Proteomic identification of new biomarkers and application in thyroid cytology

OBJECTIVE: To validate proteins identified by proteomics as potentially usable markers in thyroid pathology. STUDY DESIGN: Frozen sections of thyroid tumors were manually micro-dissected and proteins extracted. Two-dimensional (2D) gel electrophoresis and subsequent liquid chromatography/mass spectroscopy were performed, and differentially expressed proteins were identified. Validation of candidates for tumor markers (galectin-1, galectin-3, S100C and voltage-dependent anion channel 1 [VDAC1]) was done by immunohistochemistry in 21 cell blocks from fine needle aspiration biopsies (FNAB) and corresponding histology specimens (13 cases). RESULTS: Galectin-3 was negative in benign lesions and positive in FNAB from papillary carcinoma (5 of 5), follicular variant of papillary carcinoma (1 of 4) and follicular carcinoma (1 of 2). S100C was positive in some benign lesions: hyperplasia (2 of 4), goiter (1 of 3) and follicular adenoma (1 of 3), with predominantly nuclear pattern of staining. S100C was positive in malignant lesions, showing cytoplasmic location. Galectin-1 was negative in benign lesions and positive in follicular carcinoma (1 of 2), papillary carcinoma (2 of 5) and follicular variant of papillary carcinoma (1 of 4). VDAC1 was detected in benign and malignant lesions, showing a strong positivity in follicular carcinomas. CONCLUSION: Immunohistochemical validation of potential markers is a crucial step before clinical application in diagnosis. Galectin-3, galectin-1 and S100C can be used to help in discriminating benign and malignant thyroid lesions.     

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.     

Fine needle aspiration of the thyroid: can an image processing system improve differentiation?

OBJECTIVE: To differentiate thyroid nodules by means of fine needle aspiration with subsequent computer-aided diagnosis. STUDY DESIGN: Fine needle aspiration biopsies obtained from 137 patients for whom histopathologic diagnosis was available were investigated: 16 hyperplastic nodules (12%), 39 adenomas (28%), 25 follicular thyroid carcinomas (18%), 19 follicular variants of papillary thyroid carcinoma (14%) and 38 papillary thyroid carcinomas (28%). From each stained specimen 100 cell scenes were scanned and analyzed, constituting a total of 62,325 cell images. RESULTS: All the entities described could be well discriminated from each other by automated image analysis methods. Both the diagnosis of tumor type and the differentiation between benign and malignant could be achieved with a sensitivity of .98. CONCLUSION: With only 7-10 calculated cell features (texture line analysis) and classification with decision trees, a tool for high-quality cell image diagnosis is available. Subtypes of cells characterized by the calculated features could be found in all the specimens and could be assigned to the malignancies with high statistical significance. The method increases the relevance of image processing as an additional diagnostic tool for cytologic examination of thyroid nodules.     

Galectin-3 is not useful in thyroid FNA

INTRODUCTION: Previous studies have suggested that galectin-3 immunohistochemistry may be useful in the fine needle aspiration (FNA) diagnosis of thyroid carcinoma as it has been reported to selectively stain carcinomas and not adenomas or goitres. METHODS: Fifty-one patients were included in a prospective study of galectin-3 in thyroid FNA; 88.2% were female and 11.8% male, mean age 53 years, range 25-87 years. Cell blocks were prepared and stained for galectin-3 if any cells were present in needle washings from the respective FNAs. RESULTS: Twelve of 51 (23.5%) of cell blocks contained epithelial cells. One benign and one inadequate FNA were negative for galectin-3 staining. One of five non-diagnostic FNA cases, a papillary carcinoma on final histology showed positive staining. Four follicular neoplasm/suspicious of carcinoma cases showed negative staining. One malignant FNA case, a papillary carcinoma showed positive staining with galectin-3 but three further carcinomas, two papillary and one follicular were galectin-3 negative. CONCLUSION: Galectin-3 immunohistochemistry does not appear to be a useful adjunct to diagnosis in thyroid FNA as it does not reliably distinguish malignant and benign lesions. Many thyroid aspirates are of low cellularity and are not suitable for cell block immunohistochemistry.     

Ki-67 and AgNOR proliferative markers as diagnostic adjuncts to fine needle aspiration cytology of thyroid follicular lesions

OBJECTIVE: To differentiate hyperplastic nodules (HPN), follicular adenoma (FA) and follicular carcinoma (FCA) of the thyroid by cytomorphologic features combined with argyrophilic nucleolar organizer regions (AgNORs) and Ki-67 proliferative markers on fine needle aspiration cytology. STUDY DESIGN: Cytomorphologic patterns, along with two proliferation markers, Ki-67 and AgNORs, in fine needle aspirates of 123 histologically confirmed cases of thyroid follicular lesions, including 39 hyperplastic nodules, 70 follicular adenomas and 14 cases of follicular carcinomas, were recorded. RESULTS: Mean AgNOR (mAgNOR) counts and Ki-67 labelling index (LI) were consistently higher in FCA in comparison to FA and HPN irrespective of the cytologic patterns in fine needle aspiration smears. Between benign and malignant lesions, an overlap of 1.83% at the cutoff point of 4.0 was observed in cases of mAgNORs, whereas it was 11.09% at a cutoff of 5.0 in cases of Ki-67 LI. CONCLUSION: mAgNOR counting in fine needle aspiration smears is more sensitive, simple and cost effective as compared to Ki-67 LI for differentiating between benign and malignant thyroid follicular neoplasms.     

Neutrophil gelatinase-associated lipocalin (NGAL) immunohistochemical expression in follicular cell-derived thyroid tumors: a novel diagnostic tool?

Discrimination of follicular cell-derived benign and malignant tumors of the thyroid is one of the major problems encountered in surgical pathology. In the present study, we evaluated the immunohistochemical expression of NGAL, an iron-binding protein involved in the infiltrative potential of cancer cells, in a cohort of tumors including 8 follicular adenomas (FA), 2 Hurthle cell adenomas (HA), 2 atypical adenomas (AA), 8 minimally invasive follicular carcinomas (MIFC), 9 widely invasive follicular carcinomas (WIFC), 3 Hurthle cell carcinomas (HC) and 8 papillary carcinomas (PC) with 5 follicular-variant PC (FVPC) and 3 not otherwise specified (PC-NOS). Our goal was to test whether evaluation of NGAL immunoexpression may be of use in the differential diagnosis of benign and malignant thyroid neoplasias. 92% of benign tumors (specificity) were negative for NGAL, whereby NGAL immuno-expression was found in 82% (sensitivity) of malignant tumors, and, specifically, in 100% of MIFC, in 87% of WIFC, in 100% of HC, in 80% of FVPC. None of the PC-NOS displayed NGAL staining. When only tumors with a follicular architecture were considered, NGAL specificity for malignant lesions was 92%; sensitivity, positive predictive value and negative predictive value were 92%, 96% and 85%. Diagnostic accuracy of NGAL expression in the differential diagnosis between benign and malignant follicular tumors was 92%. In conclusion, NGAL protein seems to represent a marker of malignant follicular cell-derived thyroid tumors, and especially of those with follicular architecture. Hence assessment of its expression might be of use with respect to differential diagnosis from follicular benign neoplasias.     

Computerized morphometric study on fine needle aspirates of cellular follicular lesions of the thyroid

OBJECTIVE: To investigate the value of computerized nuclear morphometry in the differential diagnosis of cellular follicular lesions of the thyroid cytologically diagnosed on fine needle aspiration (FNA) smears. STUDY DESIGN: Sixty cases of FNA thyroid smears were cytologically diagnosed and classified as follows: 30 cases of follicular carcinoma, 20 cases of cellular hyperplastic nodules and 10 cases of follicular adenoma. Using an image analysis system, two morphometric variables, nuclear area and major axis length of the nucleus, were measured for each case. RESULTS: For both nuclear morphometric variables, statistical differences were found between carcinomas and hyperplastic nodules as well as between carcinomas and adenomas. No statistical differences were found between the nuclear variables in either hyperplastic nodules or adenomas. CONCLUSION: The results confirm the aim of our study, to establish nuclear morphometry by computerized image analysis as an additional tool in the differential diagnosis of thyroid follicular lesions cytologically diagnosed on FNA smears.     

Cytoplasmic and serum galectin-3 in diagnosis of thyroid malignancies

In order to address whether galectin-3 in the sera and fine needle aspirates serve as a diagnostic marker distinguishing between benign and malignant thyroid nodules, we developed an enzyme-linked immunosorbent assay. We quantified galectin-3 in fine needle aspirates from a series of 118 patients with thyroid nodules and serum galectin-3 from another series of 46 patients, which were compared with final histology after thyroidectomy. Relative galectin-3 value (ng/mg), defined as galectin-3 concentration (ng/ml) divided by total protein concentration (mg/ml) in fine needle aspirates, was significantly higher in papillary carcinoma than in the other thyroid entities. There was no significant difference in serum galectin-3 level among patients with thyroid nodules and healthy individuals. Accordingly, relative galectin-3 value allows a definitive diagnosis of papillary carcinoma independent of cellular morphology, whereas serum galectin-3 does not serve as a marker for papillary carcinoma.     

Diagnostic utility of galectin-3 in aspirates of thyroid follicular lesions

OBJECTIVE: To investigate the expression of galectin-3 in various thyroid follicular lesions, including diffuse hyperplasia, nodular hyperplasia, and benign and malignant follicular neoplasms, to clarify the diagnostic utility of galectin-3 in aspirates of follicular lesions. STUDY DESIGN: A total of 146 follicular lesions diagnosed cytologically, obtained from patients who had undergone thyroidectomy for either benign or malignant nodules, were evaluated using Papanicolaou-stained slides and cell blocks with galectin-3 immunostaining. We primarily categorized the aspirated specimens as benign, indeterminate or suspicious for a follicular neoplasm based on cellularity, architectural arrangement of the follicular cells and presence or absence of colloid. Galectin-3 immunostaining was evaluated according to the proportion and intensity of positively stained cells. Cytologic diagnoses were correlated with the results of galectin-3 and categorized into 3 groups (benign, indeterminate for malignancy, suspicious for malignancy) and compared with the corresponding histologic diagnoses. RESULTS: When the histologic diagnoses were compared with the cytologic diagnoses, the accuracy in the distinction between benign and malignant cases was 79.5% except for 8 cytologically and 3 histologically indeterminate cases. Except for 11 indeterminate cases, there were 18 (12.3%) discrepant cases. False positive cases included 8 of 62 (12.9%) nodular hyperplasias and 1 of 42 (2.4%) follicular adenomas. Of 9 false negative cases, 4 minimally invasive carcinomas and 2 widely invasive carcinomas were included. Three follicular tumors of uncertain malignant potential were cytologically categorized as malignant, and all cases showed positivity for galectin-3. CONCLUSION: Galectin-3 could be used as a useful supplementary marker for cytologic diagnosis, although it was not an absolute marker in determining whether a lesion was benign or malignant.     

Fine needle aspiration cytologic study of the solitary thyroid nodule. Profile of 308 cases with histologic correlation

The cytologic features and diagnoses of 308 solitary thyroid nodules subjected to fine needle aspiration (FNA) are presented and correlated with the histopathology. The findings reconfirmed the overall utility of FNA cytology in the differentiation of benign from neoplastic lesions and in the specific diagnosis of most types of thyroid lesions. The difficulty in the diagnosis of follicular carcinoma and in its differentiation from follicular adenoma is highlighted. Of note was the finding of Hürthle-cell populations in several types of lesions, which represents a potential diagnostic problem; this potential difficulty was lessened by sampling two or three areas within a nodule, thus obtaining a more truly representative specimen.     

AgNOR quantification in the diagnosis of follicular pattern thyroid lesions

OBJECTIVE: To evaluate the usefulness of silver staining of nucleolar organizer regions (AgNORs) in the preoperative diagnosis of follicular lesions in the thyroid with computer-aided morphometric analysis of the silver dots. STUDY DESIGN: Forty-eight cytologic smears of the thyroid were divided into 3 groups according to the results of postoperative histopathologic examination: hyperplastic nodules in nodular goiter (NG) (20), follicular thyroid adenoma (FTA) (20) and follicular thyroid carcinoma (FTC) (8). They were silver stained. The slides were analyzed with a computerized system for image analysis. Nearly 20 variables describing AgNORs were calculated (related to the area of the dots, number of dots and intranuclear localization of the dots). RESULTS: Only assessment of the total area of AgNORs in the nucleus allowed distinguishing between malignant and benign lesions. It was possible to determine the cutoff value of the total area of AgNORs in the nucleus (3.00 microns 2), limiting FTC from other lesions (observed ranges: NG, 1.64-2.87 microns 2; FTA, 1.81-2.85 microns 2; FTC, 3.01-3.97 microns 2). Evaluation of the mean number of AgNORs per nucleus did not improve the diagnosis of malignancy. CONCLUSION: Computer-aided morphometric analysis of silver dots may be useful in the preoperative diagnosis of thyroid lesions.     

Follicular Adenomas Exhibit a Unique Metabolic Profile. 1H NMR Studies of Thyroid Lesions

Thyroid cancer is the most common endocrine malignancy. However, more than 90% of thyroid nodules are benign. It remains unclear whether thyroid carcinoma arises from preexisting benign nodules. Metabolomics can provide valuable and comprehensive information about low molecular weight compounds present in living systems and further our understanding of the biology regulating pathological processes. Herein, we applied ¹H NMR-based metabolic profiling to identify the metabolites present in aqueous tissue extracts of healthy thyroid tissue (H), non-neoplastic nodules (NN), follicular adenomas (FA) and malignant thyroid cancer (TC) as an alternative way of investigating cancer lesions. Multivariate statistical methods provided clear discrimination not only between healthy thyroid tissue and pathological thyroid tissue but also between different types of thyroid lesions. Potential biomarkers common to all thyroid lesions were identified, namely, alanine, methionine, acetone, glutamate, glycine, lactate, tyrosine, phenylalanine and hypoxanthine. Metabolic changes in thyroid cancer were mainly related to osmotic regulators (taurine and scyllo- and myo-inositol), citrate, and amino acids supplying the TCA cycle. Thyroid follicular adenomas were found to display metabolic features of benign non-neoplastic nodules and simultaneously displayed a partial metabolic profile associated with malignancy. This finding allows the discrimination of follicular adenomas from benign non-neoplastic nodules and thyroid cancer with similar accuracy. Moreover, the presented data indicate that follicular adenoma could be an individual stage of thyroid cancer development.     

声触诊组织定量技术鉴别诊断甲状腺微小癌的价值

目的：声触诊组织定量技术（VTQ）是一种新的弹性成像方法,能够定量、无创地评价组织硬度信息。本文的研究目的是探讨声触诊组织定量技术在鉴别诊断甲状腺微小癌（TMCs）中的应用价值。方法：应用VTQ 技术对110 例共114 个结节（最大直径≤1 cm）进行检测,获取并分析结节及周边甲状腺组织的横向剪切波速度（SWV）。利用ROC曲线对测量结果进行分析,评价VTQ技术的诊断价值并确定最佳诊断界值。结果：114 个结节中良性结节32 个（30 个为结节性甲状腺肿,2 个为腺瘤）,恶性结节82 个（均为乳头状癌）。甲状腺良性结节及周边甲状腺组织的SWV 平均值分别为（2.30± 0.49） m/s、（2.08± 0.40） m/s,恶性结节及周边甲状腺组织的SWV 平均值分别为（3.12± 0.97） m/s、（2.06± 0.46） m/s。恶性结节的SWV 值明显高于良性结节,两者之间差异具有统计学意义（t=5.911,P＜0.0001）;恶性结节与其周边甲状腺组织比较有显著差异（P＜0.0001）;而良性结节与其周边甲状腺组织无统计学差异（P＞0.05）。ROC曲线下面积为0.833,以2.30 m/s诊断界值点时,敏感度、特异度分别为90.2%、62.5%。结论：甲状腺恶性结节的SWV 值明显高于良性结节。VTQ 技术能够提供组织硬度信息,在鉴别诊断甲状腺微小癌方面具有一定的临床应用价值。     

Beyond the suspicious thyroid fine needle aspirate. A review

Fine needle aspiration (FNA) is currently the best diagnostic tool for thyroid nodules. However, the cytologic category of indeterminate or suspicious lesion, which is found in 10-15% of cases, remains a challenge. Since neither clinical presentation nor intraoperative frozen section is often helpful in differentiating these lesions and since surgical procedures for benign and malignant lesions differ, there is a clear need to develop ancillary tests. In this review we identify 12 potential markers of thyroid malignancy that have been examined in thyroid cytologic samples. Although many of these markers hold promise as adjuncts to FNA cytology, multicenter studies have often shown limitations in the predictive value of these assays due to lack of specificity, sensitivity or both. The recent development, however, of tissue microarray techniques to validate promising new markers suggests that improvements in the approach to indeterminate thyroid FNA samples may soon be at hand. This review presents a summary of the issues facing the development of a clinically useful diagnostic test in the differential diagnosis of thyroid nodules.     

Sensitivity to radiation of human normal, hyperthyroid, and neoplastic thyroid epithelial cells in primary culture

Samples of thyroid tissue removed surgically from 63 patients were cultured in vitro and exposed to X irradiation to investigate the radiosensitivities of various types of thyroid epithelial cells. A total of 76 samples were obtained, including neoplastic cells from patients with papillary carcinoma (PC) or follicular adenoma (FA), cells from hyperthyroidism (HY) patients, and normal cells from the surgical margins of PC and FA patients. Culturing of the cells was performed in a manner which has been shown to yield a predominance of epithelial cells. Results of colony formation assays indicated that cells from HY and FA patients were the least radiosensitive: when adjusted to the overall geometric mean plating efficiency of 5.5%, the average mean lethal dose Do was 97.6 cGy for HY cells and 96.7 and 94.3 cGy, respectively for neoplastic and normal cells from FA patients. Cells from PC patients were more radiosensitive, normal cells having an adjusted average Do of 85.0 cGy and PC cells a significantly (P = 0.05) lower average Do of 74.4 cGy. After allowing for this variation by cell type, in vitro radiosensitivity was not significantly related to age at surgery (P = 0.82) or sex (P = 0.10). These results suggest that malignant thyroid cells may be especially radiosensitive.     

Diagnostic pitfalls in the evaluation of fine needle aspiration cytology of the thyroid: correlation with histopathology in 260 cases

Objectives:  Fine needle aspiration cytology (FNAC) of the thyroid is a non-invasive, cost-effective screening procedure that is valuable for distinguishing neoplastic lesions from non-neoplastic nodules. The aim of this study was to determine the diagnostic accuracy of FNACs performed at our institution by correlating FNAC results with histopathological diagnoses.
Methods:  Two hundred and seventy-one aspiration cytology specimens followed by thyroidectomy were included in the study, and the results of 260 adequate FNACs were compared with their histological diagnoses.
Results:  The sensitivity and specificity of thyroid FNAC for detecting neoplasia were 92.6% and 91.6%, respectively. There were 15 (5.7%) false positives and six (2.3%) false negatives.
Conclusions:  The results showed that follicular cells that exhibit some of the features of papillary carcinoma could be observed in a cytology slide of Hashimoto's thyroiditis, leading to a diagnostic pitfall. In addition, cellularity and overlapping cytological criteria in hyperplasia might lead to a false diagnosis.     

Cytoplasmic colloid inclusions in thyroid lesions: a cytomorphological study based on fine needle aspiration

INTRODUCTION: Intracytoplasmic lumens (ICL) with or without magenta material and transgressing vessels are features of Hürthle cell neoplasms (HCN). After detection of intracytoplasmic colloid inclusions (CIs) including targetoid (magenta) body-like structures in the Hürthle cells (HC) in a case of Hashimoto's thyroiditis (HT), we reviewed cases of HT, thyroid neoplasms, hyperplastic nodules (HN) and colloid goitres to determine the frequency of these structures. Further, an attempt was made to find out the significance of CIs. METHODS: FNA smears of 120 HT, 101 colloid goitres, 11 HN, and 76 neoplastic goitres were examined. The presence of CIs and empty ICL were noted in epithelial cells in these lesions. An attempt was made to find out the difference between HT with and without CIs in respect of various cytomorphologic features. The groups were compared using the Fisher's exact test of probability. RESULTS: The CIs were present in 36 (30.0%) of HT, 26 (34.2%) of neoplastic goitres, 3 (27.3%) of HN, and 4 (4.0%) colloid goitres. As compared to colloid goitres, CIs were present in a significant higher number of cases in HN (P = 0.0202), neoplastic goitres (P < 0.0001), and HT (P < 0.0001). Among neoplasms the frequency of CIs in HCN (75.0%) was significantly higher than that of papillary thyroid carcinoma (PTC) (33.3%, P = 0.0466), and follicular neoplasm (14.3%, P = 0.0083). The CIs were more frequent in HC in HT and HCN but in follicular cells (FC) in other lesions. The HT cases with CIs differed significantly from those without CIs in respect of HC and their cellularity, cellularity of reactive lymphoid cells, extracellular colloid and empty ICL. CONCLUSION: Care should be taken not to diagnose HT cases with an excessive Hürthle cell component and CIs, and PTC cases with Hürthlization and CIs, as HCN in FNA smears. Based on review of the literature and our findings, it is suggested that the Hürthle cell metaplasia in HT is a survival response of FC and the presence of CIs in Hürthle cell may represent their limited ability to synthesize colloid.     

Thyroid papillary microcarcinoma. Is it really a pitfall of fine needle aspiration cytology?

OBJECTIVE: To assess the role of fine needle aspiration (FNA) of the thyroid in the diagnosis of papillary microcarcinoma. STUDY DESIGN: Eight cases of papillary microcarcinoma were diagnosed by fine needle aspiration. On histologic examination they were found to be adjacent to larger nodules of interest. The microcarcinomas were inadvertently sampled when sampling the larger, dominant nodules. RESULTS: None of the eight dominant nodules were papillary carcinoma; seven were benign lesions, and one was an angioinvasive Hürthle cell carcinoma. In three cases the microcarcinomas were situated within the capsule of a hyperplastic nodule. On histologic examination, five cases had multifocal microcarcinomas, with one case having multiple lymph node metastases. Based on the clinical findings and morphologic features, there were no definitive cytologic findings that could distinguish between "incidental" microcarcinoma and clinically significant papillary carcinoma. CONCLUSION: The detection of microcarcinoma by FNA should not be considered a false positive finding since the exact nature of the lesion cannot be determined until complete histologic evaluation reveals it to be truly incidental and clinically insignificant.