Galectin-3 immunostaining in thyroid neoplasms

1. Medical doctor; resident on Pathology, Hospital de Base do Distrito Federal (HBDF). 2. Chairman professor of Pathology, PhD in Pathology, Department of Pathology, Universidade de Brasília (UnB). Work developed at the Laboratory of Pathology, Faculty of Medicine, Universidade de Brasília, and supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Empreendimentos Científicos e Tecnológicos (FINATEC) and Programa Institucional de Bolsas de Iniciação Científica (PIBIC/UnB). J Bras Patol Med Lab • v. 41 • n. 5 • p. 341-46 • outubro 2005


Introduction
Thyroid carcinoma is the most frequent endocrine malignancy.Fine-needle aspiration biopsy (FNAB), scintigraphy, and ultrasonography are procedures currently used on investigation of a nodular lesion.FNAB is the most important method in defining the malignancy of a neoplasm.It shows great diagnostic accuracy for medullary and anaplastic carcinomas, but for papillary carcinoma (the most common) false-negative results are not rare (13,23) .Nevertheless the FNAB procedure has limitations such as inadequate sampling and shared cytological morphology between malignant and benign lesions (5,14,23) .Morphological distinction between hyperplastic adenomatous nodules, well-differentiated follicular carcinomas, and follicular variants of papillary carcinoma is difficult (2,4) .Criteria for follicular carcinoma diagnosis are based on demonstration of vascular and capsular invasion (11) .These criteria can only be fulfilled in histological sections, performed after thyroidectomy.Therefore, on presurgical period, distinction between malignant and benign disease is not accurate using current available methods.This background is responsible for only 10% of resected glands being classified as malignant (15) .Patients with inconclusive cytological diagnosis are referred to surgery, and definitive diagnosis is given using the excised gland.This approach has great economical and psychological impact for the patient as well as for the health care providers.The magnitude of this problem may be realized by noting that 4% of Americans between 30 and 60 years have palpable thyroid nodules (28) .
As a result of the previous considerations, a presurgical marker for thyroid malignancy would be extremely useful for rational decision on clinical observation or surgical intervention, avoiding refer patients with benign lesions to surgery.Galectin-3, a ß-galactoside-binding lectin, has been involved in various physiologic and pathologic functions, such as cell growth, cellular adhesion, inflammation, apoptosis, and neoplastic transformation (1,12,17,19,21,24,27,33) .Xu et al. showed that galectin-3 is over-expressed in follicular and papillary carcinomas of thyroid, but not expressed in non-malignant lesions (32) .These findings were corroborated by other authors (3,8,10,18,25) .In fact, Bartolazzi et al. (3) have conducted a prospective multicentre study that showed 99% accuracy in differentiating benign and malignant lesions, using immunodetection of galectin-3.Following studies (6,9,20) , however, did not show strong relation between thyroid malignancy and galectin-3 expression.Before the application of this method on clinical practice, several trials on the expression of galectin-3, in histological and cytological samples, should be taken in order to know the sensitivity and specificity of the method in detecting malignant neoplasms.The objective of the present experiment is to describe the prevalence of galectin-3 expression on thyroid neoplasms, and to assay sensitivity and specificity of galectin-3 as a marker for malignant thyroid neoplasms.

Tissue specimens
Cases were retrieved from the archives of the Departments of Anatomical Pathology of both Hospital Universitário de Brasília (HUB) and Hospital de Base do Distrito Federal (HBDF).Specimens had been routinely fixed in formalin and processed after paraffin embedding.Fifty-seven samples, including 14 papillary carcinomas, 22 follicular carcinomas and 21 follicular adenomas, obtained by thyreoidectomy from 1992 to 2000 were selected for analysis.Normal thyroid adjacent to neoplastic tissue was also examined in 48 cases.

Immunohistochemistry
The immunohistochemical procedure followed the method described by Hsu et al. (16) .Briefly, representative sections of 4µm obtained from paraffin blocks were deparaffinized and rehydrated.Endogenous peroxidase was quenched with 3% H2O2 diluted in methanol.Sections were incubated overnight in humid chamber with mouse monoclonal antibody anti-galectin-3, clone 9C4 (Novocastra Laboratories, UK) at 4°C.The dilution used was 1:500.Sections were then incubated with biotinylated secondary antibody (DAKO, USA) for 30 minutes at room temperature.Indirect immunoperoxidase was carried out using a commercially available avidin-biotin staining kit (DAKO, USA).The immunoenzimatic activity was developed with 3,3'-diaminobenzidine tetrahydrochloride (DAB) solution.Haematoxylin was used for counterstaining.

Statistical analysis
Sensitivity, specificity, positive and negative predictive values for the immunostaining method were assessed taking histomorphological diagnosis as gold standard.Sensitivity was defined as the ratio between the number of carcinomas with positive results and the total number of thyroid cancers.Specificity was defined as the ratio between the number of benign and normal thyroid tissues with negative results and the total number of non-malignant tissues.Positive and negative predictive values were respectively calculated as follows: number of carcinomas with positive results divided by the total number of cases with positive results, and the number of benign and normal tissues divided by the total number of cases with negative results.All data were expressed in percentage.

Results
Table 1 summarizes the immunohistochemical reaction of galectin-3 in the cases studied.
Expression of galectin-3 in papillary carcinoma -All cases of papillary carcinoma showed strong (++++) or intermediate (++) immunoreaction against galectin-3.Strong cytoplasmic staining was observed in all cases and rare nuclear localization was also observed (Figure 1).
Expression of galectin-3 in follicular adenoma -Twenty cases of adenomas did not express galectin-3.Only one in 21 cases had a focal (+) immunostaining.This immunoreactive case (Figure 3), from a 40-year-old female, showed predominant nuclear expression of galectin-3.Colloid was immunostained in focal areas where follicular cells were positive.Haematoxylin-eosin preparations were revised and no vascular or capsular invasion was detected in this case.
Expression of galectin-3 in normal thyroid -Among 57 cases studied, 48 had adequate sampling of normal thyroid tissue for analysis.No expression was observed in normal tissue.
Expression of galectin-3 by stromal cells -Some stromal cells, such as nerve fibers, fibroblast, smooth muscle, inflammatory cells, and endothelial cells showed scattered immunoreaction.

Discussion
Current presurgical methods are not able to differentiate follicular adenoma from follicular carcinoma.No clinical, ecographic or scintigraphic features are reliable for labeling a follicular lesion as malignant or benign (29,31) .Although FNAB report is the most important indication for surgery, this procedure has limitations with sampling and overlapping morphological features between benign and malignant neoplasms.An FNAB report as suspect follicular nodule refers a patient to surgery, but many patients in this case will have benign diseases on paraffin preparations.A rational presurgical evaluation of these nodules would prevent surgical risk, lifetime hormone reposition and hospital cost.
In this histological study galectin-3 was specifically expressed by malignant neoplasm, showing that this molecule could be used as an adequate marker for malignant follicular cells.Our data corroborate previous works (4,8,10,18,25,32) that found consistent expression of galectin-3 by papillary carcinomas, some negative results for follicular carcinomas, and erratic expression by adenomas.High percentages for sensitivity, specificity and predictive values, obtained by our study suggest that galectin-3 expression by a follicular cell may be used as marker for its malignancy.
Adenomas expressing galectin-3, as described above, have been classified by previous works as carcinomas without morphological evidence of capsular and vascular invasion (4,7,8,25) .Thus, a new possibility of detecting early thyroid cancer can be accomplished with galectin-3 immunostaining test.However more investigation is certainly needed to certify if this finding is a true or a false positive result.
Cytoplasmic galectin-3 expression was found only in carcinomas, and therefore it may be considered an evidence of malignancy.Nuclear staining was not consistently associated with malignant neoplasm and should not be a marker for cancer, until more data are available.Cytoplasmic predominance expression of galectin-3 has been related to progression of normal tissue to adenoma and carcinoma in the colon carcinoma model (22) .Our data and previous studies suggest that the same pattern of cellular staining can be used with thyroid neoplasm.Galectin-3 may play an important role in malignant transformation, especially in cell adherence and metastasis.Several works have associated this molecule with metastasis capability (26,27) and malignant transformation (30,34) .
Our work supports previous findings that this immunodiagnostic method is a simple, cheap and important diagnostic support in distinguishing malignant and benign thyroid neoplasm.Before application of this method on cytological samples, histological data should be gathered for knowledge of diagnosis statistics.
The immunocytochemical method is preferred to other molecular biology-based methods.Because of constitutional stromal expression of galectin-3, methods like immunoblotting show false-positive results (32) .Morphological evaluation associated with immunocytostaining with galectin-3 should be the best approach for presurgical evaluation for thyroid nodules.