Galectin-3 expression in papillary microcarcinoma of the thyroid

AIMS: Galectin-3 is a beta-galactoside binding protein, recently recognized as a promising molecular marker of thyroid malignancy. As reported in several studies, galectin-3 is highly expressed in papillary thyroid carcinoma, but its expression has not been investigated in papillary microcarcinoma, which is a variant of papillary thyroid carcinoma. METHODS AND RESULTS: Using a monoclonal antibody to galectin-3 and the avidin-biotin-peroxidase complex (ABC) immunohistochemical technique, we analysed galectin-3 expression in 63 cases of papillary microcarcinoma. The results showed immunohistochemical reactivity for galectin-3 in 51 (80.9%) cases. Intensity of staining varied from strong or moderate to weak. Galectin-3 localization was mostly cytoplasmic, but also membranous or nuclear in some cells. Immunohistochemical expression of galectin-3 was not found in 12 (19.1%) cases. Most galectin-3 negative microcarcinomas (10/12) were of the non-classical type, i.e. without papillary architecture. Neither the frequency nor the intensity of a positive reaction was related to tumour size. CONCLUSIONS: Galectin-3 gene is expressed at the protein level in most papillary microcarcinomas, although with slightly lower frequency than that reported for clinically evident papillary thyroid carcinoma. The presence of galectin-3 in clinically silent microcarcinomas may indicate that galectin-3 is not related to growth or aggressiveness of papillary thyroid microcarcinomas but rather plays some other role in thyroid tumour biology.     

Cytokeratin 19 and galectin-3 immunohistochemistry in the differential diagnosis of solitary thyroid nodules

AIMS: The immunohistochemical expression of cytokeratin 19 (CK 19) and galectin-3 was evaluated in 69 thyroid lesions to assess their potential as markers in the diagnosis and classification of thyroid malignancy. The following were studied: 26 cases of papillary carcinoma, 12 of follicular carcinoma, 20 follicular adenomas, two medullary carcinomas, one anaplastic carcinoma and eight multinodular goitres. METHODS AND RESULTS: Formalin-fixed paraffin-embedded thyroid tissues were stained immunohistochemically for both CK 19 and galectin-3. CK 19 expression was found in all 26 papillary carcinomas, five of 12 follicular carcinomas, two of two medullary carcinomas and one case of anaplastic carcinoma. Only five of 20 follicular adenomas were positive for CK 19, and this was in a focal distribution. Two of eight multinodular goitres stained focally positive. Galectin-3 expression was found in 22 of 26 papillary carcinomas, 12 of 12 follicular carcinomas and one of two cases of medullary carcinoma. Only two of 20 follicular adenomas were positive. Three of eight multinodular goitres showed focal galectin-3 expression. CONCLUSIONS: Our findings suggest that the immunohistochemical localization of CK 19 and of galectin-3 is a useful adjunct to the histopathological diagnosis of a solitary thyroid lesion. The expression of CK 19 favours a diagnosis of papillary carcinoma in all its variant patterns. Galectin-3 may serve as a marker for the recognition of follicular carcinoma, particularly the minimally invasive form.     

Expression of matrix metalloproteinases in benign and malignant follicular thyroid lesions

AIMS: To examine expression of matrix metalloproteinases (MMPs) and related proteins in follicular thyroid lesions (FTLs) and to determine their usefulness for differential diagnosis of FTLs, particularly between minimally invasive carcinoma and adenoma. METHODS AND RESULTS: Six widely invasive follicular carcinomas (WIFCs), 15 minimally invasive follicular carcinomas (MIFCs), 19 follicular adenomas (FAs) and 10 adenomatous goitres (AGs) were analysed immunohistochemically for MMP-1, MMP-2, MMP-7, MMP-9, membrane-type 1-MMP (MT1-MMP) and tissue inhibitor of matrix metalloproteinase-2 (TIMP-2). MMP-1 was positive in all FTLs. MMP-2 and MMP-7 were positive in more than 80% of WIFC and MIFC cases, whereas they were negative in all FA and AG cases except one MMP-2+ FA (P < 0.001). MMP-9 stained positive significantly more in MIFC than FA or AG cases (P < 0.05, respectively). The positivity of MT1-MMP and TIMP-2 was different among some of the FTLs, but with no significant difference between MIFC and FA cases. In-situ hybridization of MMP-2 and MMP-7 mRNA in selected cases demonstrated the expression of these enzymes in the tumour cells as well as in some stromal cells. CONCLUSIONS: Our results confirm MMP expression mainly in malignant FTLs and suggest that MMP-2 and MMP-7 may be useful markers to distinguish MIFC from FA.     

Galectin-3, a marker of well-differentiated thyroid carcinoma, is expressed in thyroid nodules with cytological atypia

AIMS: The distribution of galectin-3, a widely recognized marker of well-differentiated thyroid carcinoma, was investigated in 95 thyroid lesions including nodules with foci of cytoarchitectural atypia. METHODS AND RESULTS: Twenty-eight papillary carcinomas, five follicular carcinomas, one Hurthle cell carcinoma, three poorly differentiated carcinomas, one anaplastic carcinoma, 25 nodular hyperplasias and 27 follicular adenomas, including nodules with atypical features, three neoplasms of undetermined malignant potential and two thyroiditis cases were examined. By immunohistochemistry, galectin-3 was consistently found in carcinomas; otherwise benign nodules exhibited galectin-3-positive clusters of cells with poorly developed features of differentiated carcinoma (mainly of papillary type) such as nuclear chromatin clearing, nuclear clefting, pseudoinclusions, which, in each case, were not histologically sufficient to warrant a definitive diagnosis of malignancy. In other nodules galectin-3 staining was negative. The latter were either clearly benign or showed constantly a minor degree of chromatin clearing and of other atypical features when compared with galectin-3-positive cases. CONCLUSIONS: Galectin-3, a reliable marker of differentiated thyroid carcinoma as confirmed in our series of malignant neoplasms, appears expressed in nodules with an overall benign appearance but with focal areas suspicious for malignancy. The significance of such findings needs to be further investigated.     

Computerized nuclear morphometry in thyroid follicular neoplasms

Differential diagnosis of follicular adenoma (FA) and follicular carcinoma (FC) of the thyroid can be challenging in the routine practice of surgical pathology because the diagnosis of FC is strictly defined and identification depends on the presence of invasion of the capsule or blood vessels. These features may be equivocally presented in the histological sections and interpreted subjectively by different pathologists, so an objective approach to solve this problem is essential. Computerized morphometry is a scientific tool to evaluate cellular changes and it can enhance the interpretation of morphological features by the transformation of pathological changes in cells to a qualitative form. The present study investigated the diagnostic role of objective computerized nuclear morphometry in follicular neoplasms. Thirty-six cases of thyroid FC and 36 cases of FA from patients who were matched by age and sex were studied. Four nuclear parameters were selected and analyzed: mean nuclear area, mean nuclear perimeter, largest to smallest diameter ratio of the nuclei, and coefficient of variation of the nuclear area. The results indicate that all the chosen nuclear variables were significantly correlated with the FA and FC studied. In conclusion, computerized nuclear morphometry can be considered a helpful ancillary tool for differential diagnosis of FA and FC.     

Galectin-3 and carcinoembryonic antigen expression in medullary thyroid carcinoma: possible relation to tumour progression

AIMS: Galectin-3 is a beta-galactoside binding protein involved in multiple biological processes through interactions with complementary glycoconjugates. We analysed the expression and coexpression of galectin-3 and carcinoembryonic antigen (CEA), one of the putative galectin-3 ligands, in medullary thyroid carcinoma (MTC). METHODS AND RESULTS: An immunohistochemical study using monoclonal antibodies was performed on paraffin sections of 20 cases of sporadic MTC comprising 10 cases without and 10 cases with lymph node metastases at the time of surgery. CEA expression was found in all tumours, distributed predominantly in the cytoplasm and occasionally at the cell surface. In the majority of cases (18/20) moderate to strong intensity of staining was found in most of the cells. Positive cytoplasmic staining for galectin-3 was found in 16/20 cases, but varied in intensity and distribution from weak/focal (7/16) to moderate (7/16) or strong (2/16). More intense staining for galectin-3 was mainly associated with MTC cases involving lymph node metastases. Eight out of these 10 cases showed moderate to strong galectin-3 expression concomitant with CEA expression throughout the tumour tissue. CONCLUSIONS: These findings suggest that galectin-3 might play a role in the pathobiology of MTC. Simultaneous expression of galectin-3 and CEA in the same tumour cells at an advanced stage of MTC indicates the possibility of their autocrine cooperation during tumour progression.     

Galectin-3 Expression in Tumor Progression and Metastasis of Papillary Thyroid Carcinoma

Galectin-3 plays important roles in cell adhesion, cell proliferation, apoptosis, neoplastic transformation, and metastasis. Galectin-3 expression has been evaluated in various malignant neoplasms to determine its effectiveness in differential diagnosis from benign lesions and its effects on carcinogenesis. There are few and somewhat controversial results regarding its changes through cancer progression in thyroid malignancies. We studied the presence of galectin-3 expression immunohistochemically and its relation with tumor invasiveness and lymph node metastasis in 89 cases of papillary carcinoma of the thyroid. Galectin overexpression was less frequent in cases with lymph node metastases compared with cases without lymph node metastasis (P = 0.001). Metastatic foci in lymph nodes showed a lower degree of galectin-3 overexpression than their primary lesions (P = 0.001). Degree of galectin-3 overexpression was also lower in larger tumors (P = 0.009). Additionally, a decreased level of galectin-3 overexpression was observed at the invasive edges of the tumors (P = 0.001). Galectin-3 overexpression is more profound in early stages of papillary carcinoma, and its expression intensity decreases during tumor progression. This finding is consistent with roles for galectin-3 in cell adhesion to other tumor cells and the matrix.     

Role of GPER1, EGFR and CXCR1 in differentiating between malignant follicular thyroid carcinoma and benign follicular thyroid adenoma

It is extremely difficult to discriminate between follicular thyroid carcinoma (FTC) and follicular thyroid adenoma (FTA) before surgery, because the morphologies of carcinoma cells and adenoma cells obtained by fine needle aspiration biopsy (FNAB) are similar. Molecular markers may be helpful on this issue. The purpose of this study was to assess the role of GPER1, EGFR and CXCR1 in differential diagnosis between FTC and FTA. GPER1, EGFR and CXCR1 mRNA expression levels were examined in 15 FTCs and 10 FTAs using real-time RT-PCR. FTC showed to have significantly increased mRNA levels of the three molecules compared to FTA (P < 0.001 for all the three molecules). GPER1, EGFR and CXCR1 protein expression in 106 FTCs and 128 FTAs were analyzed using immunohistochemistry. The rates of GPER1, EGFR and CXCR1 high expression were 73.6%, 72.6% and 70.8% in FTC and 30.5%, 28.1% and 27.3% in FTA, respectively. Statistical analysis showed that GPER1, EGFR and CXCR1 protein expression were correlated with one another in FTC and concomitant high expression of the three molecules had stronger correlation with the occurrence of FTC than did each alone. The positive predictive values (PPV) for concomitant high expression of the three molecules for discriminating between FTC and FTA were 91.0% for GPER1/EGFR, 93.8% for GPER1/CXCR1, 92.3% for EGFR/CXCR1 and 98.2% for GPER1/EGFR/CXCR1, respectively. These results indicated that the evaluation of GPER1, EGFR and CXCR1 concomitant high expression may be helpful in differential diagnosis between FTC and FTA.     

Capsular collagen staining of follicular thyroid neoplasms by picrosirius red: role in differential diagnosis

A key criterion in the diagnosis of thyroid follicular carcinoma is capsular invasion, but invasion cannot always be demonstrated histologically. Since invasion is likely to evoke reactions in the capsular collagen, we examined the effects of invasion on capsular collagen with the picrosirius orange-red (PSR) staining technique for collagen. Under polarized light, the color of PSR-stained collagen varies as a function of the structural and biochemical properties of the collagen fibers. Capsules of widely invasive carcinomas (n = 10), minimally invasive carcinomas (n = 10), and adenomas (n = 28) were stained with the PSR method. Carcinomas were assessed along the thickened capsule for sites of definite invasion, minimal invasion, and no evidence of invasion. In adenomas, sites of thickened capsules (similar to carcinomas) were compared to sites of thin capsules. All foci were evaluated for the color and color intensity of collagen fibers. We found a significantly higher frequency of yellow-green collagen fibers than of orange-red fibers at sites of invasion, whereas orange-red fibers significantly predominated at non-invaded sites. In a minority of cases both colors occurred but the non-dominant color was of lesser intensity in all but 1 case. There were no significant differences in staining between minimally and widely invasive carcinomas. Thick capsules of adenomas consistently stained with an intense orange-red color, although weakly stained yellow-green fibers were also observed in some of these cases. We conclude that PSR staining can provide diagnostically useful information in capsular samples of carcinomas, when both color and color intensity of PSR staining are evaluated at the same site. Specifically, intense yellow-green birefringence of collagen in a thickened capsule is additional evidence for capsular invasion.     

Characterization of dendritic cells in differentiated thyroid cancer

In this study, the types and localization pattern of dendritic cells (DCs), the expression of chemokines on carcinoma cells and of the relevant receptors on DCs, and the adhesion molecules expressed on vascular endothelial cells and DCs were examined in thyroid carcinomas. Papillary carcinoma had a higher frequency of CD1a(+) immature DCs than other thyroid tumours. Macrophage inflammatory protein (MIP)-3 alpha was expressed strongly on the majority of papillary carcinoma cells and weakly on a minority of follicular carcinoma cells. DCs positive for chemokine receptor-6 (CCR-6) were densely accumulated in papillary carcinoma. DC-SIGN(+) DCs were accumulated in papillary carcinoma but rarely in follicular carcinoma. A binding assay for DC-SIGN-mediated adhesion of isolated DCs revealed significant inhibition of DC adhesion to papillary carcinoma tissues by neutralizing antibodies against intercellular adhesion molecule-2 or DC-SIGN. These results clearly indicated marked differences between papillary carcinoma and follicular carcinoma in the accumulation of immature DCs, in MIP-3 alpha expression on carcinoma cells, and in the frequency of CCR-6(+) DCs and DC-SIGN(+) DCs.     

Follicular adenoma with papillary architecture: a lesion mimicking papillary thyroid carcinoma

AIMS: The purpose of this study was to investigate the significance of 'benign' encapsulated follicular thyroid nodules with papillary structures. METHODS AND RESULTS: Twenty-one cases of encapsulated neoplastic thyroid nodules with papillary structures and nuclear features not diagnostic of papillary thyroid carcinoma (PTC) were obtained. All cases were reviewed with particular attention to nuclear features (fine chromatin pattern, optical clearing, grooves and inclusions). Representative sections were submitted for measurement of the maximum diameter of 200 round or nearly round nuclei and for immunostaining for MIB1, CK19, HBME and Ret oncogene protein. Nine cases displayed scattered optically clear nuclei or nuclear grooves in less than 30% of total neoplastic cells. They were grouped in the category of thyroid nodules with limited nuclear features of papillary thyroid carcinoma (PTC), but not diagnostic of PTC. The other 12 cases had fine or coarse chromatin, but lacked other features of nuclei in PTC. The diameter of the nuclei ranged from 5.6 to 7.2 microm and were smaller than those of PTC (6.3-10.0 microm). Immunostaining revealed positive reactivity for MIB1 in the papillary structures. Immunostaining for CK19 and HBME varied from negative or focally weak to diffusely moderate reactivity. Ret oncogene protein immunostaining showed focal and weak reactivity in one case and was negative in other cases of the study. Clinical follow-up from 6 months to 15 years revealed no evidence of metastasis. CONCLUSIONS: The papillary structures in the study cases are unlikely to represent degenerative changes due to their proliferative activity. In view of (i) the encapsulation and the uniformity of the constituent cells, (ii) the varying degrees of immunoreactivity for CK19 and HBME and negative immunoreactivity for Ret oncogene protein, and (iii) the absence or insufficiency of nuclear criteria for the diagnosis of PTC and the absence of lymph node metastasis in all study cases, we believe that these lesions represent the papillary variant of follicular adenoma. Recognition of this pathological entity is important to avoid an over-diagnosis of PTC.     

Immunohistochemical separation of follicular variant of papillary thyroid carcinoma from follicular adenoma

The accurate diagnosis of differentiated thyroid tumors is very important for clinical management of patients. The histopathological distinction between some types of differentiated thyroid tumors can be very difficult even for experienced pathologists. We used immunohistochemical markers from published data obtained from DNA expression profiling, tissue microarray analysis, and immunohistochemistry to analyze a series of 157 thyroid tumors and 5 normal thyroids. These analyses showed that several antibodies were useful in distinguishing follicular adenomas from follicular variant of papillary thyroid carcinomas including HBME-1, CITED 1, galectin-3, cytokeratin 19, and S100A4 (p<0.0001). A combination of markers consisting of a panel of HBME-1, galectin-3, and CK19 or a panel of HBME-1, CITED1, and galectin-3 was usually most effective in distinguishing follicular adenoma from follicular variant of papillary thyroid carcinoma. Because individual tumors may not express some of these markers, the use of a panel of antibodies is recommended. These results indicate that some individual antibodies or a panel of antibodies combined with histopathological analysis can be useful in separating follicular adenoma (FA) from follicular variant of papillary thyroid carcinoma (FVPTC).     

Cell proliferation marker MCM2, but not Ki67, is helpful for distinguishing between minimally invasive follicular carcinoma and follicular adenoma of the thyroid

AIMS: To compare cell proliferation markers, minichromosome maintenance protein 2 (MCM2) and Ki67, in minimally invasive follicular carcinoma (MIFC) and follicular adenoma (FA) of the thyroid and among MIFCs with different diagnostic criteria. METHODS AND RESULTS: Twenty-two MIFCs and 20 FAs were immunohistochemically stained for MCM2 and Ki67. The MIFCs were subdivided into six Group 1 tumours with both capsular and vascular invasions, seven Group 2 tumours with vascular invasion only and nine Group 3 tumours with capsular invasion only. The MCM2 and Ki67 indices were calculated, counting more than 1000 tumour cells in the most frequently positive areas. In total and Groups 1-3 MIFCs and in FAs, the average MCM2 index was 26.7 +/- 11.0, 28.4 +/- 8.6, 26.3 +/- 14.8, 25.9 +/- 8.4 and 10.7 +/- 4.5, respectively, whereas the average Ki67 index was 2.07 +/- 1.65, 1.93 +/- 2.02, 2.49 +/-1.38, 1.84 +/- 1.5 and 1.78 +/- 0.92, respectively. There was a significant difference in the MCM2 index, but not in the Ki67 index, between each category of MIFCs and FA (P < 0.01). However, neither the MCM2 index nor the Ki67 index showed a statistically significant difference among the subgroups of MIFC. CONCLUSIONS: MCM2, but not Ki67, is a helpful marker for differentiating MIFC from FA. The tumour cell proliferative activity supports the histological criteria based on diagnosing MIFC by either capsular or vascular invasion only.     

Needle core biopsy can reliably distinguish between benign and malignant papillary lesions of the breast

AIMS: To review 21 screen-detected papillary lesions in which the core biopsy findings suggested a papillary lesion and to correlate pathological and radiological findings in order to assess the risks of associated malignancy and the need for surgical intervention. The appropriate management of non-malignant papillary breast lesions detected on needle core biopsy (NCB) is currently uncertain. METHODS AND RESULTS: Forty-seven papillary breast lesions with a histological diagnosis of papilloma, papilloma with atypical ductal hyperplasia (ADH) or ductal carcinoma in situ (DCIS), multiple papillomas, 'papillomatosis' or papillary carcinoma (invasive or in situ) were identified from records at the Leeds Breast Screening and Assessment Unit. The cases were diagnosed between between May 1995 and May 2002. In 21 cases the previous NCB contained a papillary proliferation which had been categorized as either 'B2', benign, 'B3', of uncertain malignant potential, or 'B4', suspicious of malignancy. All of the 19 'B3' or 'B4' cases and one of the two 'B2' lesions had undergone open surgical biopsy. All cases with a previous 'B4' were malignant on subsequent excision. All excised cases with a previous 'B3' or 'B2' were found benign, although four of the 'B3's derived from papillomata associated with an atypical proliferation amounting to ADH. In three of these four (75%) the papillary proliferation had been associated with epithelial hyperplasia of usual type (HUT) on the core and the radiological features were of a mass lesion detected on incident round screen which had increased in size. CONCLUSION: Our results confirm the accuracy of NCB in the diagnosis of screen-detected papillary lesions of the breast. Surgical excision may not always be necessary following a 'B3' core biopsy.     

Distinction between papillary thyroid hyperplasia and papillary thyroid carcinoma by immunohistochemical staining for cytokeratin 19, galectin-3, and HBME-1

The histopathology of papillary thyroid hyperplasia and papillary thyroid carcinoma is similar enough to cause a diagnostic dilemma in a few cases. Both lesions may have papillary fronds with fibrovascular cores, nuclear crowding, and nuclear anisocytosis. Formalin-fixed paraffin-embedded tissues from 30 randomly selected patients with papillary thyroid hyperplasia and an equal number from patients with papillary thyroid carcinoma were analyzed for expression of cytokeratin 19 (CK19), galectin-3, and HBME-1. Cases of papillary thyroid carcinoma had moderate to strong CK19, galectin-3, and HBME-1 reactivity although both CK19 and galectin-3 showed positive staining in a significant number of nonneoplastic thyroid cases. HBME-1 was uncommon in the nonneoplastic cases. These results indicate that HBME-1 may be useful in helping to distinguish papillary thyroid carcinoma from hyperplasia in diagnostically difficult cases.     

Expression of promyelocytic leukaemia protein in thyroid neoplasms

AIMS: Promyelocytic leukaemia protein (PML) is an oncoprotein involved in the pathogenesis of acute promyelocytic leukaemia and is localized in distinct PML nuclear bodies. Our previous observation of overexpression of the PML in hormone-sensitive normal tissues and malignant solid tumours, including the thyroid, led to this analysis of the PML expression in various thyroid neoplasms to characterize the importance of the PML in thyroid carcinogenesis. METHODS AND RESULTS: Immunohistochemistry was performed on paraffin-embedded tissue samples from 106 thyroid neoplasms after antigen retrieval by microwave. Immunoblotting was done with fresh frozen tissues in a few tumours. The PML was strongly expressed in all papillary carcinomas in diffuse or ball-shaped patterns. In the follicular neoplasms, the PML expression was variable, but there was no significant difference between adenomas and carcinomas. In the medullary carcinomas, the PML expression was either not detectable or was lower than in non-neoplastic thyroids. Quantitatively different expression of the PML in various thyroid neoplasms was confirmed by immunoblotting. CONCLUSION: A significant difference of the PML expression according to the type of thyroid neoplasms suggests that the PML is important in papillary thyroid carcinomas, and furthermore, that PML expression may be used in differential diagnosis of thyroid neoplasms.     

Molecular pathobiology of thyroid neoplasms

Tumors of thyroid follicular cells provide a very interesting model to understand the development of human cancer. It is becoming apparent that distinct molecular events are associated with specific stages in a multistep tumorigenic process with good genotype/phenotype correlation. For instance, mutations of the gsp and thyroid-stimulating hormone receptor genes are associated with benign hyperfunctioning thyroid nodules and adenomas while alterations of other specific genes, such as oncogenic tyrosine kinase alterations (RET/PTC, TRK) in papillary carcinoma and the newly discovered PAX8/peroxisome proliferator-activated receptor γ rearrangement, are distinctive features of cancer. Although activating RAS mutations occur at all stages of thyroid tumorigenesis, evidence is accumulating that they may also play an important role in tumor progression, a role that is well documented for p53. Environmental factors (iodine deficiency, ionizing radiations) have been shown to play a crucial role in promoting the development of thyroid cancer, influencing both its genotypic and phenotypic features. It is possible that the follicular thyroid cell has unique ways to respond to DNA damage. Similarly to leukemia or sarcomas (and unlike most epithelial cancers), numerous specific rearrangements are being discovered in thyroid cancer suggesting preferential activation of DNA repair instead of cell death programs after environmentally induced genetic alterations.