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The role of immunohistochemical markers in diagnosis and prognosis of medullary thyroid carcinoma

Fathi, Anan

Egyptian Journal of Pathology: July 2013 - Volume 33 - Issue 1 - p 13–17
doi: 10.1097/01.XEJ.0000429915.96543.7e
Original Articles

Background/objective: Medullary thyroid carcinoma may mimic wide variants of benign and malignant thyroid neoplasm. This study is designed to analyze the role of immunohistochemical markers [calcitonin, carcinoembryonic antigen (CEA), and vimentin] in the diagnosis of medullary thyroid carcinoma and to determine their prognostic value.

Material and methods: An immunohistochemical study using a panel of monoclonal antibodies for calcitonin, CEA, and vimentin was carried out on paraffin sections of 24 cases of sporadic medullary thyroid carcinoma of different stages collected from the pathology departments of Zagazig University and Cairo University.

Results: All neoplastic cells showed immunoreactivity for calcitonin, CEA, and vimentin. Strong reactivity (++++) for calcitonin was observed in all cases without lymph node metastasis (10/10; 100%), whereas its weak reaction (+ or ++) was associated with cases of advanced stages (9/14; 64%). However, strong CEA positivity was highly associated with cases of advanced stages (9/14; 64%). For vimentin, all cases showed vimentin expression with different grades of intensity and this was correlated with the stage of medullary thyroid carcinoma, but not with other clinicopathologic variables.

Conclusion: The positivity of all cases for calcitonin and CEA supports the diagnosis of medullary thyroid carcinoma. Weak immunoreactivity for calcitonin and strong CEA immunoreactivity are associated with a poor prognosis. Vimentin expression indicates epithelial mesenchymal transition phenomena with invasiveness and metastatic potential.

Pathology Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt

Correspondence to Anan Fathi, MD, Pathology Department, Faculty of Medicine, Zagazig University, 44112 Zagazig, Egypt Tel: +20 101 355 7356; fax: +20 552307830; e-mail:

Received October 31, 2012

Accepted November 14, 2012

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Medullary thyroid carcinoma (MTC) arises from calcitonin-producing C-cells of the thyroid. It represents about 3–15% of malignant thyroid tumors (Brandão et al., 2009). It behaves more aggressively than well-differentiated thyroid cancer of follicular cell origin, accounting for 13% of all thyroid-related deaths. Most cases arise sporadically but about 25% are hereditary as part of multiple endocrine neoplasia type 2 (MEN2a and 2b) or isolated familial MTC (Guerrero et al., 2011). This tumor is able to synthesize and express calcitonin. Thus, elevated basal or stimulated levels of this peptide are highly sensitive and specific indicators of MTC. It also produces a variety of serum markers such as carcinoembryonic antigen (CEA) and chromogranin A, which play an important role in the diagnosis of neuroendocrine tumors (Bockhorn et al., 2004). Considerable variability in the morphologic patterns observed in MTC results in a huge ratio of incorrect diagnosis. Thus, the confirmation of its diagnosis relies largely on immunohistochemistry for calcitonin, calcitonin-gene product, other neuroendocrine markers, and CEA (Lloyd et al., 1983; Lange et al., 2004; Gagel et al., 2005).

CEA is a tumor-associated antigen that is detected in the sera of cancer patients at increased levels. Increased expression of CEA was observed in MTC patients with recurrent or metastatic disease. Therefore, CEA is also considered an important prognostic marker (Giuffrida and Gharib, 1998). Immunohistochemical studies for calcitonin and CEA have shown a similar pattern of distribution in early MTC and a more intensive staining for CEA in patients with aggressive disseminated MTC (Bockhorn et al., 2004).

Vimentin is an intermediate filament protein, normally expressed in mesenchymal cells, but it was reported that aberrant expression of vimentin in epithelial cancer cells might be related to local invasiveness and metastasis potential (Gilles et al., 1996).

A relationship between vimentin immunostaining and metastatic potential has been suggested from studies on breast cancer, where it was associated with high proliferation, lack of differentiation, lack of estrogen receptors, and reduced survival (Heatley et al., 1993).

The association of vimentin expression with migratory status was attributed to temporary induced expression of vimentin in epithelial cells under certain conditions such as during development or wound healing. Thus, it is possible that this transient expression of vimentin in epithelial cells might be implicated in tumor invasion. This phenomenon by which epithelial cells acquire some mesenchymal features such as vimentin expression is defined as epithelial mesenchymal transition (EMT) (Gilles et al., 1996).

EMT represents the mechanism of acquisition of a migratory phenotype by epithelial cells. This leads to many genetic and epigenetic changes in carcinoma cells, affecting their motility, degradative, ability and their cohesiveness, resulting in an invasive phenotype. At the molecular level, EMT involves loss of expression of adhesion proteins such as E-cadherin and gain of mesenchymal markers such as vimentin and fibronectin (Liu et al., 2011).

The aim of this study was to evaluate the role of immunohistochemical markers (calcitonin, CEA, and vimentin) in confirming the diagnosis of MTC of different stages and predicting its prognosis and also to analyze vimentin expression in relation to clinicopathological variables at the time of surgery.

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Materials and methods

Tissue sample

Formalin-fixed paraffin-embedded tissue of 24 cases of sporadic MTC were obtained from the archival material of the pathology departments of Zagazig University and Cairo University, Faculty of Medicine. The material was selected according to their histopathological and histochemical diagnosis. Clinical data of patients as retrieved from their files showed an age range of 29–76 years, with a mean of 52.5 years, with female predominance. Nodal metastasis at the time of surgery was present in only 14 cases.

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Immunohistochemical stains were performed on 5 μm sections using monoclonal antibodies against calcitonin (dilution; 1 : 50), CEA (dilution; 1 : 100), and vimentin (dilution; 1 : 100) using the indirect immunoperoxidase technique. All antibodies were obtained from Dakopatts (Gloftrup, Denmark). Immunostaining was performed using the avidin–biotin peroxidase complex technique with the reagent supplied by Vector Laboratories (Burlingame SA, California, USA). After deparaffination and rehydration, endogenous peroxidase activity was blocked with 0.3% of H2O2/methanol, followed by nonimmune horse serum for 20 min to block nonspecific antigen binding. The sections were incubated with primary antibodies at 4°C overnight. This was followed by incubation with horse anti-mouse biotinylated IgG for 30 min and thereafter with the streptavidin–biotin peroxidase complex reagent for 30 min. After each step, sections were washed with PBS. The reaction was visualized using a diaminobendizine tetra hydrochloride solution. For each case, the primary antibody was replaced with PBS as a negative control.

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Staining evaluation

Calcitonin expression was graded as follows: −, if no staining; +, if 1–5% of cells; ++, if 5–25%; +++, if 25–50% of cells; ++++, if more than 50% of cells stained (Harach and Bergholm, 1993).

CEA expression was graded as weak, moderate, or strong (Cvejic et al., 2000).

Vimentin expression was graded 0 if no staining, 1 if up to 25% of cells reacted positively, and 2 if 26% or more of cells reacted positively (Heatley et al., 1993).

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Light microscopic examination

It was found that 21% (5/24) of tumors were circumscribed whereas the remaining 79% (19/24) of tumors were infiltrative; tumor cells were arranged in various patterns: classical, that is, sheets of cells separated by hyalinized stroma containing amyloid; insular, where cells grouped into islands surrounded by vascular stroma; and also trabecular, pseudopapillary, and papillary patterns. All cases showed polygonal tumor cells with a round or an oval nucleus, granular amphophilic cytoplasm, and inconspicuous nucleoli. Only 25% (6/24) of cases showed areas of spindle tumor cells. Amyloid was present in variable amounts in all cases. At the time of surgery, nodal metastasis was present only in 58% (14/24) of cases.

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All tumors showed a cytoplasmic reaction, but with variable grades (Figs 1 and 2). Marked staining was noted in all MTC cases without nodal metastasis (10/10; 100%), especially in areas showing a typical pattern of MTC, whereas in cases with nodal metastasis +++ was noted in five cases (5/14; 36%), and a weak reaction (+ or ++) was noted in nine cases (9/14; 64%). Normal entrapped thyroid follicles showed no immunoreactivity for calcitonin (Tables 1 and 2).

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Carcinoembryonic antigen

All tumors were immunoreactive for CEA with a membranous and cytoplasmic reaction (Figs 3 and 4). A strong CEA reaction was noted in 41% (10/24) of cases; all of them were associated with lymph node metastasis. A moderate reaction occurred in 46% (11/24) of cases. A weak staining reaction was found only in two cases.

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All cases showed perinuclear staining of vimentin, but with variable grades (Figs 5–7); grade 1 was noted in 11 (11/24; 46%) cases and grade 2 in 13 (13/24; 54%) cases. Grade 2 vimentin staining intensity was found in 11 cases of stage 3 and stage 4.

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MTC is a thyroid tumor arising from calcitonin-producing parafollicular C-cells of the thyroid (Desai et al., 2005). The neoplastic cells secrete amyloid, which is frequently present both in primary and in metastatic foci. The presence of amyloid indicates a better prognosis (Baloch and LiVolsi, 2001). There are 11 subtypes of medullary carcinoma, among them, classical, carcinoid like, papillary, giant cell types, or with squamous differentiation. Thus, medullary carcinoma may mimic wide variants of benign and malignant primary thyroid neoplasms. Thyroid paraganglioma, hyalinizing trabecular adenoma, and metastatic neuroendocrine tumors are typical differential diagnoses (Tatic, 2003).

Thyroglobulin and calcitonin are the main differentiating immunohistochemical markers for tumors with follicular and parafollicular origin, respectively (Brasanac et al., 1993). CEA is absent in follicular and papillary carcinoma and positive in medullary carcinoma (Tatic, 2003). Similarly, in our study, all cases showed variable grades of positivity for calcitonin and CEA, supporting their parafollicular origin. These results were in agreement with those of other studies (Bockhorn et al., 2004; Papi et al., 2006).

Brandão et al. (2009) and Milman et al. (2011) concluded that calcitonin and CEA are useful diagnostic and prognostic markers of MTC. Both can be identified in tumor specimens and serum of MTC patients.

Some authors have reported that immunoreactivity for calcitonin in 25% or fewer tumor cells with a marked increase in CEA positivity indicates a poor prognosis (Tatic, 2003).

In our results, marked calcitonin staining (++++) was noted in cases without lymph node metastasis (10/10; 100%), but cases with nodal metastasis at the time of surgery varied between +++ in five cases (5/14; 36%) and + or ++ in nine cases (9/14; 64%); however, the reverse was noted in CEA immunoreactivity as a strong reaction was noted in 10 cases (10/14; 71%), all of them of advanced stage (with nodal metastasis). These results are in agreement with those obtained by Machens et al. (2007), who reported that poor immunohistochemical staining for calcitonin indicates a poor prognosis, especially if associated with strong reactivity for CEA.

Fischer and Asa (2008) reported that calcitonin together with CEA are essential immunohistochemical markers for the diagnosis of MTC, especially if histologic subtypes as follicular, papillary, or encapsulated variants that have diagnostic difficulties with follicular cell-derived carcinoma and paraganglioma. Also, they found that CEA is important in the assessment of medullary carcinoma that lack or show focal reactivity for calcitonin.

Another study that was consistent with our reported that calcitonin and CEA were positive in all MTC tumors and can be considered as markers for differentiating patients with high malignancy from those with low malignancy (Takami et al., 1988).

Baloch and LiVolsi (2001) reported that a small amount of calcitonin immunoreactivity was associated with worse outcome. Thus, our study was in agreement with other studies that proved that calcitonin and CEA immunoreactivity are essential for both the identification and the assessment of progression of MTC (Wilson et al., 1986; Giraudet et al., 2008).

We also studied the expression of vimentin in MTC cases and correlated this expression with many clinicopathological variables at the time of surgery. We found that it was expressed in all cases with variable grades of intensity; grade 2 was found in 54 % (13/24) of cases. Eleven of these were in stages 3 and 4 and associated with invasion and/or nodal metastasis. This was in agreement with many in-vitro studies on breast cancer cell lines and cervical cancer cell lines that proved the association between vimentin expression and invasiveness (Thompson et al., 1992; Gilles et al., 1994; Sommers et al., 1994).

Also, this was in agreement with many studies on epithelial tumors such as renal, prostatic, pulmonary, thyroid, and ovarian tumors that found an association between vimentin expression and tumor progression as was reported by Gilles et al. (1996).

A relationship between vimentin immunostaining and metastatic potential has been suggested from studies on breast cancer, where it was associated with high proliferation, lack of differentiation, lack of estrogen receptors, and reduced survival (Heatley et al., 1993).

Vasko et al. (2007) reported that increasing vimentin staining intensity was associated with invasion, nodal metastasis, and multifocality, but no association between vimentin expression and tumor size, patient sex, or age. This was in agreement with our findings.

In our results, there were two cases of MTC stage 2 that showed grade 2 intensity of vimentin expression, where there was no nodal or distant metastasis but local invasion was present. However, three cases of MTC one in stage 3 and two in stage 4 showed grade 1 intensity of vimentin expression. This could be explained by the hypothesis that the expression of vimentin might be inducible and a transient event. Thus, potentially invasive cells would be induced to express vimentin in conjunction with the invasive phenotype. This phenotype would be reversible after the migratory cells re-establish in invasive clusters or at metastatic sites (Gilles et al., 1996).

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From this study, we concluded that:

Positive immunoreactivity for calcitonin and CEA supports the diagnosis of MTC.

A weak calcitonin reaction associated with a strong CEA reaction indicates a poor prognosis.

Vimentin expression indicates EMT phenomena with invasiveness and metastatic potential.

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Conflicts of interest

There are no conflicts of interest.

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