Increased thyroid epithelial cell proliferation in toxic thyroid nodules.

Most toxic thyroid nodules (TTN) result from clonal expansion of a single cell caused by a somatic mutation in the thyrotropin (TSH) receptor, the Gsalpha protein, or yet unknown proteins. Expanding a single cell into a TTN with thousands of cells suggests a prolonged increase in proliferation compared to nonaffected surrounding cells. To test this hypothesis, we evaluated cell proliferation in TTN. Tissue from 20 TTN and their surrounding normal thyroid tissue was studied for the occurrence of the proliferating cell nuclear antigen (PCNA) and Ki-67 epitope as markers for cell proliferation. The labeling index (number of labeled cells versus total cell number) for nodular and surrounding tissue was calculated. Nineteen samples were evaluated for PCNA immunohistochemistry. In 16 TTN, a significant (p< or =0.05%) up to 3-fold increase in the labeling index for PCNA was detectable. In only 3 toxic nodules (2 without a detectable TSH receptor or Gsalpha protein mutation), we found no significant difference in the labeling index compared to the surrounding tissue. Because labeling for KI-67 was much lower, only 16 toxic thyroid nodules were quantified. Twelve of these showed significantly (p< or =0.05%) increased labeling indices. The increase of the labeling index for both markers was similar for histologically defined adenoma versus adenomatous nodule or nodules with or without TSH receptor mutation or clonal versus polyclonal origin of toxic nodules studied. These findings are evidence that an increased thyroid epithelial cell proliferation is a uniform feature common to most TTNs, independent of their histopathological or molecular characteristics. Although increased proliferation in many TTNs is very likely the result of TSH receptor mutations, the cause of increased proliferation in TTN without a mutation is unknown.     

Cold thyroid nodules show a marked increase in proliferation markers.

Thyroid follicular adenomas and adenomatous thyroid nodules are a frequent finding in geographical areas with iodine deficiency. They occur as hypofunctioning (scintigraphically cold) or hyperfunctioning (scintigraphically hot) nodules. Their predominant clonal origin suggests that they result from clonal expansion of a single cell, which is very likely the result of a prolonged increase in proliferation compared with non-affected surrounding cells. To test whether increased cell proliferation is detectable in cold thyroid nodules, we studied paraffin-embedded tissue from 40 cold thyroid nodules and their surrounding normal thyroid tissue for the occurrence of the proliferating cell nuclear antigen (PCNA) and Ki-67 (MIB-1 antibody) epitopes as markers for cell proliferation. All 40 thyroid nodules were histologically well characterized and have been studied for molecular characteristics before. The labeling index (number of labeled cells versus total cell number) for nodular and surrounding tissue was calculated. In 33 cold thyroid nodules a significant (p < or = 0.05) increase in the labeling index for PCNA was detectable. In 19 cold thyroid nodules a significant (p < or = 0.05) increase in the labeling index for Ki-67 was detectable. Moreover, surrounding tissues with lymphocyte infiltration showed a significantly higher labeling index for both PCNA and Ki-67 compared with normal surrounding tissue. These findings are first evidence that an increased thyroid epithelial cell proliferation is a uniform feature common to most cold nodules. However, the increase of proliferation markers shows a heterogeneity that is not correlated with histopathologic, molecular, or clinical characteristics.     

An evaluation of thallium imaging for detection of carcinoma in clinically palpable solitary, nonfunctioning thyroid nodules.

OBJECTIVE: An evaluation of thallium imaging for differentiating benign from malignant lesions in clinically palpable solitary, nonfunctioning, thyroid nodules. METHODS: Seventy-eight patients presenting with a clinically palpable solitary nonfunctioning thyroid nodule were imaged with 3 mCi thallium-201 with a pinhole acquisition at 20 minutes and 3 hours after injection. Thallium uptake was assessed as grade 1, less than the rest of the gland; grade 2, same as the rest of the gland; and grade 3, more than the rest of the gland. All patients underwent surgery and the histology was compared with the thallium scan results. RESULTS: Of the 78 patients presenting with solitary thyroid nodule, 13 were malignant and 65 were benign. Twenty-four patients with benign disease showed no uptake of thallium at 3 hours (grade 1). Thirty-two patients with benign disease and 2 patients with malignant lesion had grade 2 uptake at 3 hours. Eleven patients with malignant disease and 9 with benign disease had grade 3 uptake at 3 hours. CONCLUSIONS: All malignant lesions had at least grade 2 and most had grade 3 uptake at 3 hours. All lesions with grade 1 uptake at 3 hours were benign, enabling malignancy to be excluded in one-third of cases. Thallium imaging is a useful adjunct to fine-needle cytology in evaluation of solitary thyroid nodules especially when the latter is inconclusive.     

Effect of cyclic adenosine 3',5'-monophosphate/protein kinase a pathway on markers of cell proliferation in nonfunctioning pituitary adenomas

CONTEXT: Alterations in cAMP signaling have been identified as a cause of endocrine neoplasia. In particular, activating mutations of the G(s)alpha gene and protein kinase A (PKA) overactivity due to low expression of PKA regulatory subunit 1A (R1A) have been implicated in somatotroph proliferation. OBJECTIVE: The objective of this study was to evaluate the effects of cAMP-PKA cascade activation in nonfunctioning pituitary adenomas (NFPA). DESIGN AND METHODS: By immunohistochemistry, R1A, R2A, and R2B expression was evaluated in cells obtained from eight surgically removed NFPA positive for gonadotropins. Cyclin D1 expression and ERK1/2 activity were analyzed under basal conditions and after cAMP-PKA cascade activation. RESULTS: Immunohistochemistry studies demonstrated a low R1/R2 ratio in all NFPA. Additional unbalance of R1/R2 ratio by 8-chloroadenosine cAMP (8-Cl-cAMP) and direct adenylyl cyclase stimulation by forskolin did not increase cyclin D1 expression or ERK1/2 activity in five NFPA (group 1), but even caused 74 +/- 15% and 85 +/- 13% inhibitions of cyclin D1 and ERK1/2 activity, respectively, in the remaining NFPA (group 2). Moreover, in group 2, PKA blockade by the specific inhibitor PKI increased cyclin D1 expression (96 +/- 25% over basal) and ERK1/2 activity (116 +/- 28% over basal). CONCLUSIONS: These data show that in contrast with what was previously observed in transformed somatotrophs, activation of the cAMP-PKA pathway did not generate proliferative signals in tumoral cells of the gonadotroph lineage, and in a subset of tumors even exerted a tonic inhibitory effect, thus confirming a different role for the cAMP-mediated pathway in promoting proliferation in the pituitary.     

Benign nonfunctioning thyroid adenomas are characterized by a defective targeting to cell membrane or a reduced expression of the sodium iodide symporter protein.

Nodular thyroid disease is the most common endocrine disorder. Nonfunctioning thyroid nodules are identified by their low radioiodide uptake compared with the normal extranodular tissue, which, at thyroid scintiscan, produces the typical picture of a cold thyroid nodule. Previous in vitro studies demonstrated that the majority of nonfunctioning thyroid nodules have a specific defect in iodide transport that accounts for their failure to accumulate radioactive iodide in vivo. A defect in the expression or structure of the sodium iodide symporter (NIS) gene has been hypothesized as a possible cause of the impaired iodide trapping in nonfunctioning thyroid nodules. We studied 22 patients who were submitted to surgery for a solitary nonfunctioning thyroid nodule that originated in an otherwise normal gland. Thyroid scintigraphy was performed at 1, 2, 3, 4, 6, and 24 h after the oral administration of a tracer dose of 131I (iodine). All patients showed absence of 131I uptake in the nodule, with normal uptake in the extranodular tissue and in the contralateral thyroid lobe. Eight patients with toxic adenomas who underwent lobectomy were also included in the study. We first studied the expression of human NIS (hNIS) protein by immunohistochemistry in paraffin-embedded tissue sections using a specific anti-hNIS monoclonal antibody. Subsequently, we searched for somatic mutations of hNIS gene in nonfunctioning thyroid nodules. The level of hNIS expression was determined in both the nodules and the normal tissue from the same thyroid gland. In all functioning thyroid nodules (toxic adenomas), a high expression of hNIS protein was detected with respect to normal surrounding tissue. Similar to the normal thyroid tissue, follicular cells of toxic thyroid adenomas showed an exclusive expression of hNIS protein at the cell membrane. Fifty-four percent of benign nonfunctioning thyroid nodules overexpressed hNIS protein compared with the normal surrounding tissue, but in these nodules the hNIS protein failed to target the cell membrane, being mostly localized inside the cytoplasm. hNIS protein was not detected by immunohistochemistry in 46% of nonfunctioning nodules, whereas it was expressed in the surrounding unaffected thyroid tissue. Direct sequencing of the hNIS gene in all of the nonfunctioning nodules did not reveal major genetic alterations. A silent polymorphism (GCC/GCG codon 544, exon 13) was found in one nodule. In conclusion, the results obtained in this study show that two mechanisms contribute to the reduced radioiodide uptake typical of benign nonfunctioning thyroid nodules: 1) reduced expression of the hNIS protein, and 2) defective targeting of hNIS to the cell membrane.     

Four resections for hepatic metastasis from gastric cancer: histochemical analysis of cell proliferation, apoptosis, and angiogenesis

In a patient with gastric cancer (GC) associated with one synchronous and three metachronous hepatic metastases (HM), who underwent four hepatectomies, we carried out histochemical investigations regarding cell proliferation, apoptosis, and angiogenesis in the GC and HM. Tissue samples were taken from the primary GC and four HM. Ki-67 immunostaining was performed to evaluate cell proliferation and determine the labeling index (Ki-67 LI; ie, the percentage of cancer cells with nuclei stained for Ki-67). Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) was performed to evaluate apoptosis and determine the apoptotic index (ie, the percentage of TUNEL-positive cells), and immunostaining for factor VIII-related antigen was performed to evaluate angiogenesis and measure microvessel density (MVD). The Ki-67 LI was 43.2% in the primary GC and 39.9% in the synchronous HM, and the LI increased with the number of resections of metachronous HM. The apoptotic index was 3.36% in the primary GC, and 5.30% in the synchronous HM, and the index decreased after further resections of the metachronous HM. The MVD was 35 in the primary GC, and 22 in the synchronous HM, and it increased with the number of resections of metachronous HM. The primary GC in this patient may have strongly influenced the growth of HM through effects on cell proliferation, apoptosis, and angiogenesis. Received: March 1, 1999 / Accepted: June 25, 1999     

α-Fetoprotein–producing gastric cancer: histochemical analysis of cell proliferation, apoptosis, and angiogenesis

OBJECTIVE: Alpha-fetoprotein (AFP)-producing gastric cancer has been associated with a poor prognosis. In the present study, the cell proliferation, apoptosis, and angiogenesis of this cancer were studied histochemically to determine its malignant potential. METHODS: Tissue samples were taken from four patients with AFP-producing gastric cancer and 26 patients with AFP-negative gastric cancer. Cell proliferation was evaluated by Ki-67 immunostaining, and the Ki-67 labeling index (LI) was determined. Apoptosis was studied by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling method, and the apoptotic index was determined. Angiogenesis was evaluated by measuring the microvessel density using factor VIII immunostaining, and immunostainings for vascular endothelial growth factor and thymidine phosphorylase were performed. RESULTS: The Ki-67 LI of the AFP-producing gastric cancers was significantly higher than that of the AFP-negative gastric cancers (p < 0.01). The apoptotic index of the AFP-producing gastric cancers was significantly lower than that of the AFP-negative gastric cancers (p < 0.01). The microvessel density of the AFP-producing gastric cancers was significantly higher than that of the AFP-negative gastric cancers (p < 0.01). Vascular endothelial growth factor expression was observed in all four of the AFP-producing gastric cancers, whereas thymidine phosphorylase was not expressed in any of the AFP-producing gastric cancers. CONCLUSIONS: These results suggest that AFP-producing gastric cancers have high malignant potential (high proliferative activity, weak apoptosis, and rich neovascularization) compared with that of AFP-negative gastric cancers. These biological characteristics of AFP-producing gastric cancer reflect the aggressive behavior and the poor prognosis of patients with this type of cancer.     

Inhibitory effect of thyrotropic hormone on apoptosis induced by actinomycin D in a functioning rat thyroid cell line.

Apoptosis appears to play important roles in physiological and pathological processes in the endocrine system including the thyroid, but little is known about the regulation of apoptosis in the thyroid. The functioning rat thyroid cell line (FRTL-5), a cloned cell line of differentiated thyroid cells, hardly undergoes apoptosis. In this study we examined the factors which prevent FRTL-5 cells from undergoing apoptosis. After culturing FRTL-5 cells in medium with and without TSH, actinomycin D (AMD) or cycloheximide (CHX) was added. CHX did not induce apoptosis. AMD induced apoptosis in FRTL-5 cells cultured in medium lacking TSH, as confirmed by the presence of DNA fragmentation, together with nuclear fragmentation and condensation, but AMD did not induce apoptosis in FRTL-5 cells cultured in the presence of TSH. Furthermore, the fact that AMD did not induce apoptosis in FRTL-5 cells cultured with dibutyryl cyclic AMP (Bt2cAMP), or forskolin suggests that TSH has an inhibitory effect on apoptosis in FRTL-5 cells via the TSH-cAMP pathway.     

Low-energy interstitial laser photocoagulation for treatment of nonfunctioning thyroid nodules: therapeutic outcome in relation to pretreatment and treatment parameters.

Objective: Interstitial laser photocoagulation (ILP) is a recently proposed therapeutic procedure for the ablation of benign thyroid nodules, which has already proven to be safe and effective. However, results supporting the routine use of ILP are still limited. Design: The aim of the study was to evaluate the efficacy and safety of ILP treatment in benign nonfunctioning thyroid nodules and to establish whether the therapeutic outcome may be predicted by any clinical parameter at baseline. Twenty-three patients with either a solitary nodule or a dominant nodule within a multinodular goiter underwent ILP and were evaluated 1 and 3 months later. In order to assess the efficacy of low-energy ILP, the procedure was performed with an output power of 3 W, delivering a mean energy of 33.4 +/- 12.7 Joule/mL of nodule volume, which is much lower than previously reported. Main outcome: Nodule volume significantly decreased after ILP as assessed after 1 and 3 months (analysis of variance; F = 5.37; p = 0.007). Patients with multinodular goiter showed a greater reduction at 3 months compared with patients bearing a solitary thyroid nodule (38.6 +/- 5.3 vs. 30.9 +/- 6.5%; p < 0.01). Age, sex, ultrasound pattern (isoechogenous/hypoechogenous), pretreatment volume, number of ILP treatments, and total energy delivered did not show any significant correlation with treatment outcome. Conclusions: Our results demonstrate that ILP can produce a significant reduction of thyroid nodule volume even when a much lower energy than previously reported is delivered. ILP constitutes a minimally invasive technique, which can be carried out on an outpatient basis and could represent a valid nonsurgical alternative for thyroid nodule management. Dominant nodules within a multinodular goiter appear to be more responsive to ILP compared with solitary thyroid nodules.     

甲状腺激素促进肿瘤细胞增殖及血管新生信号通路的研究

目的 探讨甲状腺激素促进肿瘤细胞增殖及血管新生的信号通路.方法 体外培养人胶质母细胞瘤细胞系（SNB19）,给予甲状腺激素（主要为T4,100 nmol/L）、四碘甲腺乙酸（tetraiodothyroacetic acid,Tetrac,100 nmol/L）、蛋白激酶C（PKC）抑制剂（2.5 μmol/L）作用后,采用Western印迹方法检测磷酸化蛋白激酶D1（PKD1）、磷酸化组蛋白去乙酰化酶（HDAC）5、磷酸化细胞外信号调节激酶（ERK）1/2的表达,ELISA方法检测细胞培养上清血管内皮生长因子（VEGF）的表达量,3-（4,5）-2-唑噻-（2,5）-二苯基溴化四氮唑蓝（MTT）比色法检测细胞增殖.结果 与对照组相比,T4干预组磷酸化PKD1、磷酸化HDAC5、磷酸化ERK1/2水平均增加（P均＜0.05）,Tetrac干预组及PKC抑制剂干预组磷酸化PKD1、磷酸化HDAC5、磷酸化ERK1/2水平均降低（P均＜0.05）.ELISA结果显示,与对照组相比,T4干预组VEGF浓度升高[（56.763±2.611） ng/L vs.（36.597±0.933） ng/L,P＜0.05],Tetrac＋T4干预组VEGF浓度降低[（22.215±1.531） ng/L vs.（36.597±0.933） ng/L,P＜0.05].MTT结果显示,与对照组相比,T4干预组OD值较高[（0.333±0.020）vs.（0.243±0.006）,P＜0.05],Tetrac干预组OD值较低[（0.060±0.016） vs.（0.243±0.006）,P＜0.05].结论 甲状腺激素通过结合整合素αvβ3,激活ERK1/2信号通路促进肿瘤细胞增殖,激活PKC/PKD1/HDAC5信号通路促进血管新生.     

Control of thyroid cell proliferation and goitrogenesis.

Human thyroid cells proliferate during development and in adults in response to physiologic and pathologic stimuli. Under normal conditions, they turn over about once every 8 years. The main physiologic regulators are thyrotropin and iodide and, in disease, thyroidstimulating and thyroid-blocking antibodies. Growth factors modulate proliferation in vitro, but their role in vivo is still unknown. Mitogenic effects are mediated via three major pathways: the cyclic AMP, protein tyrosine kinase, and the Ca(2+) phosphatidylinositol cascades. In this review, the role of these cascades in hyperthyroidism, congenital thyroid defects, and autonomous adenoma is analyzed.     

Cell proliferation,apoptosis and angiogenesis in gastric cancer and its hepatic metastases

BACKGROUND/AIMS: The levels of cell proliferation, apoptosis and angiogenesis were compared histochemically in gastric cancer and its hepatic metastases. METHODOLOGY: Tissue samples were taken from 7 patients with gastric cancer associated with synchronous and/or metachronous hepatic metastases. In the 7 gastric cancers and in 4 synchronous and 4 metachronous hepatic metastases, Ki-67 immunostaining was performed to measure the labeling index (Ki-67 LI). Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling was performed to measure the apoptotic index, and immunostaining for factor VIII-related antigen was performed to measure the microvessel density. RESULTS: The Ki-67 LI was higher in the gastric cancer and the metachronous hepatic metastasis than in the synchronous hepatic metastasis (primary lesions vs. synchronous foci vs. metachronous foci: 47.1% vs. 39.3% vs. 48.0%; P < 0.05). The apoptotic index was lower in the gastric cancer and the metachronous hepatic metastasis than in the synchronous hepatic metastasis (3.50% vs. 5.01% vs. 2.64%; P < 0.05). The microvessel density was higher in the gastric cancer and the metachronous hepatic metastasis than in the synchronous hepatic metastasis (36.0 vs. 22.2 vs. 34.2; P < 0.05). CONCLUSIONS: The present results suggest that tumor growth as indicated by cell proliferation, apoptosis and angiogenesis is less vigorous in synchronous hepatic metastasis than in primary lesion and/or metachronous hepatic metastasis.     

Prevalence of alpha-subunit hypersecretion in patients with pituitary tumors: clinically nonfunctioning and somatotroph adenomas.

Hypersecretion of the pituitary glycoprotein hormone alpha-subunit has been reported in pituitary adenomas, particularly in clinically nonfunctioning tumors and somatotroph adenomas. However, the prevalence of such hypersecretion has not been precisely defined. Using both a new highly sensitive and specific monoclonal antibody assay and a polyclonal antibody assay, serum levels of free alpha-subunit were compared in 63 unselected patients with these tumors, 19 patients with acromegaly, and 95 normal controls. In all patients the monoclonal assay detected a significantly greater number of subjects with elevated alpha-subunit levels than did the polyclonal assay (21 vs. 14; P less than 0.01). Fourteen of the 63 patients with clinically nonfunctioning tumors (22%) had elevated serum alpha-subunit levels in the monoclonal assay vs. 11 (17%) in the polyclonal assay. Among the 19 patients with acromegaly, the prevalence was 7 (37%) and 3 (16%) using the monoclonal and polyclonal assays, respectively. Twenty-eight (44%) of the patients with clinically nonfunctioning pituitary adenomas were female. Eleven (39%) of the women were under 45 yr old, as were 10 (29%) of the men. We conclude that the prevalence of free alpha-subunit hypersecretion in patients with clinically nonfunctioning and somatotroph adenomas may be higher than previously recognized, and that a sensitive and specific monoclonal antibody free alpha-subunit assay may provide a useful tumor marker in these patients. The prevalence of clinically nonfunctioning pituitary tumors among younger men and women may also have been previously under-estimated.     

Proliferating cell nuclear antigen and Ki-67 labeling in hepatocellular nodules: a comparative study

Abstract: Aims/Background: The morphologic differential diagnosis of hepatocellular nodules (HCN) is frequently difficult and objective criteria would be useful in the categorization of such lesions. This study evaluated the proliferative activity of HCN, including regenerative, macroregenerative (MRN), cirrhotic, dysplastic, and hepatocellular carcinoma (HCC), as well as intranodular cytologic changes such as bile-stained hepatocytes, eosinophilia, clear, large cell (LCC) and small cell (SCC) change, by comparing the cellular density (CD), labeling indices (LI) and density (DP) of two proliferation markers. Methods: Routinely processed tissue sections from 45 HCN from 17 adult liver explants were studied by immunohistochemistry for PCNA and Ki-67 (MIB-1). Results: A progressive increase in LI from regenerative to dysplastic nodules to HCC was observed with both proliferation markers. The values of the two markers were significantly correlated (p < 0.001). CD, PCNA and MIB-1 LI and DP values were significantly lower in regenerative compared to dysplastic nodules or HCC. MRNs had lower PCNA and MIB-1 LI and DP than regenerative nodules, but similar CD. There were no statistically significant differences in CD, PCNA, and MIB-1 LI and DP between dysplastic nodules and HCC, comparing high versus low grade dysplasia, or HCC smaller than 2 cm with those larger than 2 cm. The CD and proliferation indices LI and DP were higher in HCC than in the surrounding non-neoplastic parenchyma. Lesions with clear cell, eosinophilic and large cell change had CD, PCNA and MIB-1 indices similar to those of regenerative nodules, while these were lower in bile-stained hepatocellular lesions (p < 0.01). SCC showed CD, PCNA and MIB-1LI and DP similar to HCC and higher than surrounding regenerative lesions (p < 0.003). Conclusions: Our results suggest that PCNA and MIB-1 values are closely correlated in HCN. Regenerative nodules are characterized by low cellular proliferation, while dysplastic nodules are usually highly proliferative lesions and may represent an early stage in hepatocarcinogenesis. Hepatocellular lesions characterized by bile stained hepatocytes, eosinophilic, clear and large cell change have low proliferation rates and may not be significant for the development of malignancy.     

Fine needle aspiration cytology of benign breast disease. Markers of apoptosis and proliferation

Aim of the study was to compare the fine needle aspiration cytology findings of benign breast lesions with incidence of proliferation markers and apoptosis. This study included 37 patients with palpable breast lumps, referred for USG guided FNA. FNAC were prospectively classified as C2-benign, C4-suspicious of malignancy, and C5-malignant. The specimens were simultaneously stained for Ki-67, MPM2, Bcl2 and P53. The diagnoses in group-C2 were following: simple cyst, multiple cysts, simple cyst with apocrine metaplasia, inflammatory cyst, benign dysplasia (BD) and benign solid tumors. The final diagnoses, after histopathological verification, in cases of primary classification as C4 and C5 were as follow: proliferative fibroadenoma (FAp) and breas cancer, respectively. Great majority of C2/BD aspirates were negative for proliferative antigens Ki-67 and PCNA. These antigens were detected in part of benign solid tumors, as anticipated in suspicious solid tumor, and in all of cancer aspirates. Bcl-2 immunopositive cells were detected approximately in one quarter of C2/BD, nearly in half of C2 solid tumors and in one C4/FAp. Most of diagnosed specimens were P53-negative. Immunocytodetection of Ki67, MPM2, Bcl2, P53 might be promising, supportive method in the classification of benign breast lesions. FNAC increases the reliability of diagnosis when complemented by immunocytochemical staining. It could be helpful procedure of establishing more accurately the biology of these lesions and possibly serve as an essential factor in clinical follow-up. Nevertheless, further study on larger group of patients comparing cytological and histopathological diagnosis is required to estimate reliability of its predictive value.