Cytomorphologic features in thyroid nodules read as "suspicious for malignancy" on cytology may predict thyroid cancers with the BRAF mutation.

Some morphologic parameters have been studied to help predict the BRAF(V600E) mutation using cytopathologic specimens, which can indicate which nodules should undergo further testing. The aim of this study was to investigate the value of cytomorphologic parameters to predict the BRAF(V600E) mutation in nodules read as "suspicious for malignancy" on cytology. This study included 142 resected nodules which were diagnosed as "suspicious for malignancy" on cytology in 142 patients. At our institution, BRAF(V600E) mutation analysis was performed at the request of the referring clinicians based on the clinical features of the patients, or the judgment of the radiologists performing US-FNA because suspicious US features were observed on the targeted nodule during this study period. Cytology smears were re-reviewed to assess the presence and amount of polygonal eosinophilic (plump) cells and microfollicles, and the presence of intranuclear pseudoinclusions, irregular nuclear membranes, nuclear grooves, sickles cells, psammoma bodies, and cystic changes. We evaluated the diagnostic performances of the cytomorphologic features to predict the BRAF(V600E) mutation. Polygonal eosinophilic (plump) cells, microfollicles, intranuclear pseudoinclusions, sickle cells, and cystic changes were significantly associated with the BRAF(V600E) mutation. The mutation was not present in all 6 thyroid nodules with microfollicles larger than 20% on cytology. Additionally, polygonal eosinophilic (plump) cells larger than 20%, cystic changes, and sickle cells on cytology had a high specificity of 95%, 96.7%, and 81.7%, respectively. Excluding 6 nodules with microfollicles larger than 20% on cytology, there were 82 (60.3%) nodules with the BRAF(V600E) mutation among the 136 nodules. Among the 136 nodules, there were 95 nodules with polygonal eosinophilic (plump) cells larger than 20%, cystic changes, or sickle cells on cytology. Of the 95 nodules, 69 (72.6%) had the mutation. Cytomorphologic features can help select nodules for the BRAF(V600E) mutation test among nodules read as "suspicious for malignancy" on cytology.

Carbonic anhydrase 4 and crystallin α-B immunoreactivity may distinguish benign from malignant thyroid nodules in patients with indeterminate thyroid cytology.

BACKGROUND: Thyroid nodules are present in 19%-67% of the population and carry a 5%-10% risk of malignancy. Unfortunately, fine-needle aspiration biopsies are indeterminate in 20%-30% of patients, often necessitating thyroid surgery for diagnosis. Numerous DNA microarray studies including a recently commercialized molecular classifier have helped to better distinguish benign from malignant thyroid nodules. Unfortunately, these assays often require probes for >100 genes, are expensive, and only available at a few laboratories. We sought to validate these DNA microarray assays at the protein level and determine whether simple and widely available immunohistochemical biomarkers alone could distinguish benign from malignant thyroid nodules. METHODS: A tissue microarray (TMA) composed of 26 follicular thyroid carcinomas (FTCs) and 53 follicular adenomas (FAs) from patients with indeterminate thyroid nodules was stained with 17 immunohistochemical biomarkers selected based on prior DNA microarray studies. Antibodies used included galectin 3, growth and differentiation factor 15, protein convertase 2, cluster of differentiation 44 (CD44), glutamic oxaloacetic transaminase 1 (GOT1), trefoil factor 3 (TFF3), Friedreich Ataxia gene (X123), fibroblast growth factor 13 (FGF13), carbonic anhydrase 4 (CA4), crystallin alpha-B (CRYAB), peptidylprolyl isomerase F (PPIF), asparagine synthase (ASNS), sodium channel, non-voltage gated, 1 alpha subunit (SCNN1A), frizzled homolog 1 (FZD1), tyrosine related protein 1 (TYRP1), E cadherin, type 1 (ECAD), and thyroid hormone receptor associated protein 220 (TRAP220). Of note, two of these biomarkers (GOT1 and CD44) are now used in the Afirma classifier assay. We chose to compare specifically FTC versus FA rather than include all histologic categories to create a more uniform immunohistochemical comparison. In addition, we have found that most papillary thyroid carcinoma could often be reasonably distinguished from benign disease by morphological cytology findings alone. RESULTS: Increased immunoreactivity of CRYAB was associated with thyroid malignancy (c-statistic, 0.644; negative predictive value [NPV], 0.90) and loss of immunoreactivity of CA4 was also associated with malignancy (c-statistic, 0.715; NPV, 0.90) in indeterminate thyroid specimens. The combination of CA4 and CRYAB for discriminating FTC from FA resulted in a better c-statistic of 0.75, sensitivity of 0.76, specificity of 0.59, positive predictive value (PPV) of 0.32, and NPV of 0.91. When comparing widely angioinvasive FTC from FA, the resultant c-statistic improved to 0.84, sensitivity of 0.75, specificity of 0.76, PPV of 0.11, and NPV of 0.99. CONCLUSIONS: Loss of CA4 and increase in CRYAB immunoreactivity distinguish FTC from FA in indeterminate thyroid nodules on a thyroid TMA with an NPV of 91%. Further studies in preoperative patient fine needle aspiration (FNAs) are needed to validate these results.

The quest for diagnostic molecular markers for thyroid nodules with indeterminate or suspicious cytology.

Thyroid nodules are very common and fine needle aspiration (FNA) is a very sensitive means of diagnosis. However, its limitations include the fact that the cytology reports are often indeterminate or suspicious only. The quest for adjunctive measures to improve its specificity has been ongoing for decades, but significant results have remained elusive. The potential use of diagnostic molecular markers appears to be the most promising area of research at this time.

Bipolar radiofrequency ablation for nodular thyroid disease--ex vivo and in vivo evaluation of a dose-response relationship.

BACKGROUND: The prevalence of thyroid nodules ranges between 2% and 60% depending on the population studied. However, minimally invasive procedures like radiofrequency ablation (rfA) are increasingly used to treat tumors of parenchymatous organs, and seem to be suitable for singular thyroid nodules as well. Their successful clinical application depends on the induction of sufficiently large lesions and a knowledge of the energy parameters required for complete thermal ablation. The aim of this study was to establish a dose-response relationship for rfA of thyroid nodules. MATERIAL AND METHODS: Thermal lesions were induced in healthy porcine thyroid glands ex vivo (n=110) and in vivo (n=10) using a bipolar radiofrequency system; rf was applied in a power range of 10-20 watts. During the ablation, continuous temperature measurement at a distance of 5 and 10 mm from the applicator was performed. The transversal and axial lesion diameters were measured, and the volume was calculated. Furthermore, enzyme histochemical analysis of the thyroid tissue was performed. RESULTS: The inducible lesion volumes were between 0.91±0.71 cm(3) at 20W and 2.80±0.85 cm(3) at 14W. The maximum temperatures after rf ablation were between 44.0±9.7°C and 61.6±13.9°C at a distance of 5 mm and between 30.0±8.6°C and 53.5±8.6°C at a distance of 10 mm from the applicator. The histochemical analysis demonstrates a complete loss of nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) dehydrogenase activity in thermal lesions as a sign of irreversible cell damage. CONCLUSION: This study is the first to demonstrate a dose-response relationship for rfA of thyroid tissue. rfA is suitable for singular thyroid nodules and induces reproducible, clinically relevant lesions with irreversible cell damage in an appropriate application time.

[Puncture hystobiopsy with tissue telomerase activity measurement in preoperative thyroid nodes diagnostics].

Demonstrated, that puncture thyroid hystobiopsy is safe and informative method of preoperative thyroid nodes diagnostics. Telomerase activity in tissue samples was significantly higher in case of malignant thyroid disease, although a positive correlation of telomerase activity was also with the amount of lymphocytes in bioptates. Combination of thyroid hystobiopsy with tissue telomerase activity measurement proved to be an effective means of preoperative diagnostic of patients with nodular goiter.

Negative correlation between thyroperoxidase and dual oxidase H2O2-generating activities in thyroid nodular lesions.

Iodine incorporation into thyroglobulin is dependent on the activities of both thyroperoxidase (TPO) and thyroid dual oxidase 2 (DuOx2). Although TPO expression is decreased in some thyroid nodular lesions, DuOx1 and 2 mRNA expressions are maintained, but DuOx H2O2-generating activity has never been evaluated in such tumors. Our goal was to determine DuOx activity in hypofunctioning lesions of the thyroid. We evaluated H2O2 generation by DuOx in 12 paranodular to cold nodule samples, 17 non-toxic multinodular goiters (MNG; 33 samples), 3 papillary carcinomas (PC; 4 samples), 3 follicular carcinomas (FC; 4 samples), and 10 follicular adenomas. DuOx activity was detected in all paranodular tissues (121+/-23 nmol H2O2/h per mg protein), but was undetectable (<1 nmol H2O2 generated) in all PC, two out of four FC samples and seven out of ten adenomas. In 11 MNG at least two different areas of the goiter have been evaluated, and in 5 of these goiters one of the samples had DuOx activity below the limit of detection. The coefficient of variation in MNG samples ranged from 11.3 to 57.2%. Interestingly, in all the adenomas studied, TPO activity (486+/-142 U/g protein, n=8) was well within the range found in paranodular tissues (414+/-116 U/g protein, n=3). We found a significant negative correlation between DuOx and TPO activities, suggesting that these enzymes are regulated in opposite directions, at least in thyroid tumors.

Iodine deficiency activates antioxidant genes and causes DNA damage in the thyroid gland of rats and mice.

Because thyroid nodules are frequent in areas with iodine deficiency the aim of this study was to characterise molecular events during iodine deficiency that could explain mutagenesis and nodule formation. We therefore studied gene expression of catalytic enzymes prominent for H(2)O(2) detoxification and antioxidative defence, quantified DNA oxidation and damage as well as spontaneous mutation rates (SMR) in mice and rats fed an iodine controlled diet. Antioxidative enzymes such as superoxide dismutase 3, glutathione peroxidase 4 and the peroxiredoxins 3 and 5 showed increased mRNA expression, which indicates increased radical burden that could be the cause of additional oxidized base adducts found in thyroidal genomic DNA in our experiments of iodine deficiency. Furthermore, the uracil content of thyroid DNA was significantly higher in the iodine-deficient compared to the control group. While SMR is very high in the normal thyroid gland it is not changed in experimental iodine deficiency. Our data suggest that iodine restriction causes oxidative stress and DNA modifications. A higher uracil content of the thyroid DNA could be a precondition for C-->T transitions often detected as somatic mutations in nodular thyroid tissue. However, the absence of increased SMR would argue for more efficient DNA repair in response to iodine restriction.

Thyroid cysts: a new extra-adrenal site of aldosterone synthase expression and increased aldosterone content.

BACKGROUND: The rapid re-accumulation of fluid following aspiration of thyroid cystic lesions suggests that active transport of sodium and water may be involved in volume regulation of these lesions. In this study we address the possibility that aldosterone may take part in this process. SUBJECTS AND METHODS: Thirty-one patients (29 women and two men), with a mean age of 52.7 +/- 13.2 years (range: 27-77 years) underwent evaluation for thyroid nodules that had a sonographic cystic component. Cystic fluid obtained by FNA biopsy was sent for cytological examination and biochemical measurements. In 10 patients, material was collected for RNA extraction and determination of aldosterone synthase expression by RT-PCR amplification. RESULTS: All lesions were benign, cystic, colloid nodules. Cyst fluid aldosterone levels as measured by routine radioimmunoassay (RIA) were elevated above the normal plasma levels in all but five patients. Mean aldosterone levels were 27.1 +/- 22.9 ng/dl (SD) (range: 5.9-117.5 ng/dl). In contrast, cyst cortisol values were in the low, low normal serum range (6.2 +/- 2.9 microg/dl, range: 0.2-10.2 microg/dl). Sodium, chloride and potassium levels were 137 +/- 4.7 mEq/l, 98 +/- 5 mEq/l and 4.9 +/- 1.4 mEq/l, respectively. Plasma aldosterone levels were normal in all patients tested. To confirm these results, 12 samples were assayed after extraction and chromatography using a highly specific antibody. Cyst aldosterone levels in this group were elevated above the normal serum range in all but one patient (mean concentration: 24.5 +/- 14.6 ng/dl, range: 8.72-40.1 ng/dl). In this group, 18(OH)B levels were within the normal plasma range (12-55 ng/dl) in all but one patient (34.9 +/- 17 ng/dl). Furthermore, aldosterone synthase mRNA expression was found in aspirates of four of 10 patients. CONCLUSIONS: The increased aldosterone concentration and the presence of aldosterone synthase expression suggest that aldosterone may be locally produced and secreted in thyroid tissue. The pathophysiological implications of this finding remain to be established.

Telomerase activity in "suspicious" thyroid cytology.

BACKGROUND: Telomerase activity (TA) has been detected in most malignant neoplasms, including thyroid carcinomas. The authors studied the utility of TA detection as an ancillary tool to thyroid fine-needle aspiration (FNA) for patients with nonconclusive cytologic diagnoses. METHODS: Material obtained by FNA from palpable thyroid nodules in 167 consecutive patients was processed for conventional cytologic studies and simultaneously for TA study. Another 8 patients were excluded from TA because of the presence of lymphocytes. All patients with negative results cases were followed for > 1 year, and those who had tumors that were suspicious or positive by FNA or TA underwent resection for pathologic study of nodules. TA was analyzed by telomere repeat amplification protocol-polymerase chain reaction analysis. RESULTS: After excluding 20 patients because of insufficient material for cytologic study, 120 patients had negative results for malignant cells in cytology material, and the remaining 27 patients had results that were either suspicious (n = 21 patients) or positive (n = 6 patients). Histopathologic confirmation was obtained in 23 patients, including 18 with suspicious cytology (1 with scanty material) and 5 with positive FNA. The histopathologic diagnoses were nodular hyperplasia in 5 patients, follicular adenoma in 3 patients, papillary carcinoma in 11 patients, follicular carcinoma in 1 patient, medullary carcinoma in 2 patients, and lymphoma in 1 patient. TA was detected in 6 of 18 histologically confirmed thyroid neoplasms (1 of 3 follicular adenomas, 3 of 11 papillary carcinomas, 0 of 1 follicular carcinoma, 1 of 2 medullary carcinomas, and 1 of 1 lymphoma), including 1 neoplasm with scanty atypical cells. CONCLUSIONS: The detection of TA helped to confirm neoplasia in 6 of 23 suspicious thyroid nodules. Although it was less sensitive than FNA, TA specificity was 100% for neoplasia and 87.5% for malignancy. The sensitivity of thyroid FNA increased with the use of TA detection when cytology was nonconclusive for malignancy.

Human telomerase reverse transcriptase gene expression and the surgical management of suspicious thyroid tumors.

PURPOSE: Patients with a preoperative cytologic diagnosis of a suspicious thyroid nodule present a therapeutic dilemma because surgery differs for benign and malignant lesions. To address this issue, several molecular markers, including human telomerase reverse transcriptase (TERT), have been tested as markers of thyroid cancer. Because most studies select cases falling into well-defined categories to test new markers, they may overestimate their discriminatory power when applied to samples that are difficult to classify. Fine-needle aspirates (FNAs) of the thyroid with indeterminate cytology are an example of such cases. EXPERIMENTAL DESIGN: We examined whether assessing TERT mRNA by reverse transcription-PCR could have improved the surgical management in a cohort of 100 patients undergoing thyroidectomy for indeterminate FNA results. RESULTS: Ninety percent of 48 cancers were TERT positive, as were 35% of 52 benign lesions. When 10 cases with concomitant lymphocytic thyroiditis were excluded, the overall sensitivity of TERT was 91% (95% confidence interval, 80-98%) and specificity was 79% (64-90%). No clinical or tumor variable contributed to the predictive ability of TERT except for tumor size, which added only marginally. Basing the surgical approach on the TERT assay alone would have reduced lobectomies performed for malignant disease from 11 to 4 cases and reduced total thyroidectomies for benign lesions from to 15 to 9, an overall 50% reduction in suboptimal treatment. CONCLUSIONS: The overall performance of preoperative differential diagnosis for thyroid tumors with indeterminate FNA results can be substantially improved by the inclusion of molecular markers such as TERT.

GST profiling may be useful in the screening for thyroid nodule malignancy.

Screening tools are of utmost necessity in order to identify individuals at risk for thyroid nodule cancer. The polymorphic inheritance of human drug-metabolizing enzymes, such as those encoded by the Glutathione-S-Transferase (GST) system, plays an important role in the development of most human cancers. GSTP1 enzyme is the most important detoxification enzyme in human head and neck tissues. An aminoacid substitution (1105V) in the GSTP1 gene result in two genotypes, GSTP1AB and GSTP1BB. Those produce a variant enzyme with lower activity and less capability of effective detoxification of carcinogens than the wild type GSTP1AA. In order to look for the influence of GSTP1 enzymes inheritance pattern on thyroid cancer risk we used a PCR-SSCP-sequencing approach to compare the genotypes of 98 malignant nodules, including 77 papillary carcinomas (PC) and 21 follicular carcinomas (FC), to 44 benign nodules and to 157 healthy control individuals. Individuals with history of previous thyroid disease, exposure to radiation and antecedents of malignancy were excluded. Patients with PC and FC showed a significant over-representation of the variants of GSTP1 allele compared to the control population (p < 0.0001 The risk for thyroid cancer in individuals with the variant GSTP1 enzymes, after adjusting for gender, age, tobacco and drugs use, increased 7,092 (CI: 2,307-21,802) and 9,625 (CI: 2.484-37.291) times for PC and FC, respectively. We suggest that GST genotype may be associated with an increased susceptibility to thyroid cancer. GSTP1 profiling from peripheral blood may be a simple and useful tool in the screening for thyroid nodule malignancy. Glutathione-S-Transferase system; GSTP; Thyroid cancer; Screening.

Expression of adenylyl cyclase types III and VI in human hyperfunctioning thyroid nodules.

Hyperfunctioning thyroid nodules are characterized by the presence of spontaneous somatic mutations responsible for constitutive activation of the cAMP pathway. However, alterations affecting other elements of the cAMP signaling system may counteract the effects of the mutations. In this study, the expression of the adenylyl cyclase (AC) types III and VI was investigated by Western blot in 18 hyperfunctioning thyroid nodules; in 12 samples, we also assessed the presence of TSH receptor (TSHR) or gsp mutations and levels of AC VI and III mRNA. We found that the expression of nodular AC VI (but not AC III) was significantly lower (85.1% of normal, P=0.014) than the expression of both adenylyl cycles types of perinodular tissue from the same patients. Slightly, but not significant differences were detected in nodules with or without mutations and AC protein levels generally showed correlation with the levels of the transcripts detected by RT-PCR. In addition, AC III and AC VI expression levels within a given nodule were characterized by a significant positive correlation. These findings indicate that a diminished expression of AC type VI may be part of the mechanisms occurring in the hyperfunctioning nodules, independently of the presence of TSHR or gsp mutations, which influence the resulting phenotype.

Overexpression of type 2 iodothyronine deiodinase in follicular carcinoma as a cause of low circulating free thyroxine levels.

Thyroid function is normally undisturbed in patients with thyroid carcinoma. We have identified three patients with large or widely metastatic follicular thyroid carcinoma who had a persistently increased ratio of serum T(3) to T(4) in the absence of autonomous production of T(3) by the tumor. To investigate the possibility of tumor-mediated T(4) to T(3) conversion, we assayed types 1 and 2 iodothyronine selenodeiodinase (D1 and D2) activity in a 965-g follicular thyroid carcinoma resected from one of these patients. The V(max) for D2 was 8-fold higher than in normal human thyroid tissue. Resection of this tumor, leaving the left thyroid lobe intact, normalized the serum T(3) to T(4) ratio. In two other patients, treatment with sufficient levothyroxine to suppress TSH was associated with a high normal T(3) and a subnormal free T(4) index. In one, concomitant administration of the D1 inhibitors, propylthiouracil and propranolol, did not decrease the elevated serum T(3) to T(4) ratio. These data illustrate that increased T(4) to T(3) conversion in follicular thyroid carcinomas, probably by D2, can cause a significant perturbation in peripheral thyroid hormone concentrations.

Immunohistochemical expression of COX-2 in thyroid nodules.

BACKGROUND: Recent evidence indicates that elevated COX-2 expression is associated with the carcinogenesis of numerous neoplasms. In this study, we investigated COX-2 expression in various thyroid specimens in order to elucidate its physiological role in pathologic conditions, and to evaluate the efficiency of COX-2 protein expression as a molecular marker of malignancy in the thyroid gland. METHODS: COX-2 expression was studied immunohistochemically in 19 papillary carcinomas, 8 follicular carcinomas, 14 follicular adenomas, 2 Hürthle cell carcinomas, 4 Hürthle cell adenomas, 8 nodular hyperplasias, 3 Graves' diseases, 3 Hashimoto's thyroiditis, 2 medullary carcinomas, 1 anaplastic carcinoma, and 20 normal thyroid tissues. RESULTS: COX-2 staining was not seen in any of the normal thyroid, Graves' disease, or nodular hyperplasia specimens. In contrast, COX-2 staining was observed in all of papillary carcinomas, Hashimoto's thyroiditis, Hürthle cell carcinomas, and Hürthle cell adenomas tissues. Moreover, 7 of 8 follicular carcinomas and 11 of 14 follicular adenomas showed COX-2 staining. CONCLUSION: These results indicate that COX-2 is not useful as a marker of malignancy. Since COX-2 expression was evident in follicular adenomas and in papillary and follicular carcinomas. Thus, the enzyme may be involved in the early process of thyroid tumorigenesis.

Immunohistochemical Expression of COX-2 in Thyroid Nodules

BACKGROUND: Recent evidence indicates that elevated COX-2 expression is associated with the carcinogenesis of numerous neoplasms. In this study, we investigated COX-2 expression in various thyroid specimens in order to elucidate its physiological role in pathologic conditions, and to evaluate the efficiency of COX-2 protein expression as a molecular marker of malignancy in the thyroid gland. METHODS: COX-2 expression was studied immunohistochemically in 19 papillary carcinomas, 8 follicular carcinomas, 14 follicular adenomas, 2 H rthle cell carcinomas, 4 H rthle cell adenomas, 8 nodular hyperplasias, 3 Graves' diseases, 3 Hashimoto's thyroiditis, 2 medullary carcinomas, 1 anaplastic carcinoma, and 20 normal thyroid tissues. RESULTS: COX-2 staining was not seen in any of the normal thyroid, Graves' disease, or nodular hyperplasia specimens. In contrast, COX-2 staining was observed in all of papillary carcinomas, Hashimoto's thyroiditis, H rthle cell carcinomas, and H rthle cell adenomas tissues. Moreover, 7 of 8 follicular carcinomas and 11 of 14 follicular adenomas showed COX-2 staining. CONCLUSION: These results indicate that COX-2 is not useful as a marker of malignancy. Since COX-2 expression was evident in follicular adenomas and in papillary and follicular carcinomas. Thus, the enzyme may be involved in the early process of thyroid tumorigenesis.

APE/Ref-1 is increased in nuclear fractions of human thyroid hyperfunctioning nodules.

Apurinic/apyrimidinic endonuclease APE/Ref-1 is a multifunctional protein provided with DNA repair, transcription-factor regulation and anti-apoptotic activities. We have previously reported that, in thyroid cells, TSH regulates both the synthesis and nuclear translocation of APE/Ref-1. We have also shown that nuclear levels of this protein are reduced both in thyroid carcinoma tissues and cell lines. In the present study, APE/Ref-1 expression and cellular localization were analysed by Western blot in hyperfunctioning thyroid nodules from patients with toxic adenoma and/or toxic multinodular goiter. The total content of APE/Ref-1 protein was increased in the majority of the hyperfunctioning tissues with respect to normal adjacent tissue. There was also an increase in the nuclear levels of APE/Ref-1, suggesting enhanced cytoplasm-to-nucleus translocation of the protein in addition to its increased rate of synthesis. These results demonstrate that the phenomenon of nuclear translocation of APE/Ref-1 hypothesized on the basis of cell culture experiments does actually occur in vivo. Together with previous observations in thyroid carcinomas and tumoral cell lines, our findings suggest a two-stage model of APE/Ref-1 behaviour during malignant thyrocyte transformation: an early stage characterized by simple hyperplasia and upregulation of APE/Ref-1 in the nuclear compartment of the cell and a later stage in which nuclear levels of the protein drop to below-normal levels as the cell becomes progressively undifferentiated.

Cyclooxygenase-2 expression in thyroid nodules.

Factors contributing to the development of thyroid neoplasia remain poorly understood. Recent evidence indicates that overexpression of the inducible cyclooxygenase, COX-2, is important in the pathogenesis of epithelial carcinomas. These studies were undertaken to evaluate whether COX-2 is up-regulated in human thyroid neoplasia. Benign (n = 14), and malignant (n = 14) thyroid nodules were analyzed for expression of COX-2 mRNA by quantitative RT-PCR. Immunoblotting and immunohistochemistry were performed on representative samples. Three human thyroid cancer cell lines were similarly analyzed for COX-2 expression. Levels of COX-2 mRNA were significantly increased in thyroid nodule samples compared with adjacent thyroid tissue in the malignant specimens but not in the benign specimens. Additionally, COX-2 mRNA levels were significantly increased in malignant nodule samples compared with benign nodule samples. COX-2 protein expression was higher in 8 of 10 thyroid nodules compared with the adjacent tissue. Immunohistochemical analysis localized expression of COX-2 to the malignant epithelial cells. Immunofluorescence demonstrated COX-2 protein expression in all three thyroid cell lines. Finally, COX-2 expression could be detected by RT-PCR in fine needle aspiration specimens of thyroid nodules. These data indicate that COX-2 is up-regulated in human thyroid cancer, but not in benign thyroid nodules, and suggest that COX-2 expression may serve as a marker of malignancy in thyroid nodules.

Human telomerase reverse transcriptase expression in Diff-Quik-stained FNA samples from thyroid nodules.

Fine-needle aspiration (FNA) is a highly sensitive method in the differential diagnosis of thyroid nodules. However, 10% of thyroid FNAs are indeterminate for cancer, and thus additional markers may be useful diagnostically. The authors have demonstrated previously that human telomerase reverse transcriptase (hTERT) gene expression is useful in the distinction of benign lesions from malignant lesions. They therefore wondered whether the detection of hTERT gene expression was feasible using archival slides. To establish an experimental system, ribonucleic acid was extracted from human anaplastic thyroid carcinoma cell line (ARO) in cytologic specimens, and reverse transcription-polymerase chain reaction (RT-PCR) for hTERT expression was performed. RT-PCR analysis for hTERT gene detection was then performed using 58 Diff-Quik-stained archival FNA samples collected retrospectively. RT-PCR for human thyroglobulin (hTg) or beta-actin gene expression served as a positive control. Successful PCR results were obtained from 48 of the 58 cases. All 10 slides in which no RT-PCR products were noted were older than 3 years. hTERT gene expression was demonstrated in FNAs from two of seven cases (29%) of hyperplastic nodule, one of one case (100%) of Hashimoto's thyroiditis, three of eight cases (38%) of follicular adenoma, three of eight cases (38%) of Hürthle cell adenoma, three of four cases (75%) of follicular carcinoma, two of two cases (100%) of Hürthle cell carcinoma, and 11 of 18 cases (61%) of papillary carcinoma. All but one of the available 33 corresponding frozen samples exhibited the same RT-PCR results. This study demonstrates that Diff-Quik-stained thyroid FNA specimens less than 3 years old can be used for the detection of hTERT gene expression by RT-PCR. This test, along with careful cytopathologic examination, may improve our ability to differentiate benign lesions from malignant lesions in indeterminate FNA samples from thyroid nodules.

Human telomerase reverse transcriptase (hTERT) gene expression in thyroid neoplasms.

Ten percent of fine-needle aspirations (FNAs) of the thyroid are deemed "indeterminate" or "suspicious" for malignancy by the cytopathologist, but most of these lesions are benign. Therefore, additional markers of malignancy may prove to be a useful adjunct. The catalytic component of telomerase, human telomerase reverse transcriptase (hTERT), has been found to be reactivated in immortalized cell lines. Reverse transcription-PCR of the hTERT gene revealed expression in 15 (79%) of 19 malignant thyroid neoplasms, including 6 of 6 follicular carcinomas and 9 of 13 papillary carcinomas. In contrast, hTERT gene expression was detected in only 5 (28%) of 18 benign thyroid nodules, including 2 of 7 follicular adenomas and 3 of 11 hyperplastic nodules. All five benign thyroids exhibiting hTERT gene expression had lymphocytic thyroiditis. No normal thyroids exhibited hTERT gene expression. Telomerase enzyme activity was examined in all 37 nodules and was found to correlate with hTERT gene expression in 35 (95%) nodules. The two cases in which telomerase activity and hTERT expression results were discrepant were in two papillary carcinomas that were telomerase activity negative and hTERT positive. Finally, we have demonstrated that hTERT gene expression can be measured in in vivo FNA samples. These results suggest that hTERT expression may be more accurate than telomerase activity in distinguishing benign from malignant and may be measured in FNA samples from suspicious thyroid lesions.

Decrease of telomere length in thyroid adenomas without telomerase activity.

In somatic cells, telomeres shorten with population doubling, thus limiting their capacity to divide. Telomerase, which synthesizes telomeric repeats, can compensate for such shortening. Telomerase activity is known to be absent from most somatic differentiated cells but is present in germline cells, immortal cell lines, or a large majority of malignant tumors. Autonomous thyroid adenomas are benign tumors composed of highly differentiated cells characterized by TSH-independent function and growth. Telomere length and telomerase activity were measured in autonomous and hypofunctioning adenomas and their surrounding tissues. A significant decrease of 3.8+/-1.0 kilobases (kb) was observed in the length of the terminal restriction fragments (TRF) in 12 autonomous adenomas (8.6+/-1.1 kb), compared with the TRF length of their surrounding tissues (12.4+/-1.6 kb). The same kind of decrease, 3.5+/-1.2 kb, was also observed in 16 hypofunctioning adenomas (12.3+/-1.7 kb in surrounding tissue and 8.8+/-1.6 kb in the adenomas). No telomerase activity was detected either in the 12 autonomous adenomas studied or in most of the quiescent tissues (10 of 12). Most of the hypofunctioning adenomas tested (15 of 16) did not display telomerase activity. These results suggest that the cells have undergone a higher number of cell divisions in the adenomas than in the surrounding tissue. Moreover, there is a larger spread of the TRF length distribution in autonomous adenomas than in the collateral tissue. This could reflect the heterogeneity in proliferation status of the cells in the nodule, some of which have reached the end of their life span, whereas others are still proliferating (but with no malignant potential for the autonomous adenomas). In conclusion, benign adenomas exhibit a shorter and more variable telomere length than the normal collateral quiescent tissue, with no telomerase activity to compensate this loss in telomere length.