values overlapped partly with benign lesions. Surprisingly, only a focal and discrete galectin-3 immunoreactivity was seen in follicular cancer (1/5); no augmentation of the mRNA was found. The expression of the thyroid-specific gene thyroglobulin was highly variable in benign and malignant thyroid tissue. These results suggest that the quantitative measurement of galactin-3 mRNA is unlikely to be clinically useful and underscore the need for searching for novel markers for thyroid malignancies.

Proteomic and Postproteomic Characterization of Keratan Sulfate-Glycanated Isoforms of Thyroglobulin and Transferrin Uniquely Elaborated by Papillary Thyroid Carcinomas

Previous studies have suggested that surface components of papillary thyroid carcinoma (PTC) cells may be aberrantly glycanated, but the precise nature of these molecules has not been unveiled nor documented to be of clinical relevance. A monoclonal antibody was raised against a unique keratan sulfate (KS) determinant and used to differentially screen benign and malignant thyroid tissue for the expression of components carrying these moieties. In a total of 349 cases of benign and malignant thyroid lesions, 100% of the 115 PTC cases examined (including various histological subtypes) were found to contain KS-bearing molecules, whereas these were virtually absent from benign tissues and other thyroid tumors, with the exception of 21% of the follicular carcinoma cases analyzed. A composite immunoaffinity chromatography, immunochemistry, and mass spectrometric approach revealed that the PTC-specific KS-bearing macromolecules were unique glycoforms of thyroglobulin and transferrin. Combined, reciprocal immunoprecipitation and Western blotting further indicated that the former glycoform predominated and that most of the transferrin produced by PTC was glycanated with KS moieties. Fluorescent keratanase II-based fingerprinting of the KS moieties bound to these isoforms further demonstrated several PTC-specific peculiarities: 1) that a considerable portion of the moieties was covalently attached via a novel core protein linkage structure; 2) they had an unusual extended average length; 3) an unusual relative ratio of highly sulfated disaccharides terminating with alpha (2-3)-linked N-acetylneuraminic acid capping residues; and 4) a novel unidentified oligosaccharide moiety at the nonreducing terminus. Comparative analysis of the relative distribution of transferrin in benign versus PTC tissues highlighted a marked malignancy-associated abundance of the molecule, with a >75% frequency in expression in PTC. These findings demonstrate that PTC cells synthesize unique post-translationally modified thyroglobulin and transferrin variants in situ that may be directly exploitable for diagnosis, through histological and noninvasive cytological procedures; for devising novel strategies for antibody-guided imaging of this tumor in vivo; and for postsurgery follow-up of PTC patients.

Differential expression of mucins 1-6 in papillary thyroid carcinoma: evidence for transformation-dependent post-translational modifications of MUC1 in situ.

Mucins are primary glycoproteins of epithelia that are known to undergo major changes in their post-translational processing during neoplastic transformation. This study has examined the expression pattern of seven primary mucins, ie mucin (MUC) 1, 2, 3, 4, 5AC, 5B and 6, in normal, hyperplastic, benign neoplastic, and papillary-type carcinoma (PTC) tissues of the thyroid. MUC1 and MUC5B were the only mucins to be widely transcribed in both benign and malignant tissues. In contrast, MUC4 transcripts were undetectable in normal thyroids, and were present in only 40% of the hyperplastic and malignant thyroid tissues. In PTC, MUC1 was identified as a single mRNA transcript, rejecting the idea that this mucin may undergo transformation-dependent alternative splicing in thyroid tumours. The tissue distribution of MUC1 and MUC4 proteins was highly heterogeneous: this largely paralleled their mRNA expression profiles and supported the conclusion that whereas MUC1 was ubiquitously expressed in PTC, MUC4 was detectable in less than 20% of the cases analysed. In order to determine whether post-translational modifications of MUC1, putatively associated with malignancy, also occurred in the mucin produced by PTC, immunohistochemistry was performed with a panel of well-characterized anti-MUC1 antibodies in conjunction with digestion of the tissue sections with deglycosylating enzymes. These experiments, which were supported by immunochemical analyses of the MUC1 and MUC4 glycoforms extracted from tissues, collectively demonstrated markedly divergent MUC1 glycosylation profiles in normal and benign thyroid tissues when compared with PTC. Characteristically, these latter neoplastic cells produced mucin molecules carrying complex poly-N-lactosamine-type glycans capped with fucose and neuraminic acid residues. The present study also found evidence in PTC for the potential presence of proteolytically processed MUC1 isoforms which differ in their post-translational traits depending on whether they are retained on the cell surface or secreted into the extracellular space. It is proposed that the observed differences in the glycosylation properties of normal and neoplastic MUC1 may be exploitable as an ancillary tool in the diagnosis of PTC.

Decreased expression of catalase mRNA in thyroid anaplastic carcinoma.

A decreased expression of glutathione peroxidase mRNA, an antioxidant enzyme, was previously observed in thyroid anaplastic carcinomas. To clarify the expression of antioxidant-related enzymes in thyroid anaplastic carcinomas, the expression levels of catalase, copper and zinc superoxide dismutase and manganese superoxide dismutase mRNA in 85 benign and malignant thyroid tissues were measured by means of real-time quantitative reverse transcription-polymerase chain reaction. Decreased expression levels of catalase and copper and zinc superoxide dismutase mRNAs, but not manganese mRNA, were observed in five anaplastic carcinomas compared with normal thyroid tissues and differentiated tumors. These results suggest the possibility that anaplastic carcinoma cells are more likely to suffer damage by oxygen free radicals than normal thyroid cells or differentiated tumor cells.

Construction of cDNA Libraries from Microdissected Benign and Malignant Thyroid Tissue

cDNA libraries were constructed from thyroid epithelial cells gained by laser capture microdissection for gene expression analysis of the progression of thyroid cancer. Six histologically diverse thyroid tissue specimens were used. A mean of 93 ng of total RNA was gained per tissue sample from a mean estimated number of 25,000 microdissected cells per sample. Analysis of randomly selected clones from six libraries showed an average insert size of 600 (range, 300–1500) bp. Preliminary sequencing of clones selected from the six libraries indicates a range of 46% to 62% known genes per library, 4% to 25% anonymous expressed sequence tags per library, and 15% to 43% novel expressed sequence tags per library. Thyroglobulin was found in normal thyroid epithelium and follicular thyroid adenoma, whereas calcitonin precursor transcripts were found in medullary thyroid carcinoma. We demonstrate production of high-quality cDNA libraries of microdissected tissue of the thyroid, which should prove useful for gene expression analysis of human thyroid tumors.

Quantitative Analysis of Thymosin Beta-10 Messenger RNA in Thyroid Carcinomas

Genes that are differentially expressed in benign and malignant tissues are important for the establishment of molecular-based diagnosis of carcinomas. Our recent study on the gene expression profile of thyroid carcinomas revealed an increased expression of thymosin beta-10 mRNA. To confirm this, we measured the expression levels of thymosin beta-10 mRNA in 84 thyroid benign and malignant thyroid tissues, including five anaplastic carcinomas, by means of real-time quantitative reverse transcription-polymerase chain reaction. We found an increased expression of thymosin beta-10 mRNA in thyroid carcinomas, especially in anaplastic carcinomas. Expression levels of thymosin beta-10 mRNA relative to thyroglobulin mRNA in anaplastic carcinomas were greatly increased compared with those in differentiated carcinomas. These results suggest the usefulness of the quantitative measurement of thymosin beta-10 mRNA in molecular-based diagnosis of thyroid anaplastic carcinomas, but not of differentiated carcinomas.