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.     

Characterization of ligands for galectins, natural galactoside-binding immunoglobulin G subfractions and sarcolectin and also of the expression of calcyclin in thyroid lesions

The purpose of this study was to characterize ligands for galectins, natural galactoside-binding immunoglobulin G subfractions and sarcolectin and also the expression of calcyclin in various benign and malignant thyroid lesions. The extent of the binding of eight glycochemical probes was quantitatively assessed using computer-assisted microscopy on 76 thyroid lesions including 10 not-otherwise-specified multinodular goiters (S_MNG), 11 multinodular goiters with adenomatous hyperplasia (AH_MNG), 8 normomacrovesicular (NM_ADE) and 12 microvesicular (MIC_ADE) adenomas, and 9 papillary (P_CAR), 10 follicular variants of papillary (FvarP_CAR), 7 follicular (F_CAR) and 9 anaplastic (A_CAR) carcinomas. The 8 histochemical probes included 5 animal lectins (including galectins and sarcolectin), 1 polyclonal antibody (raised against calcyclin) and 2 immunoglobulin G subfractions from human serum with selectivity to alpha- and beta-galactosyl residues. The results show that multinodular goiters with adenomatous hyperplasia exhibited histochemical characteristics intermediate to those of normal multinodular goiters and microvesicular adenomas. Normomacrovesicular adenomas behaved very distinctly from microvesicular ones. Microvesicular adenomas were more closely related to differentiated thyroid carcinomas than any other type of benign thyroid lesions of epithelial origin. Papillary and follicular carcinomas seemed to represent the two extremes of the same biological entity with the follicular variant of the papillary carcinoma serving as a biological link between these two extremes. Anaplastic carcinomas behaved in a significantly different manner when compared to the differentiated forms of thyroid carcinomas. The results suggest that the patterns of expression of the glycoconjugates investigated in the present study may constitute useful tools for characterizing lesions in the human thyroid.     

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.     

Mutual regulation of TGF-β1, TβRII and ErbB receptors expression in human thyroid carcinomas

The role of EGF and TGF-β1 in thyroid cancer is still not clearly defined. TGF-β1 inhibited the cellular growth and migration of follicular (FTC-133) and papillary (B-CPAP) thyroid carcinoma cell lines. Co-treatments of TGF-β1 and EGF inhibited proliferation in both cell lines, but displayed opposite effect on their migratory capability, leading to inhibition in B-CPAP and promotion in FTC-133 cells, by a MAPK-dependent mechanism. TGF-β1, TβRII and EGFR expressions were evaluated in benign and malignant thyroid tumors. Both positivity (51.7% and 60.0% and 80.0% in FA and PTC and FTC) and overexpression (60.0%, 77.7% and 75.0% in FA, PTC and FTC) of EGFR mRNA correlates with the aggressive tumor behavior. The moderate overexpression of TGF-β1 and TβRII mRNA in PTC tissues (61.5% and 62.5%, respectively), counteracted their high overexpression in FTC tissues (100% and 100%, respectively), while EGFR overexpression was similar in both carcinomas. Papillary carcinomas were positive to E-cadherin expression, while the follicular carcinomas lose E-cadherin staining. Our findings of TGF-β1/TβRII and EGFR overexpressions together with a loss of E-cadherin observed in human follicular thyroid carcinomas, and of increased migration ability MAPK-dependent after EGF/TGF-β1 treatments in the follicular thyroid carcinoma cell line, reinforced the hypothesis of a cross-talk between EGF and TGF-β1 systems in follicular thyroid carcinomas phenotype.     

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.     

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.     

Demonstration of a heterogeneous transcription pattern of thyroglobulin mRNA in human thyroid tissues.

Previous reports on human thyroglobulin (hTg) modifications in thyroid carcinomas prompted us to study hTg mRNA in thyroid adenomas and carcinomas. The quantification of hTg mRNA showed a decrease in its levels of expression in both pathological conditions which differed by a factor of 2 between adenomas and carcinomas. Furthermore, PCR was used to analyse the characteristics of hTg mRNA by amplifying 4 regions of the hTg mRNA. When applied to 2 normal, 17 benign and 13 malignant pathological tissue specimens, PCR showed no modification in the size of Tg mRNA. However, abnormal sized cDNAs appeared in all tissues with no distinction between the pathologies; the Restriction Fragment Length Polymorphism study of these cDNAs suggests the existence of alternate splicing patterns in thyroglobulin mRNAs.     

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.     

In vitro splicing analysis of mini-gene constructs of the alternatively processed human calcitonin/CGRP-I pre-mRNA

The human calcitonin/CGRP-I (CALC-I) gene can be alternatively expressed into calcitonin mRNA in thyroid C-cells and into CGRP-I mRNA in particular nerve cells. Formation of calcitonin mRNA requires splicing of exons 1, 2, 3 and 4 and addition of poly(A) at exon 4, whereas splicing of exons 1, 2, 3, 5 and 6 and addition of poly(A) at exon 6 yields CGRP-I mRNA. The calcitonin and CGRP-I mRNA-specific splicing reactions were investigated in vitro, in nuclear extracts of HeLa cells, using model precursor RNAs containing the exon 3 to exon 5 region of the gene. A precursor RNA containing the full-length exon 3 to exon 5 region was only poorly spliced in vitro. Therefore, a systematic analysis was performed of the effect of deletions introduced in the intron 3, exon 4 and intron 4 of this precursor RNA on calcitonin/CGRP mRNA-specific splicing. The deletions increased the efficiency of splicing considerably. In all cases CGRP mRNA-specific splicing is strongly favoured over calcitonin mRNA-specific splicing. In addition, splicing reactions using cryptic 5' splice sites were detected which interfered with the usage of processing signals for calcitonin and CGRP mRNA-specific splicing. The results imply a major regulatory role for the exon 4 poly(A) addition reaction in the generation of calcitonin mRNA.     

Effect of thyroid-stimulating hormone and norepinephrine on cyclic AMP levels in human normal thyroids and human thyroid tumors

The effect of TSH (100mU/ml) and norepinephrine (100 muM) on the cyclic AMP levels was studied in 10 human normal tissues, 10 thyroid adenomas and 4 thyroid carcinomas (3 papillary and 1 follicular). Normal tissues responded to TSH with a marked elevation of the cyclic AMP level. Response patterns of 10 thyroid adenomas to TSH were variable; the patterns of 6 cases resembled those of normal tissues, 3 responded mildly, and one had no response to TSH. Thyroid carcinomas had a higher basal level of cyclic AMP than those of normal tissues, although they responded only slightly to TSH. Two among 4 thyroid carcinomas had no response to TSH. Norepinephrine stimulated the accumulation of cyclic AMP in 4 thyroid adenomas and 3 thyroid carcinomas, while it had little effect on normal tissues. Responses to norepinephrine was observed only in thyroid tumors, although they had low response to TSH. It is suggested from these results that tumor cells originating from thyroid follicular cells have a modified response to hormones due to neoplastic alterations.     

Evaluation of HMFG2 and thyroglobulin in the diagnosis of thyroid fine needle aspiration (FNA)

In order to appraise the usefulness of HMFG₂ and thyroglobulin (Tg) as specific markers for the diagnosis of thyroid disease, we studied 63 FNA smears. Cases tested included 30 benign (nine colloid goitres, six cases of Hashimoto's thyroiditis, six Hürthle cell adenomas, nine follicular adenomas) and 33 malignant lesions (nine follicular carcinomas, 12 papillary carcinomas, nine anaplastic carcinomas, three medullary carcinomas). All cases with malignant lesions except the anaplastic carcinomas were positive for HMFG₂. Immunoreactive cells to HMFG₂ were also found in 15 adenomas out of 30 benign cases. Positive Tg reaction was found in benign and malignant thyroid lesions, except six cases of Hashimoto's thyroiditis, nine anaplastic and three medullary carcinomas. The results obtained indicate that morphology paired with immunocytochemistry can usually depict a more specific profile of thyroid lesions for better evaluation of the pathology.