Potential diagnostic markers in nodular lesions of the thyroid gland: an immunohistochemical study

AIMS
The differential diagnosis of thyroid nodules in routine practice can be problemmatic for both pathologists and clinicians. Effective treatment requires a determination of the biological nature of the lesions. For this reason, ancilliary diagnostic markers along with histological examination of the nodules may be useful. The objective of this study was to evaluate the diagnostic usefulness of novel markers in the diagnosis of hyperplastic and neoplastic nodules.


METHODS
Forty eight thyroid lesions forming four diagnostic groups including adenomatous goiters (AS), follicular adenomas (FA), follicular (FC) and papillary carcinomas (PC) were examined using standard immunohistochemical methods. Monoclonal antibodies against galectin-3, matrix metalloproteinases (MMPs) -2 and -7 and endothelial markers CD31 and CD105 were used.


RESULTS
The cytoplasmatic expression of galectin-3 was positive in all cases of papillary carcinoma. Moreover, statistically significant differences between fused groups of benign (AS and FA) and malignant lesions (FC and PC) were found Fischer's exact test (p = 0.0001). No significant differences in cytoplasmic expression of MMPs -2 and -7 and in vascular density assessed by using of both endothelial markers between benign lesions and malignant tumors were revealed.


CONCLUSIONS
Galectin-3 appears to be a useful marker in the diagnosis of papillary carcinoma only. The matrix metalloproteinases-2 and -7 are not helpful in distinguishing hyperplastic and neoplastic thyroid nodules. Endothelial markers do not appear to be suitable for thyroid differential diagnosis. A panel of antibodies in the differential diagnosis of thyroid nodular lesions would seem most suitable and further studies with larger sets of patients are awaited.


INTRODUCTION
Thyroid nodules of various biological nature occur in approximately 10 % of persons, predominantly females.The nodules represent heterogenous group that comprises both non neoplastic and neoplastic lesions with varying biological behavior.For this reason, the diff erential diagnosis of thyroid nodules presents a number of pitfalls.Diagnostic diffi culties are caused by morphological similarities of biologically diverse lesions, such as papillary hyperplasia vs. encapsulated papillary carcinoma or hyperplastic vs. neoplastic nodule.Moreover, unusual histologic criteria of malignancy, namely capsular and/or vascular invasion of minimally invasive follicular carcinoma and nuclear characteristics of papillary carcinoma, make diff erential diagnosis challenging.Fewer problems fortunately, exist in the diagnosis of widely invasive follicular carcinoma.Diffi culties in the interpretation of histological criteria may lead to interobserver variability among pathologists in discriminating between minimally invasive follicular carcinoma and follicular adenoma 1,2 .These lesions may pose a diagnostic challenge even to more experienced pathologists [3][4][5] .Precise pathological diagnosis is essential to optimal treatment of thyroid tumors.Therefore, using ancilliary diagnostic markers along with histological examination of thyroid nodules of uncertain biological behavior may be helpful.

MATERIAL AND METHODS
Forty-eight thyroid nodule formalin-fi xed paraffi n-embedded tissue sections were examined immunohistochemically to detect the expression of three tumour markers and two endothelial markers.According to WHO classifi cation the thyroid lesions were devided into 4 groups: adenomatous goiters (12 cases), follicular adenomas (12 cases), follicular carcinomas (12 cases) and papillary carcinomas (12 cases).The clinicopathological characteristics of studied groups and cases are summarized in Table 1.
Quantitative analysis of galectin-3, MMP-2 and -7 and vascular density was carried out using a light microscope Olympus BX-40 equipped with a 40x magnifi cation objective and special eye-piece 10x10 mm grid.The percentage of galectin-3 (galectin-3 index) and MMPs positive neoplastic cells was determined by counting at least 1000 tumor cells per slide in randomly selected fi elds of tumors.Immunoreactivity for galectin -3, MMP-2 and MMP-7 was scored semiquantitatively in areas of tumor and expressed as percentage of positive cells with thresholds (absent = 0; 0.1-25 (%) = 1; 25.1-50 (%) = 2; 50.1-75 (%) = 3 and 70.1-100 (%) = 4).The number of microvessels was evaluated semiquantitatively applying method described by Weidner 21 .The microvessel density (MD) was assessed in at least 3 areas (so called "hot spots") with the highest density of CD31 and CD105 respectively.Positively stained isolated endothelial cells or cell clusters with or without visible lumina were counted as separate microvessels.The highest microvessel counts were used for statistical evaluation.

Galectin-3 expression in thyroid nodules
Galectin-3 expression was estimated in follicular cells and neoplastic cells of follicular cell origin, respectively.Galectin-3 staining pattern was pancytoplasmatic and fi nely granulated.Normal thyroid tissue revealed galectin-3 positivity only in endothelial cells and intrafollicular macrophages.The results of galectin-3 expression in studied diff erent types of thyroid nodules are shown in  Comparison of galectin-3 expression in studied groups revealed statistically signifi cant diff erences between follicular adenomas and follicular and papillary carcinomas vs. adenomatous goiter as well as between papillary carcinomas vs. follicular adenomas.Suprisingly, no signifi cant diff erences were found between follicular adenomas and follicular carcinomas (p = 0.0648) as well as between follicular carcinomas and papillary carcinomas (p = 0.3120).Diff erences in galectin-3 expression between groups of benign (AS+FA) and malignant (FC+PC) thyroid lesions were statistically signifi cant (p=0.0001).Moreover, malignant lesions presented predominantly with diff use type of galectin-3 distribution within nodule while hyperplastic and benign nodules were prone to focal galectin-3 distribution, if any.

MMPs expression in thyroid nodules
Both markers (MMP-2 and MMP-7) showed in the vicinity of tested nodules focal follicular cell expression as well as positivity in both endothelial and lymphoid cells especially in the Hashimoto thyroiditis.
The results of matrix metalloproteinase-2 immunohistochemical analysis of thyroid nodules are shown in Chart 2. All cases of adenomatous goiter as well as 11 of 12 cases of follicular carcinoma and papillary carcinoma respectively showed high expression (>75 % of cells).In the group of follicular adenoma the MMP-2 positivity (threshold >75 % od neoplastic cells) has been found in 7 of 12 cases.No statistically signifi cant diff erences in the expression of MMP-2 between various thyroid nodular lesions were revealed in our study either between benign and malignant lesions or between both types of malignant thyroid tumors.
Matrix metalloproteinase-7 expression showed similar results (Chart 3) as MMP-2.The expression of this marker in more than 75 % cells was found in the majority of lesions without respect to their biological behaviour.

Assessment of microvessel density Pan-endothelial marker CD31
Microvessels were visualized by CD31 pan-endothelial antigen and scored in three "hot spots" of every lesion of all groups.Microvessel density values (range, mean, median, standard deviation) of studied lesions are presented in Table 2.The distribution of mean MD in the individual groups is displayed in Chart 4.
No signifi cant diff erence in amount of CD-31 positive vessels was shown between the studied groups in our study.

Endothelial marker CD 105
The expression of CD105 was found in 1 case of follicular adenoma and in 3 cases of follicular carcinomas and so the results were not suitable to statistical analysis.Notably, the expression in follicular carcinomas (mean microvessel density 91.5) was more than twice as high as in follicular adenoma (mean density of vessels 43.67).

DISCUSSION
Diffi culties in diff erentiating follicular thyroid lesions have instigated large investigations of potential molecular markers that could reliably distinguish benign from malignant thyroid nodules 22,23 .Several diagnostic thyroid markers have been thus evaluated but despite the advantages they possess, most also have certain limitations.None of the markers studied to date have produced unambiguous results or usefulness in every routine situation.
Our previous studies focused on thyreoperoxidase (TPO).TPO is an enzyme specifi c for the normal follicular cells and the structural changes of enzyme are induced in malignant transformation.TPO antibody (MoAB47) strongly binds the enzyme in normal thyroid cells in benign lesions whereas in malignant tumours the enzyme is expressed weakly if present 6, 9, 24-28 .Nevertheless in combination with other thyroid tumour markers the signifi cance of thyreoperoxidase increases 16 .
The present study was designed to evaluate the utility of diagnostic markers galectin-3, matrix metalloproteinase-2 and -7 as well as angiogenesis and their combination in the histological diff erentiation of the various thyroid nodules.
Galectin-3 (gal-3) belongs to the group of human lectins with 11 types of galectins defi ned by two properties: afi nity for the beta galactosides and signifi cant sequence homology in the carbohydrate binding site.This lectin is expressed by immune cells and by a variety of epithelial cells 29 .Despite the fact that the exact function of galectin-3 is not known, it has been shown to be implicated in many biological processes including regulation of growth and apoptosis, adhesion and premRNA processing.Galectin-3 overexpression is related to neoplastic transformation and tumor spread in several carcinomas, particularly breast, colon, tongue and liver 22,23,30 .Galectin-3 has been proposed as a marker that distinguishes between benign and malignant thyroid nodules; in some reports gal-3 immunostaining was absent in benign thyroid lesions and normal thyroid tissue 22 .There is general agreement that among thyroid carcinomas PC shows the most prominent and consistent galectin-3 expression 29 .
In our study, we focused on assessement of the expression of galectin-3 in groups of both benign (collection of "benign" lesions comprised hyperplastic nodules as well) and malignant thyroid nodular lesions.Our observation confi rmed strong expression of galectin-3 in 10 of 12 cases of papillary carcinomas and in 7 of 12 cases of follicular carcinomas.Statistically signifi cant diff erence in expression of this marker was shown between groups of benign (AG + FA) and malignant lesions (FC + PC); p = 0.0001 by Fischer's exact test.In agreement with other reports 31,32,23 , we found galectin-3 immunoreactivity in follicular adenomas as well; furthermore the percentage of galectin-3 positive cells and intensity of expression was higher in atypical adenomas.This fact may mirror the potential of malignant transformation in follicular lesions without morphological signs of malignancy, e.g.transcapsular and/or vascular invasion 11,30,32 .
Matrix metalloproteinases (MMPs) are a family of zinc-dependent structurally and functionaly related endopeptidases that are capable of degrading most of the components of the extracellular matrix 2,33,34 .There is evidence, that MMPs are important for the creation and maintenance of a microenvironment that facilitates growth and angiogenesis of tumours both at primary and metastatic sites 35,36 .MMPs are expressed both in tumour cells, and in neoplastic stroma cells including fi broblasts and infl ammatory cells.MMPs have been intensively studied to elucidate their possible use in diagnosis and prognosis in some tumours.Positive correlation between most tumour aggressiveness and the expression of high levels of multiple MMP family members was shown in numerous studies.Matrix metalloproteinases and their inhibitors have been studied in many tumors and malignancies in various locations such as breast 34,37,38 , lung 39 , salivary glands 39 , colon [40][41][42][43][44] .Thus MMPs can serve as diagnostic and /or prognostic tumour markers [33][34][35][45][46][47][48] .
Campo et al was the fi rst to study MMP-2 in thyroid tumors 49 .Maeta et al published a study on the expression of MMP-2 and -9 in papillary thyroid carcinomas 50 and Cho Mar et al. examined the expression of MMP-2 and -7 in benign and malignant follicular thyroid lesions 2 .All the above studies found positive staining of matrix metalloproteinases in carcinomas, benign lesions beeing negative or minimally positive.In contrast to these results, our study failed to confi rm statistically signifi cant diff erences in expression of MMP-2 and MMP-7 between papillary and follicular carcinomas and benign lesions of thyroid gland.Percentage of cells expressing both markers in in-Potential diagnostic markers in nodular lesions of the thyroid gland: An immunohistochemical study dividual groups did not diff er signifi cantly in our study.The positivity of both markers was recorded in most of the lesions without any respect to biological behavior.We found no usefulness of MMP-2 and MMP-7 in the diff erential diagnosis of nodular thyroid lesions.
Tumour angiogenesis, the formation of tumour new blood vessels, is necessary for the growth and metastasis of solid tumours 51 .In recent years, progress has been made in the identifi cation of regulators of angiogenesis and in detection of markers of microvessels density in tumours.Von Willebrand factor (FVIII), CD34 and CD31 are panendothelial markers commonly used in the evaluation of tumor vessel density.In the last decade, studies of tumour angiogenesis have also focused on another endothelial marker -CD105 known as endoglin.As mentioned previously we chose two of listed markers -CD31 and CD105 to analyse neovascularisation.CD31 plays a role in angiogenesis through its involvement in endothelial cell-cell and cell-matrix interactions and signal transduction [52][53][54] .CD31 is involved in tumor stroma angiogenesis and its expression on endothelial cells correlates with metastatic potential in some tumors 55 .Endoglin (CD105) is a cell-surface glycoprotein most recently identifi ed as an optimal indicator of proliferation of human endothelial cells.Consistently elevated levels of CD105 expression were detected on human microvascular endothelium and on vascular endothelial cells in tissues undergoing active angiogenesis, such as regenerating and infl amed tissues or tumors 56 .In contrast to panendothelial markers, CD105 is expressed only in the newly formed blood vessels and it has been documented that its expression is limited to the newly formed blood vessels in brain, lung, breast, stomach and colon cancers 53 .
Markers of angiogenesis were viewed as a promising area of investigation as well.Early reports on breast carcinoma presented angiogenesis as one of the strongest prognostic factors 21 , but these results were not subsequently confi rmed 57 .Despite a number of studies published on angiogenesis in diff erent tumor types, endocrine neoplasms have not been subjected to extensive investigation.There are few reports on tumor angiogenesis in the hypophysis 58 , in pheochromocytomas 59 , in thyroid papillary carcinoma 60,61 and medullary carcinoma 62 .Ryska et al examined microvessel density in the thyroid gland by using endothelial marker CD31.In this study, they found no signifi cant diff erences in angiogenesis between follicular adenomas and carcinomas and between papillary carcinomas on one hand and follicular tumors on the other hand 63 .One fact that can explain the variability of the results is that there is no endothelial antigen that is absolutely specifi c and sensitive and also ubiquitous for vessels in all organs 63,64 .CD31 has been shown to be reliable marker with relatively high sensitivity, but it does not distinguish neoformed microvessels from preexisting capillary bed 63 .Endoglin (CD105) was believed to be a promising specifi c marker of newly formed vessels and it has been later shown to be an independent predictor of survival in patients with various tumor types 53 e.g.colorectal cancer 51 , endometrial cancer 65 , prostate can-cer 66 .Endoglin is currently confi rmed to be more useful marker than CD31 to identify proliferating endothelium involved in tumor angiogenesis 51 .There have been no references about using CD105 in thyroid lesions yet.
This study focused on evaluation of two diff erent angiogenetic markers CD31 and CD105 in neovascularisation of thyroid nodules.The simple technique of manual capillary counting described by Weidner et al is regarded as the "golden standard" by many investigators 67 .By this method, we found no significant differences between CD31 vascular density in benign and malignant thyroid nodules.We found large variability in vessel count in the group of adenomatous goiters (minimal count 46, maximal 188).Various degree of degeneration, character of follicular component (macrofollicular versus microfollicular nodules) and the variable proportion of stromal and follicular component can explain this interface variation.Although CD31 expression in group of follicular carcinomas was higher than in follicular adenomas the diff erence was not statistically signifi cant to allow use of this marker to diff erentiate these tumors.Expression of CD 105 exclusively on newly formed vessels has been described in brain, lung, breast, stomach, endometrium and colorectal cancer 51,53,56,65,68,69 .In relation to the earlier presented expression of CD105 in the above-mentioned tumors, the results of our thyroid study were surprising.The expression of CD105 was detected only in one case of FA (mean value of 3 "hot spots" was 43.67) and in two FC (mean value = 91.5).The signifi cance of the endoglin in thyroid tumors should be object of further investigation.

CONCLUSIONS
None of the investigated markers (gal-3, MMP-2, MMP-7, CD31 and CD105) used in our study has been validated as specifi c and sensitive enough to use in discriminating of thyroid nodules.However, expression of galectin-3 has been found in malignant thyroid neoplasms, mainly papillary carcinomas and it seems that detection of galectin-3 in thyroid tumors might helpful in the diff erential diagnosis of thyroid nodules.From these results, markers MMP-2 and MMP-7 are not helpful in distinguishing of thyroid nodules with variable biologic behavior and angiogenesis is not suitable as either a diagnostic or prognostic indicator in the evaluation of thyroid nodules either.
An ideal tumor marker has not been discovered yet, but there are many markers of note for diff erential diagnosis of thyroid lesions.Thus, we recommend using a panel of markers in routine practice.

Graph 1 .Graph 2 .Graph 3 .Graph 4 .Chart 1 .
Expression of galectin-3 in individual diagnostic groups.Expression of MMP-2 in individual diagnostic groups.Expression of MMP-7 in individual diagnostic groups.Distribution of microvessels density in individual lesions.More than 75 % of galectin-3 positive cells was found in 10 of 12 (83.3%) of papillary carcinomas, in 7 of 12 (58.3%) of follicular carcinomas, and in 4 cases (33.3 %) of follicular adenoma.All cases of adenomatous goiter expressed galectin-3 in less than 75 % of cells.

Table 1 .
Clinicopathological features of studied groups of lesions.

Table 2 .
Values of microvessel density in studied lesions.
AG, adenomatous goiter; FA, follicular adenoma; FC, follicular carcinoma; PC, papillary carcinoma; SD, standard deviation Potential diagnostic markers in nodular lesions of the thyroid gland: An immunohistochemical study