IGFBP7在甲状腺乳头状癌中的表达及意义

目的观察胰岛素样生长结合蛋白-7(IGFBP7)在甲状腺乳头状癌中的表达及意义。方法对87例甲状腺乳头癌(PTC)组织及癌旁组织(PeT)进行IGFBP7、Ki67、p53免疫组织化学分析,其中10例提取蛋白用半定量western blot方法进行IGFBP7表达水平分析。结果 (1)IGFBP7免疫组化显示,PTC癌组织IGFBP7阳性率达78.16%(68/87),明显高于癌旁组织的31.03%(27/87)(P0.05);(2)Western blot灰度值比较,癌组织IGFBP7表达水平显著高于癌旁组织(t=2.875,P0.05);(3)Ki67、p53与IGFBP7相关性分析显示,p53表达水平与IGFBP7表达水平呈显著正相关性(r=0.261,P0.05),而Ki67表达水平与IGFBP7表达水平无相关性(r=0.148,P=0.170);(4)高水平表达的IGFBP7与PTC淋巴结转移高度相关(r=0.238,P0.05)。结论 IGFBP7表达异常增高可能与甲状腺乳头状癌发生及发展有关,有望成为诊断PTC的一项新型的生物标记物。     

Technological difficulties in ghrelin and obestatin assays

In recent years we have performed more than 1,000 radioimmunoassays of ghrelin and obestatin. In these assays, we have encountered several technological obstacles. Another difficulty was the enormous discrepancy of plasma ghrelin results published by different authors. The aim of this article is to comment on these problems. Not all peptides of the hypothalamus and intestines are present in blood circulation. Several neuropeptides do not cross the blood-brain barrier, and several gastrointestinal peptides are present in extremely low concentrations in the blood. That requires time-consuming and laborious extraction. In these procedures, considerable amounts of peptides may be lost. In addition, these peptides are very unstable and prone to enzymatic degradation. This makes it mandatory to add enzymatic inhibitors to plasma samples. The peptides are also unstable in elevated temperatures, hence the assays should be performed in air-conditioned laboratories and the kits should be transported in proper low temperature conditions. Peptides may appear in several isoforms of different biological activity, but antibodies routinely used in these assays are polyclonal and do not differentiate between these forms. This complicates clinical evaluation of the results. To date, there are no international standards of ghrelin, obestatin or other active peptides, probably because of their extreme instability. Because of technological difficulties, the results of peptide assays performed in different scientific research institutions vary greatly and cannot be compared to each other. This disadvantage may be partially diminished by including samples of healthy subjects in each assay run to check whether the peptide concentrations of the patients differ significantly from that of control subjects.     

Twist及Akt2在甲状腺乳头状癌中的表达及意义

目的探讨Twist、Akt2在甲状腺乳头状癌中的表达和相互关系。方法应用免疫组化方法,对60例甲状腺乳头状癌、10例结节性甲状腺肿进行Twist、Akt2表达的研究。结果甲状腺乳头状癌Twist及Akt2阳性表达率分别为81.67%(49/60)及60.00%(36/60),结节性甲状腺肿Twist及Akt2阳性表达率分别为0.00%(0/10)及10.00%(1/10),前者与后者相比差异有统计学意义(P<0.05);Twist及Akt2在甲状腺乳头状癌中的表达水平与病人的年龄及原发肿瘤分期无关,但与有无淋巴结转移相关(P<0.05),且Akt2的表达在T3,4期肿瘤及T1,2期肿瘤相比也具有显著性(P<0.05);Twist及Akt2的表达呈正相关(r=0.492,P=0.000)。结论在大多数甲状腺乳头状癌的上皮细胞中都存在Twist及Akt2的过表达,其淋巴结转移的发生与Twist及Akt2过表达或功能的不正常可能有密切的关系;Twist与Akt2的表达呈正相关。     

Nuclear envelope organization in papillary thyroid carcinoma

Papillary thyroid carcinomas (PTCs) have characteristic nuclear shape changes compared to follicular-type thyroid epithelium. We tested the hypothesis that the altered nuclear shape results from altered distribution or expression of the major structural proteins of the nuclear envelope. Lamin A, lamin B1, lamin C, lamin B receptor (LBR), lamina-associated polypeptide 2 (LAP2), emerin, and nuclear pores were examined. PTC's with typical nuclear features by H&E were compared to non-neoplastic thyroid and follicular neoplasms using confocal microscopy, and semi-quantitative immunoblotting. Lamin A/C, lamin B1, LAP2, emerin, and nuclear pores all extend throughout the grooves and intranuclear inclusions of PTC. Their distribution and fluorescent intensity is not predictably altered relative to nuclear envelope irregularities. By immunoblotting, the abundance (per cell) and electrophoretic mobilities of lamin A, lamin B1, lamin C, emerin, and LAP2 proteins do not distinguish PTC, normal thyroid, or follicular neoplasms. These results do not support previously published predictions that lamin A/C expression is related to a loss of proliferative activity. At least three LAP2 isoforms are identified in normal and neoplastic thyroid. LBR is sparse or undetectable in all the thyroid samples. The results suggest that the irregular nuclear shape of PTC is not determined by these nuclear envelope structural proteins per se. We review the structure of the nuclear envelope, the major factors that determine nuclear shape, and the possible functional consequences of its alteration in PTC.     

Ghrelin, des-acyl ghrelin and obestatin on the gastrointestinal motility

Ghrelin, des-acyl ghrelin and obestatin are derived from a common prohormone, preproghrelin by posttranslational processing, originating from endocrine cells in the stomach. Ghrelin exerts stimulatory effects on the motility of antrum and duodenum in both fed and fasted state of animals. On the other hand, des-acyl ghrelin exerts inhibitory effects on the motility of antrum but not on the motility of duodenum in the fasted state of animals. Obestatin exerts inhibitory effects on the motility of antrum and duodenum in the fed state but not in the fasted state of animals. NPY Y2 and Y4 receptors in the brain may mediate the action of ghrelin, CRF type 2 receptor in the brain may mediate the action of des-acyl ghrelin, whereas CRF type 1 and type 2 receptors in the brain may mediate the action of obestatin.     

MicroRNA signature predicts survival in papillary thyroid carcinoma

Papillary thyroid cancer (PTC) accounts for the majority of malignant thyroid tumors. Recently, several microRNA (miRNA) expression profiling studies have used bioinformatics to suggest miRNA signatures as potential prognostic biomarkers in various malignancies. However, a prognostic miRNA biomarker has not yet been established for PTC. The aim of the present study was to identify miRNAs with prognostic value for the overall survival (OS) of patients with PTC by analyzing high-throughput miRNA data and their associated clinical characteristics downloaded from The Cancer Genome Atlas database. From our dataset, 150 differentially expressed miRNAs were identified between tumor and nontumor samples; of these miRNAs, 118 were upregulated and 32 were downregulated. Among the 150 differentially expressed miRNAs, a four miRNA signature was identified that reliably predicts OS in patients with PTC. This miRNA signature was able to classify patients into a high-risk group and a low-risk group with a significant difference in OS (P < .01). The prognostic value of the signature was validated in a testing set ( P < .01). The four miRNA signature was an independent prognostic predictor according to the multivariate analysis and demonstrated good performance in predicting 5-year disease survival with an area under the receiver operating characteristic curve area under the curve (AUC) score of 0.886. Thus, this signature may serve as a novel biomarker for predicting the survival of patients with PTC.     

BRAF initiating mutations in the papillary thyroid carcinoma

Among genetic alterations most important for the initiation of papillary thyroid carcinoma (PTC) is mutation T1799A in the BRAF gene which is the most frequent event (54.5%) in this type of thyroid cancer. It is seen in all stages, from microcarcinoma through clinically overt disease to anaplastic cancer. It has been shown that BRAF mutation is correlated with PTC histotype. It is identified most frequently in classical PTC and in tall cell variant. Moreover, BRAF mutation is described more often in older patients, whereas in young patients RET/PTC rearrangements dominate. In PTC cases with BRAF mutation V600E the prognosis is poorer, with more cancer invasiveness, metastasis and recurrence. The presence of BRAF mutation is related to the specific gene expression signature, different than in cancer cases showing RET/PTC rearrangement or no known initiating mutation.     

Expression of CD44 and cyclin D1 in fine needle aspiration cytology of papillary thyroid carcinoma

OBJECTIVE: To compare the expression pattern of CD44 and cyclin D1 immunostaining in fine needle aspiration specimens of papillary carcinoma of the thyroid and nonpapillary lesions. STUDY DESIGN: The study was performed on 80 fine needle aspiration cytologic smears of thyroid lesion retrospectively using monoclonal antibodies and on histologic material from a proportion of cases. RESULTS: Most papillary carcinomas expressed intense cell membrane or diffuse cytoplasmic staining for CD44 (97.8%). Focal immunoreactivity was observed in follicular neoplasms (28.5%) and nodular goiter (4.7%). There was no difference in CD44 immunostaining between follicular carcinoma and adenoma. Cyclin D1 was expressed in the nuclei of most papillary carcinomas (79.2%). Focal nuclear immunoreactivity was noted in nodular goiters (23.5%) and follicular neoplasms (10%). In resected specimens, all papillary carcinomas (19 cases) showed intense membranous or granular CD44 immunoreactivity. Focal cyclin D1 expression was noted in 52.6%. There was no difference in CD44 and cyclin D1 expression between the group of papillary carcinomas with regional lymph node metastasis as compared to those without metastasis. Positive staining for both CD44 and cyclin D1 would strongly favor papillary carcinoma, although further studies on cytologic material are necessary to verify this diagnostic approach. CONCLUSION: Most papillary carcinomas express CD44 and cyclin D1, whereas it is less common in follicular neoplasms and nodular goiter. This may be helpful in diagnostically difficult cases.     

Expression of cytokeratin 19 and protein p63 in fine needle aspiration biopsy of papillary thyroid carcinoma

OBJECTIVE: To analyze the immunocytochemical distribution of CK19 and p63 on archival cytologic smears of 27 papillary thyroid carcinomas (PTCs), 22 benign thyroid lesions and 5 malignant non-PTC lesions. STUDY DESIGN: Archival cytologic smears of 27 papillary carcinomas, 22 benign thyroid lesions and 5 malignant nonpapillary carcinomas were processed for immunocytochemical detection of CK19 and p63, and results were compared. RESULTS: CK19 showed strong cytoplasmic staining in 22/27 fine needle aspiration biopsies (FNABs) of PTCs, in 5 benign lesions and in 4 malignant lesions of the control group. p63 Positivity was present in FNABs of 20/27 PTC and in 1 FNAB of nodular hyperplasia. Eighteen FNABs of PTC (66.6%) showed both strong CK19 staining and p63-positive cells, whereas none of the control cases showed coexpression of CK19 and p63. CONCLUSION: Coexistence of strong CK19 positivity and p63-positive cells can enhance the cytologic diagnosis of PTC.     

Rearrangements of NTRK1 oncogene in papillary thyroid carcinoma

Papillary thyroid carcinoma (PTC) represents an example of tumour with high incidence of oncogenic sequences, such as RET/PTC and Trk. Both of them arise from the fusion of 3' terminal sequences of TK domain of RET or NTRK1 gene, respectively, with 5' terminal sequences of their activating genes. In case of NTRK1 oncogene, several rearrangement types are observed, characteristic for PTC: Trk (TMP3), Trk-T1, Trk-T2, Trk-T3 and Trk-2h, observed in human breast cancer cell line. Studies from different geographical regions, revealed significant population differences in the incidence of Trk rearrangements (0-50%), while within the same population, the frequency of Trk in spontaneous and radiation-associated PTCs is similar. The results of studies, focused on the correlation between tumour genotype and the histopathological type of tumour, involving cases of both RET/PTC and Trk rearrangements in PTC, are not unequivocal. In many studies, no correlation was observed between the presence of RET and/or NTRK1 rearrangement and such parameters, as patient's age at diagnosis, gender, histopathological type of tumour or clinical stage (TNM stage grouping), although the earliest clinical symptoms and the worst disease outcomes were observed for RET/NTRK1 rearrangement positive tumours. Differences in the rearrangement incidence between male and female patients were associated with the latency period of radiation-associated tumours, being significantly lower in women. In general, it is assumed that oncogenic Trk sequences are typical for the spontaneous type of PTC.     

Proliferative activity of papillary carcinoma and benign papillary hyperplasia of thyroid follicular cells

Papillary structures of follicular cells are observed in nodular goiter, cysts, hyperplastic areas of follicular tumors, Graves' disease, thyroiditis and carcinomas. The distinction of papillary carcinomas from benign lesions has important implication for clinical management. The aim of the study was to test a marker of proliferation activity (MIB-1) in the diagnosis of benign and malignant thyroid papillary proliferation. The study was carried out in 98 women with papillary carcinoma, nodular goiter. intracystic proliferation. Graves' disease and hyperplastic areas of follicular benign tumors. The formalin fixed, paraffin-embedded specimens were microscopically examined using HE staining and immunostaining with MIB-1 antibody (DAKO). The proliferative index (PI) was significantly higher in malignant than in benign papillary hyperplasia. Our results may provide additional information for differential papillary proliferation diagnosis by FNAB.     

DNA microarrays and papillary thyroid carcinoma gene expression profile

The paper presents gene expression profile analysis with DNA microarrays and compares two core technological platforms used for this purpose - high density oligonucleotide microarrays and cDNA microarrays. With this background recent results of papillary thyroid carcinoma analysis with DNA microarrays are presented.     

Food restriction,ghrelin, its antagonist and obestatin control expression of ghrelin and its receptor in chicken hypothalamus and ovary

The purpose of the present study was to identify the role of age, nutritional state and some metabolic hormones in control of avian hypothalamic and ovarian ghrelin/ghrelin receptor system. We examined the effect of food restriction, administration of ghrelin 1–18, ghrelin antagonistic analogue (D-Lys-3)-GHRP-6, obestatin and combinations of them on the expression of ghrelin and ghrelin receptor (GHS-R1a) in hypothalamus and ovary of old (23 months of age) and young (7 months of age) chickens. Expression of mRNAs for ghrelin and GHS-R1a in both hypothalamus and largest ovarian follicle was measured by RT-PCR. It was observed that food restriction could promote the expression of ghrelin and GHS-R1a in hypothalamus and ovary of the old chickens, but in the young chickens it reduced expression of ghrelin and did not affect expression of GHS-R1a in the ovary. Administration of ghrelin 1–18 did not affect hypothalamic or ovarian ghrelin mRNA, but significantly increased the expression of GHS-R1a in hypothalamus, but not in ovary. (D-Lys-3)-GHRP-6, significantly stimulated accumulation of ghrelin, but not GHS-R1a mRNA in hypothalamus or ghrelin or GHS-R1a in the ovary. Ghrelin 1–18 and (D-Lys-3)-GHRP-6, when given together, were able either to prevent or to induce effect of these hormones. Obestatin administration increased expression of ghrelin gene in the hypothalamus, but not expression of hypothalamic GHS-R1a, ovarian ghrelin and GHS-R1a. Furthermore, obestatin was able to modify effect of both ghrelin and fasting on hypothalamic and ovarian mRNA for ghrelin GHS-R1a. Our results (1) confirm the existence of ghrelin and its functional receptors GHS-R1a in the chicken hypothalamus and ovary (2) confirm the age-dependent control of ovarian ghrelin by feeding, (3) demonstrate, that nutritional status can influence the expression of both ghrelin and GHS-R1a in hypothalamus and in the ovary (3) demonstrates for the first time, that ghrelin can promote generation of its functional receptor in the hypothalamus, but not in the ovary, (4) show that ghrelin1–18 and (D-Lys-3)-GHRP-6 could not only be antagonists in the action on chicken hypothalamus and ovaries, but also independent regulators and even agonists, and (5) provide first evidence for action of obestatin on hypothalamic ghrelin and on the response of hypothalamic and ovarian ghrelin/GHS-R1a system to food restriction. These data indicate the involvement of both hypothalamic and ovarian ghrelin/GHS-R1 systems in mediating the effects of nutritional status, ghrelin and obestatin on reproductive processes.     

Subpopulations of cancer stem cells found in papillary thyroid carcinoma

Papillary thyroid carcinoma (PTC) is the most common form of thyroid cancer and while it has a generally good prognosis, tumor recurrence remains a major clinical challenge. Studying laboratory cell lines as well as clinical specimens indicate that PTC may follow the cancer stem cell (CSC) model. However, CSC characteristics relevant in PTC initiation and progression remain largely unknown. Here we studied a population of sphere-growing tumor cells isolated from primary cultures of clinical PTC. These sphere-growing cells consisted of aldehyde dehydrogenase positive (ALDH⁺) and ALDH negative (ALDH^-) cell subpopulations and demonstrated a hierarchical pattern of cell division. Using combinations of selective depletion, specific inhibition and cell sorting, we found that both subpopulations of the sphere cells were able to self-renew and initiate xenograft tumors independently, and fulfilled the definition of CSC. Importantly, when the subpopulations functioned together, the cancer-initiation efficiency and the xenograft tumor progression were significantly enhanced compared to either subpopulation alone. These data revealed crucial roles of ALDH^- CSC in PTC biology and suggested that CSC subpopulations function cooperatively to control PTC initiation and progression. Together, our study indicates that CSC subpopulations isolated from clinical specimens offer unprecedented opportunities for investigating PTC pathogenesis and developing effective therapies.     

Molecular analysis of structural abnormalities in papillary thyroid carcinoma gene

Rearrangements of the RET proto-oncogene (RET/PTC) and BRAF gene mutations are the major genetic alterations in the etiopathogenesis of papillary thyroid carcinoma (PTC). We have analyzed a series of 118 benign and malignant follicular cell-derived thyroid tumors for RET/PTC rearrangements and BRAF gene mutations. Oncogenic rearrangements of RET proto-oncogene was revealed by semiquantitative RT-PCR of simultaneously generated fragments corresponding to tyrosine kinase (TK) and extracellular RET domains. The clear quantitative shift toward the TK fragment is indicative for the presence of RET rearrangements. The overall frequency of RET/PTC rearrangements in PTC was 14% (12 of 85), including 7 RET/PTC1, 2 RET/PTC3, 1 deltaRFP/RET and 2 apparently uncharacterized rearrangements. The most common T1796A transversion in BRAF gene was detected in 55 of 91 PTC (60%) using mutant-allele-specific PCR. We also identified two additional mutations: the substitution G1753A (E585K) and a case of 12-bp deletion in BRAF exon 15. Moreover, there was no overlap between PTC harboring BRAF and RET/PTC mutations, which altogether were present in 75.8% of cases (69 of 91). Taken together, our observations are consistent with the notion that BRAF mutations appear to be an alternative pathway to oncogenic MAPK activation in PTCs without RET/PTC activation. Neither RET/PTC rearrangements nor BRAF muta-tions were detected in any of 3 follicular thyroid carcinomas, 11 follicular adenomas and 13 nodular goiters. The high prevalence of BRAF mutations and RET/PTC rearrangements in PTCs and the specificity of these alterations to PTC make them potentially important markers for the preoperative tumor diagnosis.