Flow cytometric DNA measurements in human thyroid tumors

By means of flow cytometry (FCM), DNA distribution pattern and the fraction of cells in the various phases of the cell cycle were studied in 52 samples of normal thyroid tissues, follicular adenomas, follicular carcinomas, medullary carcinoma and fibrosarcomas. In the normal thyroid tissues and follicular adenomas DNA diploid cell populations only were found. Among 20 follicular carcinomas in 13 cases (65%) together with the DNA diploid cells, DNA aneuploid cell lines were also observed. S-phase fraction in follicular adenomas is higher than in the normal thyroid tissues and lower than those in thyroid carcinomas. The percentage of S-phase cells in DNA aneuploid populations is significantly higher (S = 19 +/- 9.3%) than in the diploid cell lines (S = 3.7 +/- 2.6%). DNA aneuploid cell populations were predominantly observed in carcinomas with a high degree of morphological anaplasia.     

Delineation of a 150-kb breakpoint cluster in benign thyroid tumors with 19q13.4 aberrations

Structural rearrangements involving the long arm of chromosome 19 characterize a cytogenetic subgroup of benign thyroid tumors and constitute one of the most frequent specific chromosome abnormalities in epithelial tumors. Recently, we have been able to narrow down the breakpoint region affected in two cell lines to a region covered by a single PAC clone. Close to that region a candidate gene has been identified which we tentatively referred to as RITA (Rearranged In Thyroid Adenomas) now named ZNF331 according to HUGO nomenclature. However, the results had been obtained on two cell lines only making it necessary to extend the studies to a larger number of tumors including primary material. Herein, we have used four further primary tumors showing translocations involving 19q13 for fluorescence in situ hybridization (FISH) mapping studies using a variety of molecular probes from a 470-kbp cosmid/BAC contig. Ten new STSs were characterized and physically mapped within an EcoRI restriction map. The results enabled us to define an approximately 150-kbp breakpoint cluster region of the 19q13 aberrations in benign thyroid tumors flanked by two newly established STS markers.     

The Two Stem Cell MicroRNA Gene Clusters C19MC and miR-371-3 Are Activated by Specific Chromosomal Rearrangements in a Subgroup of Thyroid Adenomas

Thyroid adenomas are common benign human tumors with a high prevalence of about 5% of the adult population even in iodine sufficient areas. Rearrangements of chromosomal band 19q13.4 represent a frequent clonal cytogenetic deviation in these tumors making them the most frequent non-random chromosomal translocations in human epithelial tumors at all. Two microRNA (miRNA) gene clusters i.e. C19MC and miR-371-3 are located in close proximity to the breakpoint region of these chromosomal rearrangements and have been checked for a possible up-regulation due to the genomic alteration. In 4/5 cell lines established from thyroid adenomas with 19q13.4 rearrangements and 5/5 primary adenomas with that type of rearrangement both the C19MC and miR-371-3 cluster were found to be significantly overexpressed compared to controls lacking that particular chromosome abnormality. In the remaining cell line qRT-PCR revealed overexpression of members of the miR-371-3 cluster only which might be due to a deletion accompanying the chromosomal rearrangement in that case. In depth molecular characterization of the breakpoint in a cell line from one adenoma of this type reveals the existence of large Pol-II mRNA fragments as the most likely source of up-regulation of the C19MC cluster. The up-regulation of the clusters is likely to be causally associated with the pathogenesis of the corresponding tumors. Of note, the expression of miRNAs miR-520c and miR-373 is known to characterize stem cells and in terms of molecular oncology has been implicated in invasive growth of epithelial cells in vitro and in vivo thus allowing to delineate a distinct molecular subtype of thyroid adenomas. Besides thyroid adenomas rearrangements of 19q13.4 are frequently found in other human neoplasias as well, suggesting that activation of both clusters might be a more general phenomenon in human neoplasias.     

Fibroblast Growth Factor Receptor-2 Expression in Thyroid Tumor Progression: Potential Diagnostic Application

Fibroblast growth factor receptor-2 (FGFR-2) plays an important role in tumorigenesis. In thyroid cancer it has been observed a FGFR-2 down-modulation, but the role of this receptor has not been yet clarified. Therefore, we decided to examine the expression of both FGFR-2 isoform, FGFR-2-IIIb and FGFR-2-IIIc, in different histological thyroid variants such as hyperplasia, follicular adenoma and papillary carcinoma. Immunohistochemistry and quantitative Real-Time PCR analyses were performed on samples of hyperplasia, follicular adenoma and papillary carcinoma, compared with normal thyroid tissue. Thyroid hyperplasia did not show statistically significant reduction in FGFR-2 protein and mRNA levels. Interestingly, in both follicular adenoma and papillary carcinoma samples we observed a strongly reduced expression of both FGFR-2 isoforms. We speculate that FGFR-2 down-modulation might be an early event in thyroid carcinogenesis. Furthermore, we suggest the potential use of FGFR-2 as an early marker for thyroid cancer diagnosis.     

Immunohistochemical evidence for the overexpression of protein kinase C in proliferative diseases of human thyroid.

We report immunohistochemical evidence for the overexpression of protein kinase C in various proliferative diseases of human thyroid. Immunohistochemical characterization of various surgically removed thyroid tissues, viz., cancer tissues: papillary carcinoma and follicular carcinoma; adenoma tissues: tubular, trabecular and colloid adenomas; adenomatous goiter; and normal thyroid was done using the monospecific monoclonal antibodies MC-1a, MC-2a and MC-3a, each of which is specific for types I, II and III isozymes of protein kinase C, respectively. For protein kinase C type II, a remarkable difference in staining intensity was noted between the cancerous and normal tissues. The cytoplasm of papillary and follicular carcinoma cells stained more intensely than that of normal thyroid cells. In the benign tumor and adenomatous goiter tissues, stronger staining was noted in the papilliform-proliferating portion and cubic epithelial cells. In the normal thyroid tissues, epithelial cells of greater height were more strongly stained than simple squamous epithelial cells. These results indicated that protein kinase C type II isozyme is expressed in larger amounts in cancerous and proliferative tissues of the human thyroid.     

The mRNA levels of thyrotropin receptor, thyroglobulin and thyroid peroxidase in neoplastic human thyroid tissues

Expression levels of thyrotropin receptor (TSH-R), thyroglobulin (Tg) and thyroid peroxidase (TPO) mRNA in normal and neoplastic human thyroid tissues (6 adenomas and 7 carcinomas) were investigated by Northern-blot and slot-blot analyses. We found that TSH-R mRNA levels were significantly lower in carcinoma tissues than in normal tissues. The levels of Tg mRNA were also significantly lowered in adenoma and carcinoma tissues as compared to normal tissues. In contrast, no significant difference was observed in the expression levels of TPO mRNA between these tissues. Furthermore, TSH-R mRNA levels were well-correlated with Tg mRNA levels in neoplastic tissues. These results suggest that mRNAs of TSH-R and Tg are expressed in relation to their degree of differentiation.     

Patterns of expression of cytoskeletal proteins in human thyroid gland and thyroid carcinomas

By two-dimensional gel electrophoresis of proteins insoluble in detergents and high-salt buffer and immunofluorescence microscopy with a panel of polypeptide-specific antibodies to proteins of intermediate filaments (IF) and desmosomes, we have characterized the cytoskeletons of normal human thyroid gland, several kinds of benign lesion (goiter, Hashimoto's and Graves' diseases, adenomas), and the major thyroid carcinomas (follicular, papillary, medullary, and anaplastic). In all these tissues, desmoplakins and cytokeratins 7, 8, 18, and 19 were identified. While cytokeratins 8 and 18 occurred in all epithelial cells and cytokeratin 7 was also rather widespread, cytokeratin 19 occurred in amounts variable between the different types of tissues and in normal thyroid gland was restricted to certain clusters of follicular epithelial cells. Of all samples studied, in none did we detect cytokeratins commonly associated with stratified epithelia such as cytokeratins 4-6, 10, and 13-17, indicating that these are infrequent, if at all present, in such tissues. Coexpression of cytokeratins with vimentin appears to occur constitutively in follicular epithelial cells of normal thyroid gland and is also frequent in the diverse carcinomas, though to various degrees. Medullary carcinomas are exceptional, not only because they express neuroendocrine markers, but also because they coexpress combinations of cytokeratin IFs with neurofilaments and/or vimentin IFs in some cases, but not all. The results are discussed in relation to states of cell differentiation in normal and diseased thyroid gland and with respect to their value in tumor diagnosis.     

Deletions of the long arm of chromosome 10 in progression of follicular thyroid tumors

Previous studies of follicular thyroid tumors have shown loss of heterozygosity (LOH) on the short arm of chromosome 3 in carcinomas, and on chromosome 10 in atypical adenomas and carcinomas, but not in common adenomas. We studied LOH on these chromosomal arms in 15 follicular thyroid carcinomas, 19 atypical follicular adenomas and 6 anaplastic (undifferentiated) carcinomas. Deletion mapping of chromosome 10 using 15 polymorphic markers showed that 15 (37.5%) of the tumors displayed LOH somewhere along the long arm. Thirteen of these tumors showed deletions involving the telomeric part of chromosome 10q, distal to D1OS 187. LOH on chromosome 3p was found in 8 (20%) cases. Seven of these also showed LOH on chromosome 10q. In eight cases LOH was seen on chromosome 10q but not 3p. In comparison, the retinoblastoma gene locus at chromosome 13q showed LOH in 22% of the tumors. Most of these also had deletions on chromosome 10q. The results indicate that a region at the telomeric part of 10q may be involved in progression of follicular thyroid tumors.     

The number of nucleoli in benign and malignant thyroid lesions: a useful diagnostic sign in cytological preparations

The slides of fine needle aspiration cytology specimens from 99 cases of cold thyroid nodules with known histology were reviewed and the number of nucleoli per nucleus counted and correlated with the different histopathological groups. Significant differences were observed between benign and malignant thyroid lesions in the number of nucleoli in the cytological material. Lower values were present in nodular goitres and follicular adenomas compared to carcinomas. In benign lesions the majority of nuclei contained one nucleolus and nuclei with two, three or more nucleoli were less frequent than in follicular, papillary, medullary and anaplastic carcinomas. Only one case of follicular adenoma had cells containing three or more nucleoli compared to more than half the cases of follicular carcinoma.     

Comparison of membrane proteins from benign and malignant human thyroid tissues by two-dimensional polyacrylamide gel electrophoresis

In this study two-dimensional (2D) polyacrylamide gel electrophoresis with silver staining was used to analyze cellular membranous proteins of various normal and pathological human thyroid tissues. The aim was to understand the differences in cellular membranous proteins between these tissues, which would aid in the differential diagnosis of thyroid malignancy. Characteristic protein spots had a molecular mass of 50–64 kDa and a pI of 5.7-6.5. There were two groups of isoform protein spots in this area. The higher-molecular-mass group was found in follicular thyroid cancer tissues which and was not visible in normal thyroid tissues. The low-molecular-mass group was found in follicular carcinoma or adenoma tissues and was detected in one to three spots. The papillary thyroid carcinoma tissues gave different 2D gel maps. There were few spots of papillary thyroid carcinoma tissue membranous proteins within the examined area. The 2D gel maps may be used for differential diagnosis of follicular neoplasm. The characteristics of these protein spots require further investigation.     

Frequent epigenetic inactivation of RASSF10 in thyroid cancer

The Ras association domain family (RASSF) encodes for distinct tumor suppressors and several members are frequently silenced in human cancer. In our study, we analyzed the role of a novel RASSF member termed RASSF10 in thyroid carcinogenesis. The RASSF10 CpG island promoter was intensively methylated in nine thyroid cancer cell lines and in 66% of primary thyroid carcinomas. RASSF10 methylation was significantly increased in primary thyroid carcinoma compared to normal thyroid and follicular adenoma (0% and 10%, respectively; p     

13q14断裂重排与非小细胞肺癌转移潜能关系的研究

肿瘤转移的细胞经常存在染色体数目异常和结构畸变,在多种有转移潜能的肿瘤细胞中都涉及到13q14的异常。以往研究表明在同一组织来源但转移潜能不同的肺腺癌细胞系AGZY83-a和Anip973中存在13q14的断裂重排。采用mRNA差异展示技术（mRNA DD）分析这一对细胞系得到的差异表达基因BRI基因位于13q14。为了进一步分析肺癌细胞的转移潜能与13q14断裂重排间的关系,采用13q涂染探针对具有不同转移潜能的非小细胞肺癌细胞系PAa、SPC-1-A和95D中期分裂相进行G显带后的荧光原位杂交分析。结果发现在3个肺癌细胞系中有多种13号染色体长臂的结构异常,其中此3个细胞系均涉及13q32-33的频发断裂。但是低转移肺癌细胞系PAa、SPC-1-A均未涉及13q14的断裂,而高转移肺癌细胞系95D的两种细胞克隆均可见13q14的断裂。提示13q14断裂点与肺癌细胞的转移能力有一定的相关性,两者之间的遗传学意义需要进一步研究探索。     

Detection of cathepsin B up-regulation in neoplastic thyroid tissues by proteomic analysis

Nodular or multinodular goiter is the most common non-neoplastic thyroid disease and may be difficult to distinguish from true neoplastic thyroid diseases using microscopic criteria. We have used two-dimensional gel electrophoresis to study the protein patterns of thyroid tissues including normal thyroid, multinodular goiter, diffuse hyperplasia, follicular adenoma, follicular carcinoma and papillary carcinoma. Specific proteins, in the region of molecular mass 15-30 kDa and isoelectric point 4.5-6.5, were identified by electrospray tandem mass spectrometry and protein sequencing. The most distinctive protein found is cathepsin B, which could be detected as four spots, with differential expression in different thyroid diseases. In particular, two of these cathepsin B spots CB2 and CB3 are strongly up-regulated in neoplastic diseases, compared to non-neoplastic diseases. In addition, overexpression of ATP synthase D chain and prohibitin were observed in papillary carcinoma, which should allow it to be differentiated from follicular carcinoma. Changes in expression of other proteins were also observed in disease states compared to normal tissues, namely translationally controlled tumor protein, thioredoxin peroxidase 1, glutathione-S-transferase P, DJ-1 protein, superoxide dismutase (Cu, Zn), and heat shock protein 27, but these changes are less characteristic, so they do not allow the differentiation between neoplastic and non-neoplastic tissues. Thus, the proteomic approach is a useful diagnostic tool for studying diseases involving the thyroid nodule.