Molecular genetic alterations of FHIT and p53 genes in benign and malignant thyroid gland lesions

Several oncogenes and tumor-suppressor genes are involved either as early or late event in thyroid gland carcinogenesis. Human FHIT (fragile histidine triad) gene is highly conserved gene whose loss of function may be important in the development and/or progression of various types of cancer. We undertook this study to analyze FHIT and p53 gene status in different benignant and malignant thyroid tumors. Status of these genes as well as intensity of apoptosis was analyzed in tumor tissues by molecular genetic methods, immunohistochemistry, and FACS-scan analysis. The majority of the malignant thyroid cancers displayed aberrant expression of FHIT gene, concominant with p53 gene inactivation. This is followed by low rate of apoptosis, which may be important in the development and/or progression of thyroid cancer. We found higher incidence of p53 mutation and aberrant processing of FHIT mRNA in malignant tumors (papillary, follicular, medullary and anaplastic carcinomas) and in those tumors with distant metastasis. The growth of p53(-)/FHIT(-) follicular carcinoma of human origin was much faster in nude mice than p53(+)/FHIT(+) follicular carcinoma, and mice had shorter survival rate. Our results show a correlation between aberrant FHIT and p53 expression, low rate of apoptosis, and malignancy. Concomitant aberration of FHIT gene and p53 could be responsible for development of highly malignant types of thyroid cancer and may be considered as a prognostic marker for these tumors.     

Biomarcadores moleculares implicados en el proceso de desdiferenciación tumoral del carcinoma de tiroides de origen epitelial: perspectivas

Although papillary or follicular well-differentiated thyroid carcinoma usually has a good prognosis, a proportion of well-differentiated thyroid carcinomasshow a more aggressive behavior with local recurrence and metastases, either at diagnosis (in less than 5% of cases) or over time. Although there are several scoring systems to assess prognosis of well-differentiated thyroid carcinoma, mainly based on clinical and pathological data, there is currently no valid criterion to define an adequate, differential treatment for patients with low risk carcinomas as compared to those with more aggressive tumors. Identification of patients with a high risk at the time of diagnosis would be essential to develop new therapeutic strategies and to improve follow-up, and molecular biomarkers could be a highly useful tool for this purpose.     

Intraoperative Diagnostic Strategy to Distinguish Parathyroid Adenomas from Metastatic Thyroid Cancer

ObjectiveTo report the limitations of frozen section examination and the value of intraoperative tissue aspiration for parathyroid hormone assay to distinguish parathyroid adenomas from metastatic thyroid carcinoma.MethodsWe describe 2 patients with a biochemical diagnosis of primary hyperparathyroidism who underwent intraoperative frozen section analysis of suspected parathyroid tumors. Parathyroid gland aspiration for parathyroid hormone was also performed for confirmation.ResultsThe intraoperative frozen section examination of the suspected parathyroid tumors inaccurately identified the tumors as follicular carcinomas. The parathyroid gland aspirate, however, accurately substantiated the presence of parathyroid adenomas, rather than follicular cancers.ConclusionAspiration of a suspected parathyroid tumor for parathyroid hormone assay accurately determines whether a nodule is a parathyroid gland and facilitates intraoperative decision making, especially when frozen section diagnosis is misleading. (Endocr Pract. 2009; 15:454-457)     

The significance of paravacuolar granules of the thyroid. A histologic, cytologic and ultrastructural study

The presence of the so-called "paravacuolar granules" in thyroid follicular cells has been associated with increased metabolic activity of the gland, regressive changes, degeneration, phagocytic activity and benign papillary hyperplasia. During the course of a review of the intraoperative cytologic preparations and corresponding histologic sections from 73 thyroid cases, the presence of granules within follicular cells was noted in 25 cases (18 adenomatous or colloid goiters, 3 follicular adenomas, 2 papillary carcinomas, 1 follicular carcinoma and in thyroid tissue surrounding a follicular adenoma in 1 case). Histochemical and ultrastructural studies showed the granules to consist of lysosomes containing hemosiderin or lipofuscin pigments. These findings indicate that the presence of paravacuolar granules in thyroid cells is a common nonspecific finding that simply reflects: (1) the erythrophagocytic capability of the follicular epithelial cells, which results in the accumulation of iron within lysosomes, and (2) the accumulation of lipofuscin pigments within lysosomes as a result of degradation of endogenous cellular material.     

Apoptosis-inducing activity of vitamin C and vitamin K.

Apoptosis-inducing activity of vitamins C and K and of their analogs are reviewed. Vitamin C shows both reducing and oxidizing activities, depending on the environment in which this vitamin is present. Higher concentrations of vitamin C induce apoptotic cell death in various tumor cell lines including oral squamous cell carcinoma and salivary gland tumor cell lines, possibly via its prooxidant action. The apoptosis-inducing activity of ascorbate is stimulated by Cu2+, lignin and ion chelator, and inhibited by catalase, Fe3+, Co2+ and saliva. On the other hand, at lower concentrations, ascorbic acid displays an antioxidant property, preventing the spontaneous and stress or antitumor agent-induced apoptosis. Sodium 5,6-benzylidene-L-ascorbate, intravenous administration of which induces degeneration of human inoperable tumors and rat hepatocellular carcinoma in vivo, induces apoptotic or non-apoptotic cell death, depending on the types of target cells. On the other hand, elevation of intracellular concentration of ascorbic acid by treatment with ascorbate 2-phosphate or dehydroascorbic acid makes the cells resistant to the oxidative stress-induced apoptosis. Vitamin K2, which has a geranylgeranyl group as a side chain,and vitamin K3 induces apoptosis of various cultured cells including osteoclasts and osteoblasts, by elevating peroxide and superoxide radicals. Synergistic apoptosis-inducing actions have been found between vitamins C and K, and between these vitamins and antiproliferative agents. The possible therapeutic application of these vitamins is discussed.     

Sensitivity and specificity of fine needle aspiration cytology in the diagnosis of tumors of the thyroid gland

A study of the preoperative fine needle aspiration cytologies in consecutive patients with primary malignant tumors (203 cases) or benign thyroid tumors (217 cases) showed a sensitivity of 0.57 and a specificity of 0.98. The sensitivity of FNA cytology in medullary and undifferentiated carcinomas was 0.82 and 0.84, respectively; none of these were microscopically misdiagnosed. The sensitivity was only 0.58 for papillary carcinomas (excluding occult carcinoma) and 0.42 for follicular carcinoma. Four reasons for these low sensitivities were identified: tumors missed at aspiration, microscopic misinterpretations, diagnoses of cellular atypia and indeterminate diagnoses. Reevaluation of the false diagnoses once more emphasized the problem of distinguishing follicular adenomas from follicular carcinomas. Microscopically undiagnosed papillary carcinomas were either the result of misinterpretations of the characteristic cytomorphologic features or of the smears being misdiagnosed as showing cellular atypia when papillary formations were missing and only one or two of the other cellular criteria were evident. The specificity of FNA cytology of thyroid tumors was found to be high enough to permit surgical intervention after a cytodiagnosis of malignancy.     

Role of the insulin-like growth factor system in adrenocortical growth control and carcinogenesis.

Clinically silent adrenocortical adenomas are the most frequent abnormalities in the adrenal gland. In contrast, adrenocortical carcinoma is a rare tumor with an extremely poor prognosis. The factors responsible for the frequent occurrence of benign adrenocortical tumors on one hand and the rare malignant transformation on the other are not known. Several genetic alterations such as loss of imprinting or loss of heterozygosity of the 11p15 gene locus causing a strong IGF-II overexpression have been demonstrated in the majority of adrenocortical carcinomas. In addition to IGF-II overexpression, increased levels of the IGF-I-receptor and IGFBP-2 have been found in advanced human adrenocortical carcinomas, suggesting an important role for the IGF-system in adrenocortical carcinogenesis. IGFs are potent mitogens regulating growth and apoptosis through interaction with the IGF-I-receptor, and overexpression of the human IGF-I-receptor promotes ligand-dependent neoplastic transformation in a variety of different cell systems. It is evident, therefore, that high levels of IGF-II in combination with overexpression of the IGF-I-receptor can provide a significant growth advantage for adrenocortical carcinoma cells and thus contribute to the highly malignant phenotype of this rare type of cancer. Additionally, it has been shown that overexpression of IGFBP-2 can promote malignant transformation of Y1 mouse adrenocortical tumor cells through unknown IGF-independent mechanisms. As one possible mechanism, we have recently found altered expression of catalase in IGFBP-2-overexpressing tumor cells, thus implicating IGFBP-2 in influencing intracellular peroxide levels. However, since transgenic mice with IGF-II or IGFBP-2 overexpression in the adrenal gland do not show an increased frequency of adrenal tumors, IGF-II or IGFBP-2 may act as progression factors but not as initiation factors in adrenocortical tumorigenesis.     

Does the tumor microenvironment influence radiation-induced apoptosis?

Cytotoxic anti-cancer agents induce apoptosis in tumor and normal tissues. Therefore, it is important to investigate which factors determine these apoptotic processes and hence their likely impact on therapeutic gain. Radiation-induced apoptosis in tumors may be inhibited due to mutations of apoptotic elements or to tumor microenvironmental conditions arising from vascular insufficiency. Tumors typically contain regions of hypoxia, low glucose and acidosis. Hypoxic cells compromise treatment partly because of reduced fixation of damage during radiotherapy and partly because they promote a more malignant phenotype. There is also evidence that hypoxia may inhibit apoptosis. For some cell types, concurrent hypoxia may modulate radiation-induced apoptosis while, for others, post-irradiation hypoxia may be required. This may reflect the activity of different apoptotic pathways. Pathways involving mitochondrial components as well as regulation of SAPK and Fas have been implicated. In addition, several key stages in apoptosis are sensitive to depletion of cellular energy reserves, which results from hypoxia and low glucose conditions. There is also evidence that low pH in tumors can interfere with radiation-induced apoptosis, partly through cell cycle arrest and other undefined mechanisms. Conclusions: Hypoxia, low glucose and acidosis influence radiation-induced apoptosis and thus may be detrimental to radiotherapy.     

Glial fibrillary acidic protein and desmin in salivary neoplasms

The presence of intermediate filament proteins (IFP) in normal salivary gland tissue and in a number of salivary gland neoplasms has been investigated by immunohistochemical techniques on frozen sections. Cytokeratins (CKs) were seen in almost all normal epithelial cells. In the parotid gland and in palatal gland tissue, a co-expression of cytokeratin and glial fibrillary acidic protein (GFAP) was seen in some myoepithelial cells, but this was not apparent in the submandibular gland. In some pleomorphic adenomas, carcinomas in pleomorphic adenomas, one mucoepidermoid carcinoma, one mucus-producing adenopapillary carcinoma and one adenoid cystic carcinoma, cells expressing three different IFP classes were found (CKs, vimentin, GFAP). These cells were most often situated peripherally in the tumour cords or ducts. The cytokeratin pattern in these cells, as revealed by mAbs PKK1-3, was similar to that in normal myoepithelial cells. Furthermore, reactivity for a fourth class of IFP, desmin, could be seen in this cell type in two carcinomas in pleomorphic adenomas, and also in a few cells in a pleomorphic adenoma and an adenoid cystic carcinoma. Thus the pattern of IFP expression in salivary gland neoplasms, is very complex, and cannot always be related to the normal tissue.     

Comparative localization of cathepsin B protein and activity in colorectal cancer.

Cathepsin B is a lysosomal cysteine proteinase that may participate in cancer progression. We compared localization of its protein and activity during progression of human colorectal cancer. In adenomas and carcinomas, protein expression and, particularly, activity were elevated compared with those in normal colorectal mucosa. In normal mucosa, cathepsin B protein expression was moderate in stroma and variable in epithelium, whereas activity was mainly present in distinct areas of stroma directly underneath the surface of the colon and in epithelium at the surface of the colon. Stroma in adenomas and carcinomas contained moderate to high protein levels but little activity except for areas of angiogenesis, inflammation, and necrosis, in which activity was high. In adenomas and the majority of well-differentiated carcinomas and moderately differentiated carcinomas, cathepsin B protein and activity were found in granular form in the epithelium, close to the basement membrane. Protein and activity levels were low and diffusely distributed in cancer cells in the remainder of the well-differentiated and moderately differentiated carcinomas and in all poorly differentiated carcinomas. Invasive fronts in most cancers contained moderate protein levels but high activity. We conclude that (a) activity localization is essential to understand the role of cathepsin B in cancer progression, and (b) cathepsin B activity in human colon is associated with invasion of cancer cells, endothelial cells, and inflammatory cells, and in cell death, both apoptotic and necrotic.     

Gene expression profiling reveals two separate mechanisms regulating apoptosis in rectal carcinomas in vivo

The level of apoptosis in rectal carcinomas of patients treated by surgery only predicts local failure; patients with intrinsically high-apoptotic tumors develop less local recurrences than patients with low levels of apoptosis. To identify genes involved in this intrinsic apoptotic process in vivo, 47 rectal tumors with known apoptotic phenotype (24 low- and 23 high-apoptotic) were analyzed by oligonucleotide microarray technology. We identified several genes differentially expressed between low- and high-apoptotic tumors. Unsupervised clustering of the tumors based on expression levels of these genes separated the low-apoptotic from the high-apoptotic tumors, indicating a gene expression-dependent regulation. In addition, this clustering revealed two subgroups of high-apoptotic tumors. One high-apoptotic subgroup showed subtle differences in mRNA and protein expression of the known apoptotic regulators BAX, cIAP2 and ARC compared to the low-apoptotic tumors. The other subgroup of high-apoptotic tumors showed high expression of immune-related genes; predominantly HLA class II and chemokines, but also HLA class I and interferon-inducible genes were highly expressed. Immunohistochemistry revealed HLA-DR expression in epithelial tumor cells in 70% of these high-apoptotic tumors. The expression data suggest that high levels of apoptosis in rectal carcinoma patients can be the result of either slightly altered expression of known pro- and anti-apoptotic genes or high expression of immune-related genes.     

What if Many Follicular Variant Papillary Thyroid Carcinomas are not Malignant? a Review of Follicular Variant Papillary Thyroid Carcinoma and a Proposal for a New Classification

ObjectivesTo review the relevant literature concerning follicular variant of papillary thyroid carcinoma (FVPTC) with an emphasis on the heterogeneity of this disorder and to propose a new classification for FVPTC on the basis of molecular diagnostics and apply the classification to a typical case.MethodsEnglish-language articles pertaining to FVPTC published between January 1990 and December 2010 were reviewed.ResultsFVPTC is particularly vexing. The criteria for diagnosing FVPTC appear to have changed over the years. Pathologists often disagree about the diagnosis of FVPTC. The clinical behavior of these tumors is variable. Molecular diagnostic studies suggest that FVPTC represents a heterogeneous group of disorders rather than a single entity.ConclusionsOn the basis of the available data, it is proposed that individual cases of FVPTC be reclassified as papillary thyroid carcinoma, follicular thyroid carcinoma, or follicular adenomas, after appropriate molecularbiologic studies have been completed. Long-term followup studies to validate this classification are necessary. (Endocr Pract. 2011;17:768-787)     

Increased expression of the enzyme beta 1-4-galactosyltransferase is associated with human parotid neoplasms

Human biopsy samples of parotid gland neoplasms were examined for the level of enzyme activity of the glycosyltransferase, beta 1-4-galactosyltransferase. An analysis of an adenoid cystic carcinoma, Warthin's tumor, mucoepidermoid carcinoma, and five pleomorphic adenomas all revealed elevated levels of enzyme activity. Evidence for plasma membrane beta 1-4-galactosyltransferase activity was provided by membrane fractionation as well as intact cell enzyme assays. On the other hand, the major protein of human saliva, salivary alpha-amylase, was substantially reduced in the same tissue compared with adjacent normal parotid gland tissue. The trichloroacetic acid-soluble proteins isolated from gland homogenates were also reduced in two of the carcinoma samples but increased in the pleomorphic adenomas. Additionally, the proliferation of these cells, in vitro, could be retarded by culturing in media containing the galactosyltransferase specific modifier protein, alpha-lactalbumin, or the nucleotide sugar, UDP-galactose.     

Comparison of CD15, galectin-3 and HBME-1 expression in follicular thyroid neoplasms

INTRODUCTION: Estimation of malignancy in thyroid follicular neoplasms is a common diagnostic problem, thus revealing of differences in expression of some antigens in both benign and malignant lesions seems to be essential. The aim of this study is to evaluate the immunohistochemical expression of CD15, galectin-3 and HBME-1 in follicular adenomas and carcinomas. MATERIAL AND METHODS: Samples of 38 follicular adenomas (23 "classical", 5 with intracapsular invasion, 10 oncocytic) and 15 follicular carcinomas (9 "classic", 6 oncocytic) were stained immunohistochemically with anti-CD15, galectin-3 and HBME-1. RESULTS: In the whole group we found statistically significant differences in CD15 expression between follicular adenomas and carcinomas. "Classic" follicular carcinomas (without oncocytic tumors) showed stronger CD15 and HBME- 1 expression than "classic" adenomas. Adenomas with intracapsular invasion differed from "classic" adenomas only in HBME-1 expression. In oncocytic tumors the expression of examined antigens was similar. CONCLUSIONS: 1. In the group of nonoxyphilic tumors positive reaction with HBME-1 was more common in adenomas with intracapsular invasion and carcinomas, but positive reaction with anti-CD15--only in carcinomas. We suggest that reactivity with these antibodies could mark malignancy. 2. Oncocytic tumors had similar expression of CD15 and HBME-1 and galectin-3.     

Telomerase activity in thyroid fine needle aspirates

OBJECTIVE: To study the utility of telomerase activity (TA) detection in thyroid fine needle aspirates (FNAs). STUDY DESIGN: One hundred two thyroid nodules were studied: 70 in FNA samples and 32 in frozen tumors. From among FNA samples, there were 57 nodules of the goiter, 1 adenoma, 7 papillary carcinomas, 1 medullary carcinoma and 1 lymphoma. Three cases of thyroiditis were excluded. The 32 frozen tissue tumors consisted of 5 follicular adenomas, 4 follicular carcinomas and 23 papillary carcinomas. TA was analyzed by Telomerase PCR ELISA (Roche Diagnostics, Indianapolis, Indiana, U.S.A.). RESULTS: TA was negative in the 57 nonneoplastic nodules and 6 follicular adenomas and positive in 3 of 4 follicular carcinomas and 10 of 30 papillary carcinomas. TA sensitivity was 41.4% and specificity 100%. Sensitivity for malignancy was higher (85.7%) in FNAs than in TA. CONCLUSION: TA seems highly specific for neoplasms of the thyroid. Further studies are needed to confirm whether TA detection could contribute to identifying neoplasms when FNAs are inconclusive for malignancy and in cases of scanty material.     

An Immunohistochemical Study of Leu 7 and PCNA Expression in Thyroid Neoplasms

Monoclonal Antibody (MoAb) HNK, or anti-leu-7, is reactive with several neuroendocrine and nonneuroendocrine tumors. The aim of this study is to examine anti-leu-7 reactivity in thyroid neoplasms and its relationship to cellular proliferation as determined by anti-PCNA reactivity. The expression of anti-leu-7 in 56 thyroid neoplasms (24 papillary carcinomas, 14 follicular carcinomas, two medullary carcinomas and 16 follicular adenomas) was examined immunohistochemically. Papillary and follicular thyroid carcinomas reacted with anti-leu-7 in a membranous and cytoplasmic pattern in 88% and 93% of cases, respectively. The adjacent benign tissues were nonreactive. Only eight cases diagnosed as follicular adenomas were reactive with anti-leu-7. Furthermore, the mean proliferative index (PI), as measured by the percentage of nuclei immunoreactive with anti-PCNA, was greater than 30% in all thyroid neoplasms reactive with anti-leu-7. The PI was 58% for papillary carcinomas and 68% and 48% for follicular carcinomas, and follicular adenomas, respectively. Lesions originally classified as follicular adenomas that were nonreactive with anti-leu-7 had a PI of 24% and were reclassified as hyperplastic nodules. These data suggest that anti-leu-7 may be useful for characterizing thyroid neoplasia.     

Mitochondrial D-Loop instability in thyroid tumours is not a marker of malignancy

Despite the numerous studies describing a high frequency of mitochondrial DNA (mtDNA) somatic mutations in many types of human primary tumors the mechanisms that generate such mutations and the role of mtDNA mutations in tumor development remain unclear. We present the results obtained in the study of mtDNA displacement-loop (D-Loop) region in a series of 66 thyroid tumors, and respective adjacent parenchyma, including benign (adenomas, n=30) and malignant tumors (follicular carcinomas, n=17 and papillary carcinomas, n=19). Three repetitive regions were analyzed [two mononucleotide repetitive (D310 and D568) and one dinucleotide repetitive (D514)]. Thirty-two (48.5%) of the 66 tumors [15/30 (50.0%) adenomas, 8/17 (47.1%) follicular carcinomas and 9/19 (47.4%) papillary carcinomas] harbored somatic insertions in D-Loop repetitive regions. Twenty (30.3%) of the 66 tumors [12/30 (40%) adenomas, 3/17 (17.6%) follicular carcinomas and 5/19 (26.3%) papillary carcinomas] harbored somatic insertions at the D310 mononucleotide repeat. Three (4.6%) of the 66 tumors [1/30 (3.3%) adenomas and 2/17 (11.8%) follicular carcinomas] harbored somatic insertions at the D568 mononucleotide repeat. Fifteen (22.7%) of the 66 tumors [3/30 (10.0%) adenomas, 5/17 (29.4%) follicular carcinomas and 7/19 (36.8%) papillary carcinomas] harbored somatic insertions at the D514 dinucleotide repeat. Five (7.6%) of the 66 tumors [1/30 (3.3%) adenomas, 1/17 (5.9%) follicular carcinomas and 2/19 (10.5%) papillary carcinomas] harbored somatic insertions in more than one region, and in one of them (a carcinoma) alterations were detected in the three regions. We conclude that mutations in the mtDNA D-Loop region are frequent in benign and malignant thyroid tumors and cannot be considered a marker of malignancy. Our study shows, furthermore, two repetitive regions (D310 and D514) that appear to be susceptible to mutation in thyroid tumors.     

An Immunohistochemical Study of Leu 7 and PCNA Expression in Thyroid Neoplasms

Monoclonal Antibody (MoAb) HNK, or anti-leu-7, is reactive with several neuroendocrine and nonneuroendocrine tumors. The aim of this study is to examine anti-leu-7 reactivity in thyroid neoplasms and its relationship to cellular proliferation as determined by anti-PCNA reactivity. The expression of anti-leu-7 in 56 thyroid neoplasms (24 papillary carcinomas, 14 follicular carcinomas, two medullary carcinomas and 16 follicular adenomas) was examined immunohistochemically. Papillary and follicular thyroid carcinomas reacted with anti-leu-7 in a membranous and cytoplasmic pattern in 88% and 93% of cases, respectively. The adjacent benign tissues were nonreactive. Only eight cases diagnosed as follicular adenomas were reactive with anti-leu-7. Furthermore, the mean proliferative index (PI), as measured by the percentage of nuclei immunoreactive with anti-PCNA, was greater than 30% in all thyroid neoplasms reactive with anti-leu-7. The PI was 58% for papillary carcinomas and 68% and 48% for follicular carcinomas, and follicular adenomas, respectively. Lesions originally classified as follicular adenomas that were nonreactive with anti-leu-7 had a PI of 24% and were reclassified as hyperplastic nodules. These data suggest that anti-leu-7 may be useful for characterizing thyroid neoplasia.