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Department of Clinical and Biological Sciences (E.S., A.M., G.P., P.C., A.A., F.O.), University of Turin, and Department of Pathology (S.C., P.D.G.), San Luigi Hospital, 10043 Orbassano, Turin, Italy
Address all correspondence and requests for reprints to: Fabio Orlandi, M.D., Department of Clinical and Biological Sciences, University of Turin, San Luigi Hospital, Regione Gonzole 10, 10043 Orbassano (TO), Italy. E-mail: fabio.orlandi{at}unito.it
Abstract
Thyroid nodules are a common occurrence in the general population, but only a small number of them are eventually diagnosed as cancers. Fine-needle aspiration biopsy (FNAB) is the most accurate and cost-effective method for the presurgical management of thyroid nodules, but it misses the differential diagnosis between thyroid follicular adenomas and follicular carcinomas. Among them, minimally invasive follicular carcinoma (MIC), also defined as encapsulated tumor, only differs from follicular adenoma for the exhibition of minimal, but entire thickness, infiltration of the capsule and/or vascular invasion. This feature cannot be assessed in FNAB and can occasionally be hard to recognize in surgical specimens. As reported in several studies, galectin-3 is a reliable marker of thyroid malignancy, but no data are available on MICs. We analyzed the immunohistochemical expression of galectin-3 in 17 MICs and 52 follicular adenomas in both preoperative paraffin-embedded cytological human thyroid sediments (cell blocks) obtained by FNAB and in the corresponding surgical specimens. Among the MICs, all surgical samples showed galectin-3 immunoreactivity in the cytoplasm, whereas 16 of 17 corresponding FNAB cell blocks were positive. No evidence of cytoplasmic galectin-3 expression was observed in 48 of 52 adenomas in both cell blocks and histological tissues. These findings indicate that galectin-3 is a reliable presurgical molecular marker of MIC, improving the accuracy of conventional FNAB. It also proves to be useful in the histopathological assessment of resected tumors having suspected malignant features.
FINE-NEEDLE ASPIRATION biopsy (FNAB) is a well established diagnostic technique for the preoperative investigation of thyroid nodules, allowing a significant reduction in the number of thyroid surgical operations (1, 2, 3). However, an important limitation of FNAB is the lack of sensitivity in the evaluation of follicular neoplasms due to its inability to differentiate benign follicular lesions from their malignant counterparts (4, 5). On the basis of the extent of the invasiveness, follicular carcinomas are classified as widely invasive or minimally invasive (MIC) tumors. Widely invasive tumors show widespread infiltration of blood vessels and/or adjacent thyroid tissue and often lack complete encapsulation, whereas MICs are grossly encapsulated tumors that unequivocally infiltrate the vessels located within or immediately outside the capsule and/or penetrate the full thickness of the capsule (6, 7). There is debate as to what extent of capsular invasion is necessary to define a tumor as being MIC. In fact, some researchers accept a limited extent of capsule invasion for this definition (8), whereas others, including us, require the full thickness infiltration of the capsule (6, 9).
MIC accounts for about 50% of all follicular cancers and shows cytological features overlapping those of follicular adenomas (6, 9). To date, no effective and easy method has been established to preoperatively identify this malignant tumor. In fact, as its distinction requires the unequivocal demonstration of entire capsular invasion and/or vascular infiltration, the differential diagnosis cannot be performed by FNAB, and some difficulties may also arise in the histopathological assessment (6).
We here report the effectiveness of galectin-3 immunostaining as a marker of minimally invasive follicular thyroid cancer in samples obtained both by FNAB and surgical excision.
Galectin-3 polypeptide is a member of a growing family of ß-galactoside-binding animal lectins (Mr, 29–35 kDa) consisting of an amino-terminal domain, rich in proline and glycine, and of a COOH-terminal carbohydrate recognition domain (10). Galectin-3 is expressed in many tissues and cell types, where it is localized in the nucleus and/or the cytoplasm (11), on the cell surface (12, 13), or in the extracellular environment (11, 14). Galectin-3 has been suggested to play a role in different physiological and pathological processes, including cell-cell and cell-matrix adhesion (13, 15), cell growth (16), neoplastic transformation (17, 18), metastatization (19, 20), cell cycle regulation (21), cellular damage reparation (22), and apoptosis (23, 24). Galectin-3 expression is modulated by oncogenic and viral transformation stimuli (25, 26, 27) and is increased in several human tumors (28, 29). Furthermore, it has been detected in human thyroid carcinomas, but not in benign tumors or the normal gland (30, 31, 32, 33). In particular, we previously reported that cytoplasmic galectin-3, selectively expressed in malignant thyroid cells, is easily detectable by immunohistochemistry on both cytological paraffin samples (cell blocks) obtained by FNAB and histological sections (34, 35).
The aim of this study was to investigate the expression of galectin-3 in minimally invasive follicular thyroid cancer, to verify whether this lectin could be considered a reliable presurgical marker of MIC, and to determine whether it could improve the accuracy of histopathological assessment.
Subjects and Methods
Patients
Surgical specimens and cytological sediments were routinely collected and stored, allowing the retrospective selection of 69 consecutive patients who had undergone surgery at San Luigi Hospital, Orbassano (Turin, Italy), in the period from 1998–2000, due to nodular thyroid disease with a histological diagnosis of minimally invasive follicular thyroid carcinoma or follicular adenoma. All cases had a preoperative FNAB diagnosis that was reported as follicular neoplasm. Surgery was performed to confirm or exclude a malignant lesion. Patients included 9 males and 60 females, with a median age of 42 yr (range, 19–75 yr). Written informed consent was obtained from each subject with the approval of the San Luigi Hospital review board.
Surgical specimens
The surgical specimens were in part immediately embedded in OCT 4583 (Miles Scientific, Naperville, IL) and snap-frozen for frozen section intraoperative diagnosis. The remaining tissue was formalin-fixed and paraffin-embedded for both routine histopathological examination and immunohistochemical staining.
Histopathological classification
Hematoxylin- and eosin-stained slides were used for review and classification of the tumors. The histological findings of full thickness capsular invasion and/or infiltration of the blood vessels located within or immediately outside the capsule and of neoplastic cells attached at some point to the vessel wall were adopted as diagnostic criteria to define tumors as MICs according to Rosai et al. (6). Tumors showing invasion of four or more vessels were excluded from the study.
The series included 52 follicular adenomas (2 of 52 showing piecemeal infiltration of the capsule, without evidence of entire thickness capsular invasion) and 17 MICs. Minimally invasive follicular carcinomas consisted of 9 Hürthle cell carcinomas, 5 well differentiated follicular carcinomas, and 3 trabecular cancers.
FNAB
Before surgery, all patients underwent FNAB. Biopsies were performed with a 22-gauge, 1.5-in. needle attached to a 30-ml plastic syringe (36). After aspiration, a small amount of fluid was expelled from the needle, smeared in part onto polylysine-coated slides, fixed, and stained using the Papanicolaou method for a rapid specimen adequacy assessment. The remaining material was used for cell block preparation, in which galectin-3 expression was subsequently evaluated by the immunoperoxidase technique.
Cell blocks were obtained by fixing the aspirated fragments into an alcoholic solution (alcohol at 95 C) for 2 h at room temperature and centrifuging for 10 min at 3000 r/min. After an additional 3-h incubation in 95 C alcohol, the sedimented specimens were dehydrated and paraffin-embedded. Finally, paraffin-embedded sections were serially cut in a microtome (Top Sledge, Pabisch, Italy) and transferred onto poly-L-lysine-coated slides for the cytomorphological and immunocytochemical galectin-3 evaluation.
Antibodies
The mouse monoclonal antibody against human galectin-3 (9C4 clone, IgG1 subclass) used in this study was purchased from Novocastra Laboratories (Newcastle, UK) and was used at dilutions of 1:100 and 1:500, respectively, for immunoperoxidase staining of alcohol-fixed cytological and formalin-fixed histological samples. For control purposes, irrelevant antibodies were used.
Indirect immunoperoxidase technique
The cell blocks and tissue sections (4 µm) were first overlaid on polylysine-coated slides, dewaxed in xylene, rehydrated in decreasing ethanol concentrations, and incubated for 10 min in PBS (pH 7.4). Endogenous peroxidase activity was quenched with 3% hydrogen peroxide (H2O2) for 15 min at room temperature. Slides were then washed twice in PBS for 5 min. For the formalin-fixed tissue sections, microwave-based antigen retrieval was performed by placing slides in 0.01 M sodium citrate buffer (pH 6.0) in a microwave oven set at high power for three consecutive cycles of 5 min each. These slides were left to cool for 20 min at room temperature and rinsed in PBS. Cell blocks and tissue sections were then incubated in the blocking solution (ChemMate Buffer Kit, DAKO Corp., Copenhagen, Denmark). The serial sections were overlaid with 50 µl of the primary antibody anti-galectin-3 diluted in antibody diluent (DAKO Corp.) and incubated in a humidified chamber at 4 C overnight. After a thorough wash in PBS, the sections were incubated with the biotinylated goat antimouse IgG (Vector Laboratories, Inc., Burlingame, CA) for 45 min, and processed for streptavidin-conjugated peroxidase (DAKO Corp.). The sections were then washed three times in PBS, and incubated with 3',3'-diaminobenzidine tetrahydrochloride (DAKO Corp., Denmark) for 10 min, to develop enzymatic activity.
Slides were subsequently rinsed in tap water, counterstained with Mayer’s hemalum solution, mounted in Entellan (Merck & Co., Rahway, NJ) and examined with an Axioskop photomicroscope (Carl Zeiss, Jena, Germany). Negative controls were obtained by omitting the primary antibody or substituting it with an unrelated monoclonal antibody of the same isotype. Sections from human colonic mucosa were used as positive controls, whereas histiocytes as internal positive controls.
Cell block staining evaluation
Galectin-3 immunostaining was blindly evaluated by two independent observers, without knowledge of the previously established histological diagnosis, using an arbitrary semiquantitative scale: -, no reactivity or some neoplastic cells with nuclear reactivity; +, focal areas with weak positivity in the cytoplasm or in both the cytoplasm and the nucleus; ++, 30–60% of the neoplastic cells with a middle level of positivity in the cytoplasm or in both the cytoplasm and the nucleus; and +++, more than 60% of the neoplastic cells with a strong positivity in the cytoplasm or in both the cytoplasm and the nucleus.
Results
Follicular thyroid adenoma
Immunohistochemical analysis of adenoma cell blocks with
antigalectin-3 antibody did not show any cytoplasmic immunoreactivity
in 48 of 52 cases (Fig. 1A
), whereas
nuclear staining was observed in focal areas or in sporadic neoplastic
cells of most adenoma samples. The same pattern of cell block staining
was observed in the histological counterpart (Fig. 1B).
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, C and D).
Of these, only two, diagnosed as Hürthle cell adenomas, showed
piecemeal infiltration of the inner
border of the capsule without any evidence of full thickness capsular
invasion and/or vascular infiltration (Fig. 2, A–C, and Table 1). This feature was not observed in any
other follicular adenoma, independently of galectin-3 expression.
Normal thyroid follicular cells were negative (Fig. 3A).
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. Minimally invasive follicular thyroid carcinoma
Sixteen of 17 MIC cell blocks positively stained in the cytoplasm
(Fig. 3, C, E, and G), whereas 1 sample did not show any
immunoreactivity (Table 2, case FC10). In
MIC cell blocks, galectin-3 intensity and distribution (focal/diffuse,
cytoplasmic/cytoplasmic, and nuclear) varied according to the different
subhistotypes. Minimally invasive Hürthle cell carcinomas showed
a strong cytoplasmic and nuclear immunoreactivity (+++; Fig. 3C),
whereas minimally invasive, well differentiated follicular cancers
presented a middle level of positivity in the cytoplasm (++; Fig. 3E)
and some nuclei. Finally, minimally invasive trabecular subhistotypes
more frequently showed a weak cytoplasmic expression of galectin-3 in
focal areas (+), with no nuclear reactivity (Fig. 3G). Some stromal
cells (fibroblasts) were positive.
|
) expressed
galectin-3, including the case with negative FNAB. The positive
surgical cases showed the same labeling pattern as the corresponding
cell blocks (Fig. 3, A, D, F, and H).
Preoperative cytological evaluation and histological diagnosis,
galectin-3 expression, and additional data for each case are summarized
in Table 2.
Discussion
We analyzed galectin-3 expression as well as its subcellular distribution to evaluate the potential role of this lectin as a presurgical molecular marker of MIC. The distinction between follicular adenoma and minimally invasive thyroid carcinoma requires histological demonstration of full thickness capsular invasion of the nodule and/or vessel invasion (6). This assumption intrinsically excludes the possibility of making a preoperative diagnosis of adenoma vs. carcinoma by a cytomorphological approach. To this purpose, molecular markers differentially expressed in benign vs. malignant neoplasms could be useful (37, 38, 39, 40, 41).
As reported in other studies, galectin-3 expression is a sensitive marker of follicular thyroid cancer that proves very useful for the presurgical diagnostic evaluation of thyroid tumors (34, 35, 42, 43, 44). However, to date, no focused data have been reported on the sensitivity of cytological galectin-3 expression in minimally invasive follicular thyroid carcinoma.
The present results clearly showed significant differences in the
cytoplasmic expression of galectin-3 between follicular adenoma and
MIC. These differences were also detectable in preoperative FNAB
cytological material. In particular, considering cytoplasmic
immunoreactivity only, galectin-3 expression was found in 16 of 17
MICs, and no reaction was seen in 48 of 52 follicular adenomas. As far
as galectin-3 subcellular distribution in thyroid neoplastic lesions is
concerned, nuclear immunoreactivity was disregarded. It is, in fact,
possible that galectin-3 nuclear localization is related only to
cellular proliferation, whereas its cytoplasmic accumulation would
represent the true sign of malignant cell transformation. This
observation is in agreement with other studies (45, 46, 47)
and is supported here by the presence of nuclear galectin-3
immunoreactivity in some adenoma (Fig. 1, A and B) and in normal
thyroid cells (data not shown).
Furthermore, in minimally invasive thyroid carcinomas we also identified different patterns of galectin-3 expression for each follicular subhistotype that showed a gradual decrease in cytoplasmic and nuclear intensity of stain, starting from a high level in the Hürthle cell type, passing through a mid level in the well differentiated subtype, and ending with a low level in trabecular subhistotype. This is in agreement with our previous work, in which poorly differentiated follicular subhistotypes showed lower levels of galectin-3 compared with well differentiated cancers (34).
Among the MICs, one case of trabecular subhistotype lacked galectin-3
immunoreactivity in the FNAB cell block, as opposed to the positive
immunostaining of the corresponding surgical specimen. Considering the
typical focal galectin-3 expression in trabecular cancer subhistotype
(Fig. 3H), this could be due to FNAB sampling in galectin-3-negative
areas. Although the FNAB technique is supposed to aspirate material
from all over the tumor node, the limited amount of tissue
fragments/cells available for cytological examination cannot always be
representative of the whole lesion.
As far as follicular adenomas are concerned, four cases showed nuclear
and cytoplasmic positivity in both cell block and tissue samples (Table 1). Histological evaluation of these cases demonstrated features
suspicious of malignancy, such as hypercellularity, increased
nucleus-cytoplasm ratio, and mitoses. However, no full thickness
infiltration of the capsule or blood vessel invasion was detected in
multiple histological serial sections, thus leading to a diagnosis of
follicular adenomas with atypia. We do not know whether these cases
were true false positive or, rather, cases undergoing malignant
transformation with lack of clear-cut morphological signs of
malignancy, according to the currently accepted criteria proposed in
the literature (6). The agreement between cytological and
histological galectin-3 positivity seems to support the second
hypothesis. In this respect, emblematic results arose from two of these
positive adenomas, in which piecemeal infiltration of the capsule
without evidence of entire thickness capsular invasion was found (Fig. 2). These findings suggest that galectin-3-positive adenomas with cell
atypia may represent real early thyroid cancers still lacking the
invasiveness features of malignancy. In this context, the presence of
positive adenomas should be regarded as an index of galectin-3’s
extreme sensitivity as a marker of thyroid malignant transformation. As
this lectin is early expressed during thyroid malignant transformation,
it could play a prevailing role in cell growth, cell cycle, and
antiapoptotic mechanisms of the thyroid. Nevertheless, further studies
are required to confirm or exclude this hypothesis.
The immunodetection of galectin-3 can also prove useful for the
histopathological evaluation of doubtful cases (follicular adenoma
vs. MIC). In one of our cases of carcinomas (Table 2, case
FC7), the original histological diagnosis was Hürthle cell
follicular adenoma. Upon revision, a strong galectin-3 immunoreactivity
was found, and vascular invasion was observed in the new tissue
sections. The diagnosis was thus changed into minimally invasive
carcinoma. Therefore, a cytoplasmic galectin-3 positivity should always
alert the pathologist toward a deeper search for malignant criteria, by
means, for example, of an increased number of tissue sections.
In summary, the data presented above indicate that cytoplasmic galectin-3 expression is a reliable presurgical molecular marker of minimally invasive thyroid carcinoma, improving the accuracy of the conventional cytological evaluation of thyroid lesions and allowing a better selection of patients requiring surgery.
Acknowledgments
We thank Dr. Luciano Gubetta for revising all histological diagnoses, and Prof. Mauro Papotti (Department of Biomedical Sciences and Oncology, University of Turin) for his suggestions. We are also indebted to P. Borasio, S. Conticello, and P. Mello Teggia for surgical management of our patients.
Footnotes
This work was supported by Grant 570 (December 21, 2000) from Regione Piemonte, the Ministero per l’Università e la Ricerca Scientifica e Tecnologica (Rome, Italy) and Fondazione La Stampa-Specchio dei Tempi (Turin, Italy).
Abbreviations: FNAB, Fine-needle aspiration biopsy; MIC, minimally invasive follicular carcinoma.
Received April 23, 2001.
Accepted July 23, 2001.
References
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