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Dysadherin: Expression and Clinical Significance in Thyroid Carcinoma

Pathology Division (H.S., Y.I., A.M., S.H.), National Cancer Center Research Institute, Tokyo 104-0045, Japan; Department of Medicine (Y.A., H.S.), Tokai University School of Medicine, Kanagawa 259-1193, Japan; and Ito Hospital (K.I.), Tokyo 150-8308, Japan

Address all correspondence and requests for reprints to: Setsuo Hirohashi, M.D., Director, National Cancer Center Research Institute, 5-1-1, Tsukiji, Chuo-ku, Tokyo 104-0045, Japan. E-mail: shirohas{at}ncc.go.jp.

	Abstract

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Dysadherin is a cancer-associated cell membrane glycoprotein. Its cDNA encodes 178 amino acids, including a putative signal sequence, a potential O-glycosylated extracellular domain, a single transmembrane domain, and a short cytoplasmic tail. Dysadherin is believed to down-regulate the expression of E-cadherin, the prime mediator of cell-cell adhesion in epithelial cells, by a posttranscriptional mechanism and promote the metastasis of carcinoma cells. To evaluate the association between dysadherin expression and E-cadherin expression in thyroid carcinoma, immunostaining for dysadherin and E-cadherin was performed in 51 papillary, 10 follicular, and 31 undifferentiated carcinomas. Immunoreactivity for dysadherin, localized at cell-cell boundaries, was detected in 39 of the 51 papillary carcinomas and all 31 undifferentiated carcinomas but not in the follicular carcinomas or normal thyroid tissue controls. Dysadherin expression was significantly higher in undifferentiated carcinoma than in papillary carcinoma and follicular carcinoma and showed significant negative correlation with E-cadherin expression. The degree of dysadherin expression was significantly associated with the prognosis, occurrence of secondary undifferentiated carcinomas, size of the primary tumor, and metastasis to the regional lymph nodes and lungs. In conclusion, a process involving increased dysadherin expression may lead to an adverse clinical outcome.

	Introduction

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DYSADHERIN IS A novel cancer-associated cell membrane glycoprotein (1). Its cDNA encodes 178 amino acids, which include a putative signal sequence, a potential O-glycosylated extracellular domain, a single-transmembrane domain, and a short cytoplasmic tail. Dysadherin immunoreactivity to the monoclonal antibody NCC-3G10 has been detected in a wide variety of carcinoma cells but in only a limited number of normal cells, including lymphocytes, endothelial cells, and the basal cells of stratified squamous epithelium. Transfection of dysadherin cDNA into the liver carcinoma cell line PLC/PRF/5 reduces cell-cell adhesiveness as assessed by morphology and by a Ca²⁺-dependent cell aggregation assay (1).

Cadherins are members of a large family of transmembrane glycoproteins that mediate Ca²⁺-dependent homophilic cell-cell adhesion and play an important role in the maintenance of normal tissue architecture (2). E-cadherin is the prime mediator of cell-cell adhesion in epithelial cells. In PLC/PRF/5 cells transfected with dysadherin cDNA, levels of E-cadherin protein were markedly decreased in inverse proportion to the expression level of dysadherin, and the expression of E-cadherin mRNA remained unaffected. Moreover, when metastatic potential was examined by injecting dysadherin-transfected PLC/PRF/5 cells into the spleens of mice with severe combined immunodeficiency, the transfectants formed many more metastatic nodules than controls. Thus, dysadherin appears to be involved in the down- regulation of E-cadherin by a posttranscriptional mechanism and play an important role in tumor development and metastasis (1).

Thyroid carcinomas are divided into a wide range of morphologic subgroups according to histological criteria (3). Among those arising from the follicular cells of the thyroid, the main categories include papillary carcinoma (PC), follicular carcinoma (FC) and undifferentiated carcinoma (UC). PC and FC, which are differentiated carcinomas, are among the most curable of thyroid carcinomas (4, 5), whereas UC is among the most aggressive. Many UCs have been found to coexist with a differentiated component (either PC or FC), leading to the assumption that UCs commonly arise from differentiated carcinomas (6, 7, 8).

At present, nothing is known about dysadherin expression in thyroid carcinomas. Therefore, in the present study, we examined the expression of dysadherin and its association with the expression of E-cadherin in various types of thyroid carcinoma.

	Materials and Methods

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Patients and histological classification

Thyroid carcinoma specimens were obtained from 92 patients (80 women and 12 men, with a mean age of 55 yr) undergoing clinically indicated surgery at Ito Hospital, Tokyo, Japan. The study was conducted in accordance with the Declaration of Helsinki. Informed consent was obtained from all patients, and the study protocol was approved by the equivalent of an internal review board.

We defined carcinomas diagnosed for the first time as primary carcinomas, and those occurring more than 6 months after initial surgery as secondary carcinomas. Regional recurrence was defined as new evidence of regional disease occurring more than 6 months after initially successful therapy.

The thyroid carcinoma specimens were classified as 51 PCs, 10 FCs, and 31 UCs, according to the histological typing criteria of the World Health Organization (3). Primary carcinomas were further categorized according to the tumor-node-metastasis (TNM) classification of malignant tumors of the International Union Against Cancer (9). Forty-two of the 51 PCs and all 10 FCs were primary carcinomas. Nine of the PCs were secondary carcinomas. Of the 31 UCs, 23 were primaries and 8 were secondaries. Of the 51 patients with PC, 21 developed UC as a secondary carcinoma during follow-up. The remaining 30 experienced neither relapse (regional recurrence or distant metastasis) nor development of UC during their clinical courses. Thus, PCs were classified into two groups with regard to the occurrence of UC: PC with UC and PC without UC. All 10 FCs observed during this study were of the minimally invasive type (3) and have shown no relapse throughout follow-up.

All 31 patients with UC and all 21 of those with PC with UC died from their thyroid carcinoma. The other 30 patients with PC and the 10 with FC remain alive without relapse.

Follow-up of the patients

After diagnosis, all patients underwent routine scintigraphic evaluation using radioactive iodine and computed tomography to detect distant metastases. Biochemical evaluations (including thyroglobulin determination) were repeated, accompanied by sonographic follow-up of any regional recurrences. Except at the time of scintigraphy, thyroid-stimulating hormone was suppressed by thyroxine treatment.

Follow-up duration was calculated up to the time of the last evaluation or the time of death. The mean follow-up duration for all patients was 60 months (range 1–235 months). The mean follow-up duration for survivors (the 30 patients with PC and the 10 with FC) was 60 months (range 48–141 months). The mean survival durations of patients with UC and PC with UC were 8 months (range 1–42 months) and 69 months (range 2–235 months), respectively.

Monoclonal antibodies

An undiluted culture supernatant preparation of cloned NCC-3G10, which recognizes dysadherin, was used as the primary antibody for dysadherin detection (1). An undiluted culture supernatant preparation of a mouse antihuman E-cadherin monoclonal antibody, HECD-1, which recognizes the extracellular domain of E-cadherin, was used as the primary antibody for E-cadherin detection. Both NCC-3G10 and HECD-1 belong to the Ig G1 subclass.

Immunohistochemistry and evaluation

For each specimen, 10% formalin-fixed, paraffin-embedded blocks containing both the carcinoma and its adjacent nonneoplastic thyroid tissue were prepared. This enabled an internal staining control to be included for each individual. Consecutive sections (3 µm thick) from each block were mounted on adhesive-coated slides, deparaffinized, and hydrated with xylene and ethanol. Endogenous peroxidase was blocked by soaking in 0.3% hydrogen peroxide in methanol for 30 min. The slides were placed into plastic jars containing 10 mM citrate buffer, which were then heated in a H2800 microwave processor (Energy Beam Inc., Agawam, MA) for 20 min to unmask the antigen. After cooling, the sections were preincubated in PBS containing 2% (vol/vol) normal swine serum (Dako, Glostrup, Denmark) for 10 min. The slides were then incubated at 4 C overnight with the primary antibody, washed with PBS and incubated for 30 min with biotinylated antimouse IgG (Vector Laboratories, Burlingame, CA) as a secondary antibody. The sections were then incubated for 30 min with avidin-biotinyl-peroxidase complex using a Vectastain ABC kit (Vector Laboratories). The peroxidase reaction was performed using 0.02% (wt/vol) 3,3'-diaminobenzidine tetrahydrochloride and 0.007% (vol/vol) hydrogen peroxide in Tris-HCl buffer (pH 7.6) as a chromogen, and was allowed to proceed for 5–10 min. The cell nuclei were then counterstained with hematoxylin. The slides were washed three times with PBS between each step.

Negative controls were performed by incubating the slides with normal mouse Ig G1 (Dako) instead of the primary antibody and yielded negative results in all cases. Focal aggregates of lymphocytes were occasionally observed in the thyroid in patients with focal lymphocytic thyroiditis and were classed as nonspecific lymphocytic thyroiditis (10). The intensity of dysadherin immunostaining in the tumor cells was evaluated by comparison with lymphocytes or endothelial cells on the same slide as an internal positive control, as previously described (1). The intensity of E-cadherin immunostaining in the tumor cells was evaluated by comparison with normal epithelial cells present on the same slide as an internal positive control.

Dysadherin and E-cadherin expression were semiquantified as follows: absent (-), 0% expression; low (1+), 1–20% of cells positive; intermediate (2+), 21–50% of cells positive; and high (3+), more than 50% of cells positive. The intensity of the staining in the tumor tissues was graded into three categories: weaker than, equal to, or stronger than that in the adjacent nonneoplastic thyroid tissue. However, detailed statistical evaluation by logistic regression analysis with or without various combinations of the percentages of positive cells revealed this grading to be irrelevant. Therefore, staining intensity is not considered further in this study.

Statistical analysis

The SPSS program package (SPSS, Inc., Chicago, IL) was used for all statistical analyses. The following nonparametric tests were performed using distribution-free methods: the Kruskal-Wallis test, the Mann-Whitney U test, Wilcoxon’s rank sum test, and calculation of Kendall’s rank correlation coefficient (11). P < 0.05 were considered statistically significant.

	Results

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Analyses of the associations between dysadherin and E-cadherin expression and clinicopathological data are summarized in Table 1, A and B.

Immunoreactivity for dysadherin was completely absent from normal thyroid follicular epithelial cells but was observed on the cell surfaces of some lymphocytes (Fig. 1A).

View larger version (130K): [in this window] [in a new window]   FIG. 1. A, Immunoreactivity for dysadherin was completely absent from normal thyroid follicular epithelial cells. However, the lymphocytes included as positive controls showed dysadherin immunoreactivity on their surfaces (x100). B, Immunoreactivity for E-cadherin was observed in the membranes of normal thyroid follicular epithelial cells. This was less intense in the flat cells lining large follicles than in the cuboidal or columnar cells lining medium-sized or small follicles. The lateral borders of the epithelial cells exhibited more intense staining than their basal borders; immunoreactivity was faint or absent in the apical borders facing the follicular lumen (x100).

PC

Immunoreactivity for dysadherin was observed at the cell-cell boundaries in PCs (Fig. 2A). The expression of dysadherin showed high variability in the PC specimens, with no expression in 12, low expression in 28, intermediate expression in seven, and high expression in four.

View larger version (160K): [in this window] [in a new window]   FIG. 2. Immunoreactivity for both dysadherin (A) and E-cadherin (B) was observed at the cell-cell boundaries in papillary carcinomas (x100).

FC

None of the 10 FC specimens showed any immunoreactivity for dysadherin (Fig. 3A).

View larger version (157K): [in this window] [in a new window]   FIG. 3. A, None of the 10 follicular carcinoma specimens showed any immunoreactivity for dysadherin, whereas endothelial cells included as a positive control showed dysadherin immunoreactivity on their surfaces (x100). B, Expression of E-cadherin was observed at the cell-cell boundaries in follicular carcinoma (x100).

UC

UCs are composed of varying proportions of spindle, polygonal, and giant cells. Immunoreactivity for dysadherin was observed in all specimens, with intermediate expression in 12 and high expression in 19 (Fig. 4A). Dysadherin expression was observed on the surfaces of UC tumor cells.

View larger version (151K): [in this window] [in a new window]   FIG. 4. A, Expression of dysadherin was apparent on the surfaces of the tumor cells in undifferentiated carcinoma (x100). B, A small number of polygonal and giant tumor cells showed immunoreactivity for E-cadherin on their membranes in undifferentiated carcinoma (x100).

Correlation between dysadherin expression and E-cadherin expression

To evaluate the correlation between dysadherin expression and E-cadherin expression in each of 92 specimens of thyroid carcinoma, the results of immunohistochemistry were categorized into 16 classes. The classified data are summarized in Table 2. Kendall’s value was calculated to be -0.523 (P < 0.001, by Kendall’s rank correlation coefficient), indicating a significant negative correlation between dysadherin expression and E-cadherin expression.

Histopathology. In both PCs and FCs, E-cadherin was expressed to a significantly higher extent than dysadherin (P < 0.001 and P = 0.003, respectively, by Wilcoxon’s rank sum test). On the other hand, dysadherin was expressed to a significantly higher extent than E-cadherin in UCs (P < 0.001 by Wilcoxon’s rank sum test).

When all three histopathological types were considered, dysadherin expression was highest in UCs, next highest in PCs, and lowest in FCs (P < 0.001 by the Kruskal-Wallis test). Conversely, E-cadherin expression was highest in FCs, next highest in PCs, and lowest in UCs (P < 0.001 by the Kruskal-Wallis test).

UC as a secondary carcinoma

Dysadherin expression was significantly higher in PC with UC than in PC without UC (P = 0.001 by the Mann-Whitney U test). However, E-cadherin expression did not differ significantly between PC with UC and PC without UC (P = 0.664 by the Mann-Whitney U test).

Prognosis

Dysadherin expression was significantly higher in patients who died from thyroid carcinoma than in those who remained alive (P < 0.001 by the Mann-Whitney U test). In contrast, E-cadherin expression was significantly higher in patients who remained alive than in those who died (P < 0.001 by the Mann-Whitney U test).

Gender

Neither dysadherin expression nor E-cadherin expression were significantly related to gender (P = 0.371 and P = 0.136 by the Mann-Whitney U test, respectively).

Dysadherin expression and primary tumor status

Associations between the TNM categories of the tumors and dysadherin expression are summarized in Table 3. According to the TNM classification of malignant tumors, nine specimens were classified as pT1, 20 as pT2, 22 as pT3, and 24 as pT4. As shown in Table 3, the degree of dysadherin expression was highest in pT4 specimens, second highest in pT3 specimens, third highest in pT2 specimens, and lowest in pT1 specimens (P = 0.001 by the Kruskal-Wallis test). Thus, dysadherin expression was significantly associated with tumor size.

Sixty-nine of the tumors were graded as M0 and six as M1. Among the M0 specimens, dysadherin expression was absent from 22, low in 24, intermediate in 11, and high in 12, and among the M1 specimens it was intermediate in four and high in two. None of the M1 specimens showed absent or low expression. Thus, dysadherin expression was significantly higher in M1 than M0 tumors (P = 0.011 by the Mann-Whitney U test).

The TNM classification of malignant tumors divides the stage grouping of PC and FC into two age groups: younger than 45 yr and 45 yr and older. In patients aged younger than 45 yr, dysadherin expression was absent from four tumors, low in nine and intermediate in three. None of the tumors in this age group showed high expression. In patients 45 yr and older, dysadherin expression was absent from eight tumors, low in 15, intermediate in two, and high in one. Dysadherin expression did not differ significantly between these groups (P = 0.57 by the Mann-Whitney U test).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In this study, we showed that PCs and UCs express dysadherin at their cell-cell boundaries, whereas FCs and normal thyroid do not contain dysadherin. In addition, significant negative correlation was observed between dysadherin expression and E-cadherin expression. These findings are compatible with the hypothesis that dysadherin is a transmembrane protein that may down-regulate E-cadherin expression (1).

In UCs, dysadherin expression was significantly higher than E-cadherin expression, whereas in PCs and FCs, E-cadherin expression was significantly higher than dysadherin expression. When UCs, PCs, and FCs were compared, dysadherin expression was significantly higher in UCs. In contrast, E-cadherin expression was significantly lower in UCs than in PCs and FCs. When PC was classified with respect to the occurrence of UC as a secondary carcinoma, dysadherin expression was significantly higher in PC with UC than in PC without UC. Thus, it is possible that increased dysadherin expression is associated with UC and PC with UC.

The prognosis of FC varies tremendously, depending on the type of tumor, and FC generally has a worse prognosis than PC (12, 13). However, in the present study, the FCs exhibited lower expression of dysadherin than the PCs. This may explain why all 10 of the FCs encountered in this study were of the minimally invasive type.

We further showed that dysadherin expression correlates significantly with tumor size, regional lymph node metastasis, and distant metastasis of the primary carcinoma. A significant association between dysadherin expression and death from thyroid carcinoma was also observed. These findings indicate that dysadherin expression is associated with an adverse clinical outcome in thyroid carcinoma.

Several possible mechanisms for the irreversible and reversible inactivation of E-cadherin in human tumors have been reported (14). It appears that transcriptional or posttranscriptional mechanisms, rather than structural abnormalities of the E-cadherin gene, are responsible for the down-regulation of E-cadherin expression in thyroid carcinomas (15, 16). Thus, it is likely that increased dysadherin expression is one of the mechanisms underlying the posttranscriptional down-regulation of E-cadherin expression in thyroid carcinomas.

In conclusion, dysadherin expression was observed in PCs and UCs of the thyroid. Our results suggest that a process involving increased dysadherin expression may lead to an adverse clinical outcome in thyroid carcinoma.

	Acknowledgments

 
We thank Mr. Toshimichi Fujisawa of Ito Hospital for technical assistance.

	Footnotes

 
This work was supported by a Grant-in-Aid for the Second Term Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health, Labor, and Welfare of Japan and 2002 Tokai University School of Medicine Research Aid.

Abbreviations: FC, Follicular carcinoma; PC, papillary carcinoma; TNM, tumor-node-metastasis; UC, undifferentiated carcinoma.

Received November 8, 2002.

Accepted May 20, 2003.

	References

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