CBX7 Expression in Oncocytic Thyroid Neoplastic Lesions (Hürthle Cell Adenomas and Carcinomas)

Introduction

CBX7 is a chromobox family protein and a member of the polycomb repressive complex 1 (PRC1) that, together with the PRC2, maintains developmental regulatory genes in a silenced state [1,2,3]. We have previously found that the CBX7 gene was drastically downregulated in thyroid carcinoma cell lines versus normal thyroid cells. Subsequently, we demonstrated that there is progressive reduction in CBX7 expression levels that was well related with the malignant grade of the thyroid neoplasias [4]. Indeed, its expression is slightly reduced in papillary and follicular carcinomas compared to normal thyroid tissues, whereas its expression is not detected at all in anaplastic thyroid carcinomas. Re-expression of CBX7 in thyroid cancer cells reduced their proliferation rate indicating a critical role of CBX7 in the regulation of thyroid carcinoma cell proliferation [4].

Several studies have also confirmed a critical role of CBX7 in the progression step of carcinogenesis. Indeed, a drastic downregulation of CBX7 expression has been reported by several groups also in other malignant neoplasias including pancreatic [5], colon [6], lung [7], gastric [8], bladder [9] and breast [10] carcinomas. Recently, the tumor suppressor role of CBX7 has been validated by the phenotype of Cbx7-null mice since they develop liver and lung adenomas and carcinomas [7].

Hürthle cell tumors of the thyroid are unusual neoplasms characterized by the presence of oncocytes (also called Askanazy or oxyphil cells), which are large polygonal cells with hyperchromatic, often bizarre, nuclei and an eosinophilic granular cytoplasm. Such a peculiar aspect of the cytoplasm is related to the presence of a large number of mitochondria. Hürthle cell adenoma is a rare form of thyroid neoplasia that disproportionately affects women. Like all adenomas, Hürthle cell adenomas are benign, but they have the potential to become malignant and metastasize to other body organs. Hürthle cell carcinoma of the thyroid gland is an unusual and relatively rare type of differentiated thyroid cancer. Hürthle cell cancer accounts for only about 3-10% of all differentiated thyroid cancers and has the highest incidence of metastasis among the differentiated thyroid cancers. Indeed, metastatic disease is reported at the time of initial diagnosis in 10-20% of patients and in 34% of the patients overall. Lymph node metastasis frequently involves the regional lymph nodes, even though metastases usually occur hematogenously. The overall mortality rates range from 9 to 28% [11,12,13,14,15].

According to the World Health Organization (WHO), these neoplasms are considered a variant of follicular carcinoma of the thyroid and are referred to as follicular carcinoma, oxyphilic type. Distinguishing a benign neoplasm from a malignant neoplasm based on cytologic analysis of fine-needle aspiration biopsy is not possible. Features such as pleomorphism, anaplasia, hyperchromatism, and atypia are also observed in benign follicular adenomas. Therefore, definitive differentiation of Hürthle cell carcinoma from Hürthle cell adenoma is based on capsular and/or vascular invasion. Regardless of whether or not the tumor is malignant, Hürthle cell adenomas and carcinomas are both removed promptly to prevent the disease from progressing further and total thyroidectomy has been suggested as the treatment of choice for patients affected by Hürthle cell neoplasms [12].

Therefore, the development of new tools for a more accurate diagnosis of Hürthle cell tumors may be envisaged. Here, we report the analysis of CBX7 expression by immunohistochemical techniques in a series of Hürthle adenomas and carcinomas. CBX7 expression was low or null in 68% of Hürthle adenomas, whereas it was not found reduced in follicular adenomas, as already reported [4]. According to previous published data on papillary and follicular thyroid carcinomas, we have found reduction of CBX7 expression in about 74% of Hürthle carcinomas. Therefore, these results seem to confirm the peculiar identity of Hürthle adenomas.

Materials and Methods

Results

We carried out an immunohistochemical analysis of paraffin-embedded tissues using polyclonal antibodies raised against the carboxy-terminal region of human CBX7 protein [4]. The results are summarized in table 1.

Table 1

Immunohistochemical analysis of CBX7 expression in thyroid Hürthle hyperplasias, adenomas and carcinomas (thyroid Hürthle lesions)

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CBX7 is expressed at high level in normal thyroid tissues as already reported. No changes in its expression, compared to normal thyroid tissues, were detected in goiters, in classical follicular adenomas (apart from only 1 case that showed low CBX7 expression), and Hürthle hyperplasias. Conversely, CBX7 expression is significantly reduced in 68% (34/50) of the Hürthle adenomas analyzed (table 1), with 28% (14/50) of cases completely negative for CBX7 expression (table 1). Similar results were obtained when we analyzed CBX7 expression in Hürthle carcinomas with a total 74% (20/27) of CBX7 low expressors, with 37% (10/27) of cases completely negative for CBX7 expression (table 1).

Some representative results are shown in the figures 1 and 2. Indeed, in figure 1a we show CBX7 expression in normal thyroid tissue: a strong nuclear signal was present in the follicular thyroid cells. The same result was obtained analyzing the follicular thyroid adenomas (fig. 1c). In figure 2 we show the analysis of three Hürthle cell adenomas and one Hürthle cell carcinoma for CBX7 expression. In figure 2a, we observe no staining in an area where Hürthle cells are present. In figure 2b and c the Hürthle cells show a weak and a strong staining, respectively. In figure 2d we show a representative Hürthle carcinoma where no staining was observed. It is noteworthy that no staining was observed when normal thyroid gland samples were stained with antibodies preincubated with the peptide against which the antibodies were raised (fig. 1b) or in the absence of the primary antibodies (data not shown).

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Immunohistochemical analysis of CBX7 protein expression in normal and benign neoplastic thyroid tissues. Paraffin sections from normal and neoplastic thyroid tissues were analyzed by immunohistochemistry using antibodies raised against a carboxy-terminal region of human CBX7 protein. a Immunostaining of a normal thyroid (×200). A strong nuclear staining was observed. b Immunostaining of the same normal thyroid (×200) with antibodies preincubated with the peptide against which the antibodies were raised. c Immunostaining of a thyroid adenoma (×200). A strong nuclear staining was observed.

Citation: European Thyroid Journal 3, 4; 10.1159/000367989

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Immunohistochemical analysis of CBX7 protein expression in neoplastic thyroid tissues. The tissue samples have been treated as above. a Immunostaining of a Hürthle cell adenoma (×200). No CBX7 protein expression was observed. b Immunostaining of a Hürthle cell adenoma (×200). Low CBX7 protein expression was observed. c Immunostaining of a Hürthle cell adenoma (×200). High CBX7 protein expression was observed. d Immunostaining of a Hürthle cell carcinoma (×200). No CBX7 expression was observed.

Citation: European Thyroid Journal 3, 4; 10.1159/000367989

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To investigate whether the decreased expression of CBX7 also occurs at mRNA level, we evaluated its expression in six Hürthle cell adenomas by quantitative RT-PCR analysis. A marked reduction in CBX7 expression, in comparison to a pool of three normal thyroid tissues used as normal control, was observed in four out of the six analyzed samples (fig. 3). Hence, this result confirms that CBX7 expression is regulated at transcriptional level.

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Expression analysis of CBX7 mRNA in six Hürthle cell adenoma samples. Quantitative RT-PCR was performed on six Hürthle cell adenoma samples. Relative expression value represents CBX7 mRNA expression level of each Hürthle cell adenoma sample compared to the CBX7 level observed in the mean of three normal thyroid samples, as control set equal to 1. GAPDH mRNA expression was evaluated as internal control. Reactions were performed in duplicate. NT = Normal thyroid; H1-H6 = Hürthle cell adenomas.

Citation: European Thyroid Journal 3, 4; 10.1159/000367989

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Discussion

CBX7 has been reported as a tumor suppressor gene in most of the human neoplasias since its loss of expression seems to occur in advanced stages of carcinogenesis. Indeed, a reduced CBX7 expression has been correlated with an advanced N stage and poor outcome of colorectal carcinoma patients. Moreover, a considerable progressive decrease of CBX7 protein expression has been found associated with increasing malignancy grade of pancreatic adenocarcinoma, gradually going from normal pancreatic tissue to pancreatic intraepithelial neoplasias (PanINs) and invasive ductal adenocarcinoma. Conversely, the maintenance of CBX7 expression showed a trend toward a longer survival [5].

The critical role of the loss of CBX7 expression in human cancer progression has recently been confirmed by the identification of several CBX7-regulated genes. We have, in fact, demonstrated that CBX7 is able to negatively regulate the expression of a set of genes involved in the acquisition of a fully malignant phenotype, such as SPP1, SPINK1, STEAP1 [17]. Conversely, CBX7 positively regulates the expression of FOS, FOSB, EGR1 [17] and E-cadherin [18], whose loss of expression is considered a hallmark of the epithelial-mesenchymal transition. Consistently, we found that in pancreatic carcinomas the E-cadherin reduced expression resulted associated with the loss of CBX7 expression and with a trend towards a poorer patient survival [5]. Equally, E-cadherin expression is weakly reduced in differentiated thyroid carcinomas, not aggressive and still expressing CBX7, even at a reduced level, whereas it is drastically reduced in poorly differentiated and anaplastic carcinomas, that are highly aggressive and do not express CBX7 at all [18]. A similar correlation has been found in thyroid and lung carcinoma samples between CBX7 and its regulated genes [17]. Finally, the inhibition of cell growth following the restoration of CBX7 expression in thyroid cancer cells [4] has further demonstrated the critical role of the absence of CBX7 expression in carcinogenesis.

In the present study, prompted from the strict correlation found between CBX7 expression and the aggressiveness of thyroid neoplasias [4], we have analyzed CBX7 expression in thyroid lesions containing oncocytic cells. Immunohistochemical analysis evidenced a reduced CBX7 expression in Hürthle adenomas and carcinomas with respect to the normal thyroid tissue, whereas no changes in CBX7 expression were observed in Hürthle hyperplasias, that are considered non-neoplastic hyperproliferative lesions. The lack of CBX7 expression in a significant number of Hürthle adenoma samples is of interest. Indeed, 68% of Hürthle adenomas showed a reduced or absent expression of CBX7, that is, conversely, high in all classical follicular adenomas. The latter, at odds with the Hürthle adenomas, very rarely progress to follicular carcinomas. This result is then consistent with different behavior of these neoplasias, confirming a certain correlation between the loss of CBX7 expression and tumor aggressiveness [4,5,6,7,8,9,10].

Unfortunately, we have been able to analyze CBX7-specific mRNA by quantitative RT-PCR only in a limited number of Hürthle adenoma samples due to the unavailability in our laboratory of a significant number of surgically removed specimens: the results shown in figure 3 are consistent with the immunohistochemical data.

Interestingly, we have previously shown that CBX7 is target of miR-181b [10], that is overexpressed in papillary carcinomas, but not in follicular adenomas. The components of the miR-181 family (miR-181a, miR-181b and miR-181c) are upregulated in several malignant neoplasias other than thyroid with a correlation between high miR-181 expression with a poor prognosis [19]. Moreover, we have also demonstrated that CBX7 is negatively regulated by HMGA1, a protein whose overexpression is associated with cancer progression in various malignancies including thyroid tumors (where it is abundantly expressed), whereas its expression is absent in follicular thyroid adenomas [20]. Therefore, it would be helpful to associate the analysis of CBX7 expression with that of HMGA1 and miR-181 in Hürthle adenomas.

In conclusion, the evaluation of CBX7 expression might be considered a potential tool for the differential diagnosis between classical and Hürthle adenomas, but further studies analyzing CBX7 expression in surgically removed neoplasias and fine-needle aspiration biopsies using other more quantitative techniques are still required to finally assess the utility of CBX7 evaluation in thyroid pathology.

Acknowledgements

This work was supported by grants from Associazione Italiana per la Ricerca sul Cancro (AIRC) (IG 11477, A.F.) and (IG 12962, G.C.), and from the Ministero dell'Università e della Ricerca Scientifica e Tecnologica (MIUR).

Disclosure Statement

The authors have no conflicts of interest to disclose.