Endocr Pathol (2008) 19:197–202 DOI 10.1007/s12022-008-9035-1
Differential Expression of Dysadherin in Papillary Thyroid Carcinoma and Microcarcinoma: Correlation with E-cadherin Anna Batistatou & Konstantinos Charalabopoulos & Yukihiro Nakanishi & Constantine Vagianos & Setsuo Hirohashi & Niki J. Agnantis & Chrissoula D. Scopa
Published online: 3 August 2008 # Humana Press Inc. 2008
Abstract Dysadherin is a novel glycoprotein, with an anti-cell–cell adhesion function. The aim of the present study was to examine the expression of dysadherin in thyroid papillary microcarcinoma (PMC), to associate it with the expression of E-cadherin and to investigate whether there are differences with papillary carcinoma (PC). A statistically significant difference in dysadherin and E-cadherin expression between PC and PMC and a negative correlation between E-cadherin and dysadherin expression regardless of tumor size were noted. Based on these findings it is hypothesized that retained cell–cell adhesion, through maintenance of the E-cadherin adhesion system, in PMC prevents neoplastic cells from dissociating easily from each other and metastasizing. Increased dysadherin expression is possibly one of the postA. Batistatou (*) : N. J. Agnantis Department of Pathology, University of Ioannina Medical School, University Campus, P.O. Box 1186, 451 10 Ioannina, Greece e-mail:
[email protected] K. Charalabopoulos Department of Physiology, Clinical Unit, University of Ioannina Medical School, Ioannina, Greece Y. Nakanishi : S. Hirohashi Pathology Division, National Cancer Center Research Institute, Tokyo, Japan C. Vagianos Department of Surgery, University of Patras Medical School, Patras, Greece C. D. Scopa Department of Pathology, University of Patras Medical School, Patras, Greece
transcriptional mechanisms responsible for E-cadherin downregulation in thyroid papillary neoplasia. Keywords dysadherin . E-cadherin . thyroid . papillary carcinoma . microcarcinoma
Introduction Papillary carcinoma (PC) is the most common malignant neoplasm of the thyroid gland. It is a well-differentiated neoplasm and usually has an indolent clinical course. However, larger tumors (>2 cm, or greater than T1) have higher prevalence rates of regional lymph node and distant metastases [1–3]. A thyroid carcinoma measuring 1 cm or less is defined as microcarcinoma. The most common subtype is papillary microcarcinoma (PMC). PMC is almost always an incidental finding at autopsy or in thyroid glands removed for other reasons [3, 4]. Microscopically, PMC shares common architectural, cytologic, and immunohistochemical features with the larger PC; however the overwhelming majority never gives clinically evident metastases [4–6]. This lack of clinical significance has led to the “Porto proposal”, of renaming PMC to papillary microtumor in order to avoid overtreatment [7]. The molecular pathogenesis of thyroid carcinoma has started to be elucidated. Rearrangements of Ret/PTC and mutations of the Ras and BRAF genes have been associated with the development of PC and possibly with its prognosis [8–10]. PMC is believed to develop in adolescence or young adulthood and remains stable. Very rarely, unknown additional molecular events may take place, which will speed up its rate of growth and lead to its clinical presentation [7].
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The cadherin superfamily is a group of Ca2+-dependent cell–cell adhesion molecules, which are essential for the induction and maintenance of tissue structures. Epithelial Ecadherin is present in most epithelial cells and changes in its expression during development play a crucial role in morphogenesis [11]. Furthermore, E-cadherin appears to play an important role in carcinogenesis, since its reduction/ loss has been associated with the development and progression of human carcinomas [12, 13]. Several studies have investigated the expression of E-cadherin in thyroid malignancies [14–19]. In general, it appears that E-cadherin expression is retained in follicular neoplasms; it is reduced in papillary carcinomas and lost in anaplastic and poorly differentiated carcinomas. There is no specific information so far for the differences in E-cadherin expression between PC and PMC. Furthermore, the mechanism of E-cadherin reduction remains obscure. Somatic mutations in the Ecadherin gene are rare in thyroid tumors [18]; while it has been suggested that hypermethylation of the promoter of this gene contributes to E-cadherin inactivation in thyroid neoplasms [19]. Recently, a part of our research team has reported the cloning and characterization of dysadherin (FXYD5), a cancer-associated cell membrane glycoprotein. Dysadherin has an anti-cell–cell adhesion function and downregulates E-cadherin, in a post-transcriptional manner, either exclusively, or in part, in several human neoplasms [20]. The dysadherin gene is upregulated in cells transformed by oncogenes, i.e., v-Ras, v-Src and neu [21]. Dysadherin expression has been detected in several neoplasms such as breast, gastric, colorectal, pancreatic, esophageal, thyroid, head and neck, testicular, and cervical carcinomas as well as in malignant melanoma and associated with tumor aggressiveness [22–34]. In thyroid tumors, dysadherin expression was found to be significantly higher in undifferentiated, than in papillary and follicular carcinomas and to correlate negatively with E-cadherin expression [28]. PMC were not included in this study. The aim of the present study, extending the previous one [28], is to examine the expression of dysadherin in thyroid PMC, to associate it with the expression of E-cadherin and to investigate whether there are differences with PC.
Materials and Methods Eighty formalin-fixed, paraffin-embedded archival tissue blocks of primary papillary carcinomas from the Departments of Pathology of University Hospitals of Patras and Ioannina, Greece, were included in the current study. The material consisted of 40 PC (24 pT1, 14 pT2, two pT3) and 40 PMC (mean diameter, 0.42 cm; range, 0.2–0.8 cm) from an equal number of patients, without clinically evident
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nodal metastases. All PC and PMC were of the classical type. The classification was made by categories. Immunohistochemistry Immunostaining was performed on formalin-fixed, paraffinembedded tissue sections using the EnVision System (DAKO Corp, Netherlands), and the monoclonal antibodies: NCC-M53 against dysadherin and E-cadherin (CM170B, Biocare Medical, CA, USA). Briefly, 4-μmthick tissue sections were deparaffinized in xylene; rehydrated through graded concentrations of alcohol and heated in a microwave oven for 2 cycles of 15 min each at 300 W, in citrate buffer, for antigen retrieval. Endogenous peroxidase activity was blocked with H2O2 solution in methanol (0.01 M), for 30 min. After washing with phosphatebuffered saline (PBS) for 5 min, the primary antibodies NCC-M53 (dilution, 1:1000) and CM170B (dilution, 1:50) were applied for incubation (30 min at room temperature and overnight at 4°C, respectively). Then the slides were washed for 10 min with PBS and were visualized with the EnVision system (DAKO) using diaminobezidine tetrahydrochloride as a chromogen (Sigma Fast DAB tablets, St. Louis, MO, USA). Finally, all sections were counterstained with hematoxylin. As a negative control, the first antibody was substituted with normal mouse immunoglobulin of the same class. Two pathologists (AB and CDS) without knowledge of the clinical data, performed independently semiquantitative evaluation of the staining, as previously described [28]. Where disagreement arose, slides were reviewed together and a consensus view was obtained. Staining with both antibodies was graded as 0, if no cells were stained; low (1+) if 1–20% of cells were stained; intermediate (2+) if 21–50% of cells were stained; high (3+) if >50% of cells were stained [34]. Dysadherin staining, when present, was complete membranous, and of moderate to strong intensity. The staining for E-cadherin was more heterogeneous, since in several cases only granular cytoplasmic staining was noted, while in others a weak incomplete membranous staining was observed. Such staining patterns were considered aberrant. For E-cadherin, membranous staining intensity was compared to the adjacent non-neoplastic thyroid tissue, and was equal or moderately decreased. As described previously, the grading of the intensity of staining
Table 1 Immunohistochemical expression of dysadherin Histologic type
0
1+
2+
3+
PC PMC
10 (25%) 20 (50%)
17 (42.5%) 14 (35%)
8 (20%) 6 (15%)
5 (12.5%) 0
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for dysadherin and E-cadherin was found to provide no additional information to the percentage of positive cells [28], therefore it was not considered further in this study. Statistical Analysis All analyses were performed by the statistical package STATA (v.8, StataCorp LP, TX, USA). Two-tailed statistical significance was set at 5%. All comparisons were based on χ2 test.
Results Dysadherin Expression
Fig. 1 Papillary carcinoma: a high immunohistochemical expression of dysadherin (3+), b absent expression of E-cadherin (−), (DABX400)
Fig. 2 One case of papillary microcarcinoma: a and c absent expression of dysadherin (−), in areas with papillary (a) and follicular architecture (c), b and d intermediate immunohistochemical expression of E-cadherin (2+) in areas with papillary (b) and follicular architecture (d). (DABX400)
Dysadherin immunostaining was observed in the membranes of the neoplastic cells and it was heterogenous throughout the neoplasm. No dysadherin expression was detectable in adjacent normal thyroid follicular cells. Positive staining of lymphocytes and endothelial cells was used as an internal positive control. In PC low membranous expression was detected in 42.5% (17/40) of the cases, intermediate expression in 20% (8/40), and high expression in 12.5% (5/40) (Table 1, Fig. 1a). In PMC low expression was detected in 35% (14/40) of the cases, intermediate expression in 15% (6/40), and high expression in none (0/ 40) (Table 1, Fig. 2a). Occasionally, within a classical papillary tumor small areas of follicular growth pattern
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Table 2 Immunohistochemical expression of E-cadherin
Discussion
Histologic type
0
1+
2+
3+
PC PMC
1 (2.5%) 0
15 (37.5%) 4 (10%)
16 (40%) 18 (45%)
8 (20%) 18 (45%)
were noted. In these areas the expression of dysadherin was similar to that in papillary areas (Fig. 2c). There was a statistically significant difference in dysadherin expression between PC and PMC (p value<0.05). Furthermore, in PC high dysadherin expression was statistically significant different between tumor sizes (p value< 0.001). E-cadherin Expression Immunoreactivity for E-cadherin was detected in the membranes of non-neoplastic follicular cells. In PC low expression was detected in 37.5% (15/40) of the cases, intermediate expression in 40% (16/40), and high expression in 20% (8/40) (Table 2, Fig. 1b). In PMC low expression was detected in 10% (4/40) of the cases, intermediate expression in 45% (18/40), and high expression in 45% (18/40) (Table 2, Fig. 2b). In the small areas of follicular growth pattern, within the classical papillary tumors, the expression of E-cadherin was similar to that in papillary areas (Fig. 2d). There was a high statistically significant difference in Ecadherin expression between PC and PMC (p value= 0.001). E-cadherin expression was not statistically significant different between tumor sizes (p value>0.05). Occasionally, within a classical papillary tumor small areas of follicular growth pattern were noted. In these areas the expression of E-cadherin and dysadherin was similar to papillary areas. A highly statistical significant difference was observed between E-cadherin and dysadherin expression regardless of tumor size (p value<0.001) (Table 3). E-cadherin and dysadherin are not proportionally expressed. High values of one of the above proteins are associated with low values of the other.
Dysadherin is a novel anti-cell–cell adhesion molecule, whose expression has been studied in several carcinomas, as well as in testicular neoplasms and cutaneous malignant melanomas [20, 22–34]. In all these cancer types, dysadherin expression seems to reflect tumor aggressiveness, being furthermore a marker of poor prognosis. Regarding thyroid carcinoma, the only published study indicates that dysadherin expression is associated with an adverse clinical outcome in thyroid carcinoma [28]. In the present study, in accordance with the above-mentioned one, we have found that dysadherin is not expressed in non-neoplastic thyroid follicular cells. PC expresses dysadherin, and this expression correlates significantly with tumor size. Herein, the role of dysadherin in the neoplastic transformation of thyroid gland was further investigated, by focusing on its expression in PC and PMC. PMC has been defined as a papillary neoplasm measuring 1 cm or less in diameter [7]. It is the most common subtype of thyroid cancer and is often identified incidentally in surgically removed thyroid glands for other reasons (e.g. nodular hyperplasia, thyroiditis) [4]. It usually remains clinically silent, however recurrences and clinically evident metastases have been reported, albeit very rarely. The optimal therapy is still under debate, with some authors favoring conservative and others aggressive therapy. It appears that the absolute size of PMC is important since tumors >0.5 cm have higher recurrence rates, if treated by partial thyroidectomy alone. A corollary to this notion is that PC, which although not highly aggressive has definitely a less indolent course than PMC, differs from PMC only by size. Furthermore, within the group of PC size is important, since the cumulative risk of distant metastasis increases once the primary tumor is >2 cm [1]. The exact mechanism by which size influences the behavior of papillary thyroid neoplasms is not clear yet [8]. Dysregulation of the expression of proteins associated with the cell cycle appears to be linked to more aggressive behavior of PMC. Examples include overexpression (but not gene amplification) of cyclin D1 [10, 35], overexpression of the protein p130 (of the retinoblastoma gene family [36], phosphorylation and inactivation of Tob (a novel antiproli-
Table 3 Correlation between dysadherin and E-cadherin expression E-cadherin expression
0 1+ 2+ 3+
Dysadherin expression 0
1+
2+
3+
0 1 (1.25%) 12 (15%) 17 (21.25%)
0 5 (6.25%) 18 (22.5%) 8 (10%)
0 10 (12.5%) 3 (3.75%) 1 (1.25%)
1 (1.25%) 3 (3.75%) 1 (1.25%) 0
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ferative protein) [37]. Currently, research for additional markers for identifying the subset of PMC and PC with a potential for aggressive clinical course is underway [38]. Furthermore, the local immunoresponse to papillary neoplasms might also contribute to their behaviors [39]. Based on the data available from other carcinomas, it is conceivable that if dysadherin expression reflects tumor aggressiveness, then its expression should differ significantly between PC and PMC. We sought to examine this hypothesis by investigating the expression of dysadherin in PC and PMC. Indeed, as shown herein, dysadherin expression is inversely correlated with the size of the papillary tumor and is statistically significant different between PC and PMC. This finding provides the first molecular basis for the difference in clinical behavior between PC and PMC. High dysadherin expression has been associated with reduced cell–cell adhesiveness in in vitro systems as well as in cases with the development of lymph node metastases [20]. Thus, the indolent clinical course of PMC, which metastasizes very rarely [7], is possibly due, at least in part, to the lack of high dysadherin expression. E-cadherin is normally expressed in the thyroid gland and represents the main cell–cell adhesion molecule of the thyroid follicular cells. It has been proposed that reduced/ aberrant expression of E-cadherin is critical for the pathogenesis and biological behavior of certain thyroid carcinomas [14–19]. Reduction or loss of E-cadherin has been correlated with poorly differentiated or undifferentiated/anaplastic thyroid tumors, widely invasive growth and lymph node metastases. In papillary carcinomas lack of Ecadherin expression has been considered as an adverse prognostic factor for survival. However, a specific investigation of E-cadherin expression in PMC is lacking so far. Herein, in agreement with previous studies, we have shown that E-cadherin expression is reduced in PC. The results of the present study also expand the published information on E-cadherin expression in papillary thyroid tumors. We have shown that E-cadherin expression is retained in about half of the PMC and is low in only 10% of them. Reduction of E-cadherin expression is significantly higher in PC. As noted previously PMC metastases are an extremely rare phenomenon, while PC has a higher metastatic potential. Based on our findings it is conceivable that in PMC retained cell–cell adhesion, through maintenance of the Ecadherin adhesion system, prevents neoplastic cells from dissociating easily from each other and expanding locally or from entering the lymph vessels and metastasizing to regional lymph nodes. The possible mechanisms of E-cadherin inactivation are still under investigation. In human neoplasms there are several mechanisms for the reversible and irreversible inactivation of E-cadherin. It has been reported that irreversible molecular alterations of the E-cadherin gene
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are infrequent in thyroid tumors, while methylation is frequent in papillary carcinoma [18, 19]. Translational pathways may also be involved in the reduction of Ecadherin expression in the thyroid [8]. One of the posttranscriptional pathways involved in E-cadherin regulation is the expression of dysadherin. Since dysadherin expression was negatively correlated with E-cadherin, it is likely that increased dysadherin expression is one of the posttranscriptional mechanisms responsible for E-cadherin downregulation in thyroid papillary neoplasia. In summary, this is the first study showing differences in the cell–cell adhesion system between PC and PMC, which may contribute to the differences in their biological behavior. Acknowledgements statistical analysis.
We thank Dr A. Karakosta for aid in the
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