Thyroid nodules are a common clinical problem. Thyroid hyperplasia is a physiologic response of the follicular epithelium to hormonal changes that result in disturbances in the feedback mechanism of thyrotropin-releasing hormone and thyroid-stimulating hormone (Perez-Montiel and Suster, 2008).
Thyroid cancer is the most common malignant endocrine tumor (Shahebrahimi et al., 2013). It represents about 5–24% of thyroid nodules. Papillary thyroid carcinoma constitutes about 80% of all thyroid malignancies (Mokhtari et al., 2013).
The incidence of papillary thyroid carcinoma has been increasing recently, and a precise diagnosis is essential for optimal treatment (Kyung et al., 2011). Papillary carcinomas that have an exclusive or almost exclusive follicular pattern are designated as a follicular variant of papillary carcinoma (Scopa, 2004).
The diagnosis of papillary carcinoma is based on nuclear morphology, which is characterized by enlarged and elongated nuclei with crowding and overlap, irregular nuclear contour, chromatin clearing with peripheral margination of chromatin, giving rise to what is known as orphan annie eye nuclei, multiple micronucleoli located immediately underneath the nuclear membrane, nuclear grooves, and intranuclear cytoplasmic pseudoinclusions from the accumulation of cytoplasm in prominent nuclear grooves (Al-Brahim and Sylvia, 2006) (Fig. 1).
Thyroid hyperplasia consists exclusively of either microfollicles or macrofollicles. The hyperplastic follicular cells become high cuboidal or columnar. Sometimes the hyperplasia of the cells is so great that it forms intra-acinar papillary infoldings (Baloch and LiVolsi, 2002).
A study conducted by Perez-Montiel and Suster (2008) showed that sometimes thyroid hyperplasia, although a completely benign condition, can display nuclear features that are similar to papillary thyroid carcinoma, a condition that makes the differentiation between both lesions a challenging task for the pathologists (Fig. 2).
In the great majority of cases, the pathological diagnosis of surgically removed thyroid nodules is possible by morphological examination in routine hematoxylin–eosin-stained sections. However, there are cases in which pathological criteria do not allow the differentiation between benign hyperplasia and papillary thyroid carcinoma, particularly follicular variant, making the distinction between these two groups quite subtle and challenging (Kakudo et al., 2012).
Accordingly, histopathology cannot always differentiate between hyperplasia and papillary carcinoma. Therefore, can immunohistochemistry achieve what histopathology had failed in? This is what we tried to do in this study using antibodies against CD56 and cyclin D1.
CD56 is a neural cell adhesion molecule that plays a role in cell–cell adhesion. It is normally expressed in follicular epithelial cells of the thyroid gland (Lee et al., 2008). Cyclin D1 is a positive regulator of the cell cycle. It encodes a nuclear protein that forms complexes with CDKs 4 and 6, which phosphorylate and inactivate the retinoblastoma protein. This allows cell cycle progression from G1 to S phase (Wang et al., 1998).
Materials and methods
This is a preliminary study that consisted of 30 paraffin blocks of thyroid nodules, 15 diagnosed as follicular variant of papillary thyroid carcinoma and 15 diagnosed as benign thyroid hyperplasia, collected from Kasr Al-Ainy Hospital and from private laboratories.
The study was done on paraffin blocks already stored in the archives of Pathology Department, Kasr Al-Ainy Hospital. The blocks are registered by number and the patients are unknown. We have no contact with patients at all.
Each paraffin block was recut using a rotatory microtome at 4 µm thickness and then mounted on glass slides to be stained by hematoxylin and eosin for routine histopathological examination and on charged slides for immunostaining using the standard immunoperoxidase method.
For the assessment of immunohistochemical expression, two representative slides from each case were stained, one using an antibody against CD56 (CD56, mouse anti-human monoclonal antibody, clone 123C3, code M7304, dilution 1 : 50; DAKO Denmark A/S, Glostrup, Denmark), whereas the other using an antibody against cyclin D1 (mouse anti-human monoclonal antibody, clone DCS-6, code NP 005, dilution 1 : 50; DAKO Denmark A/S). A section of tonsil was used as a positive control for cases stained for CD56, whereas a section of Mantle cell lymphoma was used as a positive control for cases stained for cyclin D1. Every section was carefully examined at power magnification (×100) for the presence of tumor cell immunostaining using Olympus microscope CX21 (Olympus America Inc., Pennsylvania, USA).
A positive membranous immunostaining with or without cytoplasmic staining in 10% or more of the neoplastic cells qualified the case as ‘positive (+)’ for CD56 (Mokhtari et al., 2013).
Immunostained sections were evaluated by estimating the percentage of tumor cells stained with monoclonal anti-cyclin D1 antibody. Only a distinct brown nuclear staining of tumor cells was considered positive. The percentage of positive cells was calculated, and positive staining was scored as follows: 1+=less than 10%, 2+=11 to 50%, and 3+=more than 50% tumor cells stained positive (Wang et al., 2000).
Data were collected, coded, and analyzed by SPSS software, version 9 under windows XP. Data were represented in the form of mean, SD, and percentage. The Fisher exact test was used to compare tumor cell immunoexpression between papillary thyroid carcinoma and benign thyroid hyperplasia. One-way analysis of variance test was used to determine whether the difference was significant. Significance was established at P value less than 0.05.
Our study consisted of 30 Egyptian cases of thyroid nodules: 15 (50%) cases were diagnosed as benign thyroid hyperplasia and the remaining 15 (50%) cases were diagnosed as papillary carcinoma, follicular variant. In benign thyroid hyperplasia, the age ranged from 19 to 56 years with a mean of 35.3±11.1. On the other hand, the age in papillary carcinoma ranged from 40 to 58 years with a mean of 48.8±6.1.
Female predominance was observed in both diseases. Women represented 80% (12 cases) of thyroid hyperplasia cases and 66.7% (10 cases) of papillary carcinoma cases.
Interpretation of CD56 immunohistochemical staining, as shown in Table 1, revealed positive expression in 13 cases of benign hyperplasia, representing 86.7% of its total cases (Fig. 3). On the contrary, only one papillary carcinoma case was CD56 positive (6.7%). These results were highly significant (P<0.0001).
As for cyclin D1, positive immunostaining, shown in Table 2, was observed in 46.7% of hyperplasia cases (seven cases) (Fig. 4) and 40% of papillary carcinoma cases (six cases). However, these results were insignificant (P>1).
As universally known, thyroid lesions are generally more common in women than in men and are more prevalent in the middle-age group. Therefore, not surprisingly, our study showed that benign thyroid hyperplasia was more prevalent in women, representing 80% of its total cases. The age of the patients ranged from 19 up to 56 years with a mean of 35.3±11.1.
As expected as well, in papillary thyroid carcinoma, women represented 66.7% of its total cases, and the age of patients ranged from 40 to 58 years with a mean of 48.8±6.1.
Our study revealed a highly significant difference in CD56 immunoexpression between benign hyperplasia and papillary carcinoma. CD56 was positive in 86.7% of benign hyperplasia cases but in only one single papillary carcinoma case.
Similar to our results, the study conducted by El Demellawy et al. (2008) showed positive CD56 expression in all cases of benign hyperplasia and in none of the papillary carcinoma cases. In addition, agreeing with our results, Kyung et al. (2011) in their study found that CD56 expression was negative in 76/80 of papillary carcinoma cases.
These results were different from those stated by Tong et al. (2011) who found that CD56 expression was negative in both lesions. In addition, the study conducted by Hülya et al. (2010) found that CD56 expression was present in 35% of papillary carcinoma cases, showing no significant difference in CD56 expression between benign and malignant follicular lesions of the thyroid gland.
Negative CD56 expression in papillary thyroid carcinoma was not surprising; being an antigen related to the differentiation of follicular cells, it was expected to be underexpressed in malignancy. Moreover, CD56 plays a role in cell–cell adhesion, and thus its negative expression in papillary thyroid carcinoma helps in tumor cell metastasis and invasion. CD56 has also been reported to decrease tumor invasion by suppressing vascular endothelial growth factor VEGF-D (Hülya et al., 2010).
By studying cyclin D1, we observed no significant difference in its immunoexpression between benign hyperplasia and papillary carcinoma, as it was positively stained in 46.7 and 40% of cases, respectively.
Similar to our results, Wang et al. (2000) observed positive cyclin D1 expression in 28/34 papillary carcinoma cases. In addition, a study conducted by Saiz et al. (2002) showed concurrent overexpression of cyclin D1 in the majority of benign and malignant thyroid lesions.
Cyclin D1 expression in both benign hyperplasia and malignant papillary carcinoma was not actually against our expectations, because cyclin D1 is a positive cell cycle regulator, which is expected to be overexpressed in any lesion showing cell proliferation, regardless of whether it is benign or malignant.
The only difference between benign and malignant cell proliferation could be in the cyclin D1 gene expression, and not the protein expression. Therefore, cyclin D1 gene mutation should be searched for and that is what we encourage other studies to do.
We conclude that CD56 can be used as a useful marker to differentiate between benign hyperplasia and papillary carcinoma. CD56 is considered a strong negative marker for papillary carcinoma diagnosis. Cyclin D1 is not a marker to be used to differentiate between benign hyperplasia and papillary carcinoma.
Conflicts of interest
There are no conflicts of interest.
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©2016Egyptian Journal of Pathology
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