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Department of Pathology (C.S., C.C., M.A.J.) and Endocrinology Unit (M.A.M.-B., A.S., E.N.), Hospital Universitario Virgen del Rocío, Seville, 41013 Spain; and Department of Microbiology, Faculty of Biology, University of Seville (M.T.), and Instituto de Recursos Naturales, Consejo Superior de Investigaciones Cientificas (J.A.P.-T.), Seville 41080, Spain
Address all correspondence and requests for reprints to: Dr. Miguel A. Japón, Department of Pathology, Hospital Universitario Virgen del Rocío, Avenida Manuel Siurot s/n, Seville 41013, Spain. E-mail: mjapon{at}cica.es.
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Abstract |
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 Top Abstract IntroductionPatients and MethodsResultsDiscussionReferences |
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Objective: The objective of this study was to examine the immunohistochemical expression of hPTTG in DTC and its association with known prognostic factors.
Design: hPTTG expression was analyzed by immunostaining on paraffin-embedded tissues. Clinical data were used to determine any associations between the expression of hPTTG and prognostic variables of DTC. A median follow-up of 43 months allowed us to analyze the persistence of disease and the response to radioiodine therapy.
Setting: The study was conducted at a tertiary university hospital.
Patients: Ninety-five patients undergoing surgical resection for DTC (n = 60) or benign nodular thyroid disease (n = 35) were studied.
Main Outcome Measure: The main outcome measure was the association between hPTTG expression and prognostic factors in DTC.
Results: Among DTC cases, 21 (35%) had low and 39 (65%) had high hPTTG immunostaining. Adjacent nonneoplastic thyroid tissue was largely unstained. Among benign nodular thyroid disease cases, immunostaining was detected focally in eight (22.8%). A significant association was found between hPTTG expression and the presence of nodal (P < 0.01) or distant metastases (P < 0.05). A significant association with TNM was also found, because 83.3% of advanced TNM stages showed elevated hPTTG (P < 0.05). The association between hPTTG overexpression and decreased radioiodine uptake during follow-up was also significant (P < 0.05). The expression levels of hPTTG were confirmed as an independent prognostic factor for persistent disease (relative risk, 3.0; 95% confidence interval, 1.1–8.7; P < 0.05).
Conclusions: Immunohistochemical analysis of hPTTG is of potential value in the determination of tumor aggressiveness in DTC.
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Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Human pituitary tumor-transforming gene (hpttg) product is a 22-kDa protein that is abundantly expressed in many tumor cell lines and is tumorigenic in vivo (7, 8). The expression of hPTTG is regulated through the cell cycle, peaking at mitosis, and is phosphorylated by cell division cycle 2 (9). hPTTG has been identified as a vertebrate securin on the basis of its analogy to yeast securins Pds1 and Cut2 (10), and therefore, primary hPTTG function is related to the control of sister chromatid separation to opposite spindle poles. Based on this functional activity, genomic imbalance as a result of chromosome missegregation is a rationale for the oncogenic potential of dysregulated hpttg expression. hpttg is expressed at high levels in human pituitary adenomas (11, 12), but also in other epithelial malignant tumors, including breast, lung, ovary, and colorectal tumors (12, 13, 14), as well as in hemopoietic neoplasias (7, 15). Most importantly, hpttg expression has been correlated with lymph node invasion in colorectal cancer; therefore, it might be a prognostic molecular marker (14). Recently, increased hpttg expression has been demonstrated in thyroid carcinomas (16, 17), and an association between hpttg mRNA and early tumor recurrence has been found in a series of patients (17). In the present work we aimed at analyzing the expression of hPTTG in a large series of thyroid tumors comprising DTCs, follicular adenomas, and multinodular goiters, using a specific rabbit polyclonal anti-hPTTG antibody and immunohistochemical methods. We also aimed at studying the association of tissue hPTTG overexpression with established prognostic factors and its usefulness as a prognostic marker of persistent disease in DTC.
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Patients and Methods |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Thyroid tissue samples were retrieved from archival material of a series of 95 patients undergoing surgical resection for DTC (n = 60) or benign nodular thyroid disease (BNTD; n = 35). All procedures were conducted in accordance with the guidelines of the local ethical committees. DTC included 47 papillary carcinomas (PTC; 33 of conventional histology, eight of the tall cell variant, three of the follicular variant of PTC, and three diffuse sclerosing PTC) and 13 follicular carcinomas (FTC; 10 of conventional histology, two of the insular variant, and one oxyphilic FTC). BNTD cases included 22 multinodular goiters and 13 follicular adenomas. All specimens were formalin fixed and paraffin embedded according to routine protocols. Patients’ demographics, thyroid function, and the presence of autoantibodies were recorded. TNM staging of the tumor at presentation (18) was recorded in all cases of DTC inasmuch as precise information about tumor size, tumor extent beyond the thyroid capsule, lymph node status, and distant metastasis was available from medical records (Table 1
). Follow-up protocols were conducted according to international standards (19). For patients who had undergone total thyroidectomy and radioiodine remnant ablation, a TSH-stimulated 131I diagnostic whole body scan (DWBS) was performed 12 months after ablation. Thyroglobulin (Tg) was measured every 6 months after thyroid hormone suppression of TSH and in every DWBS, after T4 withdrawal. Tg and anti-Tg autoantibodies (TgAb) assays were performed simultaneously. Serum Tg was measured by immunoradiometric assay (DiaSorin, Saluggia, Italy) with a functional sensitivity of 0.3 ng/ml. TgAb levels were measured by immunoradiometric method (DiaSorin); according to our laboratory reference range, TgAb values above 100 IU/ml were considered positive. An elevated Tg level was defined as Tg higher than 3 ng/ml, and hypothyroid was considered a TSH level of 30 µIU/ml or more after T4 withdrawal. After two negative DWBS, additional DWBS were indicated whenever serum Tg levels increased or tumor recurrence was suggested by clinical findings. Because the presence of TgAb can falsely lower immunometric Tg results, TgAb-positive patients were evaluated by DWBS even with undetectable Tg levels on T4 therapy or whether rising Tg was detected. Computerized tomography (CT), [99mTc]methoxyisobutyl-isonitrile scan (MIBI), and/or [18F]deoxyglucose positron emission tomography (FDG-PET) examination were performed when Tg was elevated after T4 withdrawal, but a negative TSH-stimulated DWBS scan was found. In AbTg-positive patients, evidence of increasing levels of serial Tg, but negative TSH-stimulated DWBS, was also an indication for CT, MIBI, and/or FDG-PET examination. Remission was defined as low Tg levels (undetectable on T4 therapy or 
3 ng/ml after T4 withdrawal) and negative DWBS. In AbTg-positive patients, the absence of imaging for disease by CT, MIBI, and/or FDG-PET in the cases in which they were indicated was necessary for remission. Criteria for persistent disease were Tg levels greater than 3 ng/ml after T4 withdrawal and/or imaging for disease (DWBS, CT, MIBI, or FDG-PET examination) after total or near-total thyroidectomy and radioiodine ablation of postoperative thyroid remnants. Recurrence was defined as evidence of rising Tg and/or imaging for disease after a period of remission. In patients with detectable Tg levels during T4 therapy and who did not meet the definition of persistent or recurrent disease, a follow-up neck ultrasonography, Tg measurement after T4 withdrawal and DWBS were performed to correctly classify their status as remission, persistent disease, or recurrent disease. For persistence and recurrence analysis, only cases with data from DWBS after ablation of postoperative thyroid remnants were evaluated (n = 48). The median follow-up was 43 months (range, 12–120 months) after radioiodine ablation.
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Tissue paraffin tissue sections (5 µm thick) were dewaxed in xylene and rehydrated in a series of graded alcohols. Sections were immersed in 3% H2O2 aqueous solution for 30 min to quench endogenous peroxidase activity, then covered with 10% normal serum in Tris-buffered saline to block nonspecific binding sites. A rabbit polyclonal anti-hPTTG antibody was available for this study. Specificity control experiments for this antibody have been described previously (7, 9). Sections were incubated overnight with a 1:500 dilution of primary anti-hPTTG antibody. After several washes in Tris buffer, secondary biotinylated antirabbit IgG and streptavidin-biotin-peroxidase complex were applied according to the manufacturer’s protocols (DakoCytomation, Glostrup, Denmark). Visualization of the immunoreaction was completed using 3,3'-diaminobenzidine as the peroxidase chromogenic substrate. The slides were then counterstained with hematoxylin and mounted in DPX (BDH Laboratories, Poole, UK). Sections in which primary antibody was omitted were used as negative controls. Immunostaining was evaluated by two independent pathologists on at least 10 microscopic fields (x200), and unequivocal moderately to intensely stained cells were counted. Immunohistochemical expression of hPTTG was scored as high (>25% of the carcinoma cells stained), low (10–25%), or absent (<10%). In selected cases with available frozen material, the expression of hPTTG was confirmed by immunoblot.
Statistical methods
The association of immunohistochemical expression of hPTTG with other tumor variables was assessed by 
2 test or Fisher’s exact test, when indicated. Student’s t test was used for comparison of tumor-related quantitative variables between two categories of hPTTG immunostaining. Tumor variables included patient age, gender, tumor size, histological type and variants, lymph node status, presence of distant metastases, and TNM stage. Among histological variants, the following were regarded as predictive of high risk (4): tall cell and diffuse sclerosing variants of PTC, and insular and oxyphilic variants of FTC. Persistent disease curves were estimated by the actuarial method of Kaplan and Meier, and the differences were analyzed by the log-rank test of Mantel and Cox. The Cox proportional hazards model was used to estimate the effect of hPTTG expression on persistence of disease, simultaneously with several prognostic factors. All tests were two-sided, and P < 0.05 was considered significant. Calculations were made with the aid of SPSS 12.0 statistical package (SPSS, Inc., Chicago, IL).
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Results |
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All cases were examined by immunohistochemistry using archival paraffin-embedded tissues. Of the 60 DTC evaluated, 21 (35%) were low or absent, and 39 (65%) were intensely immunostained with anti-hPTTG polyclonal antibody (Fig. 1, A–C). The staining was mainly cytoplasmic, but nuclei were also occasionally stained. hPTTG expression in the intensely stained category was not associated with DTC type (PTC or FTC). Among histological subtypes of high risk (n = 14), hPTTG immunohistochemical expression was high in 90.5% (Fig. 1, D and E); among nonhigh-risk histological subtypes (n = 46), hPTTG staining was unequivocal in 58.7%. Nonneoplastic thyroid tissue was largely unstained, with the exception of focally stained follicular cells. Among BNTD cases, the expression of hPTTG was low or absent in 27 (77.1%) and high in eight (22.8%; seven of 22 multinodular goiters and one of 13 follicular adenomas). Eleven multinodular goiters and three follicular adenomas showed thyroiditis foci. Among unequivocally stained BNTD cases, the expression of hPTTG was focal and associated with thyroiditis foci (Fig. 1F).
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The association between immunohistochemical staining for hPTTG in thyroid carcinoma tissue and predictive factors of high risk is described in Table 2. hPTTG was significantly associated with regional lymph node (P < 0.01) and distant metastasis (P < 0.05). However, neither tumor size nor aggressive histological variants were significantly associated with immunohistochemical overexpression. TNM stage was also associated with hPTTG overexpression (P < 0.05), because 83.3% of tumors of advanced TNM stages, i.e. TNM III and IV, showed unequivocal immunostaining for hPTTG. Neither age nor gender was significantly associated with hPTTG overexpression. The association between hPTTG overexpression and positive TgAb at diagnosis was also assessed; no significant association was found.
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Seventeen patients with rising Tg levels after T4 withdrawal, but negative TSH-stimulated DWBS, were found in the persistent disease group. In nine of these patients, distant metastases were confirmed by CT, MIBI, or FDG-PET. Immunohistochemical analysis showed unequivocal staining for hPTTG in 16 tumors, and a significant association between immunohistochemical staining for hPTTG in primary thyroid carcinoma tissue and persistent disease with negative TSH-stimulated DWBS was found (P < 0.05).
Association of hPTTG expression with persistent disease in DTC
After a mean follow-up of 51.0 months after radioiodine ablation, persistence of the disease was demonstrated in two of 13 patients with low hPTTG tumors (15.4%) and in 23 of 35 patients with high hPTTG tumors (65.7%). The mean time for achieving no evidence of disease after radioiodine ablation was 22.0 months [95% confidence interval (CI), 10.7–33.4] in patients with low hPTTG tumors and 81.4 months (95% CI, 63.9–98.8) in patients with intense hPTTG tumors. Kaplan-Meier curves demonstrated a significantly longer time of persistent disease after radioiodine ablation for patients who had primary tumor with unequivocal expression of hPTTG (P < 0.01) (Fig. 2). Three patients (two PTC and one FTC) showed criteria for recurrent disease (28, 55, and 78 months after radioiodine ablation, respectively); immunohistochemical analysis at diagnosis showed unequivocal staining for hPTTG in two of them and negative staining in the last one. We also investigated whether hPTTG expression maintained its prognostic value for persistent disease in the presence of all of the above-mentioned variables by means of a multivariate regression analysis (Table 3). In the series, the expression level of hPTTG was confirmed as an independent prognostic factor for persistent disease. For high hPTTG carcinomas, the adjusted relative risk was 3.0 (95% CI, 1.1–8.7; P < 0.05) in the multivariate analysis.
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Discussion |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Heaney et al. (16) first reported increased hpttg mRNA expression in a subset of FTC, but not in four of eight PTC, examined by Northern blot analysis, suggesting that hpttg may play a role in the molecular events implicated in the divergence of FTC vs. PTC phenotypes. Contrarily, we found no difference in the pattern of hPTTG expression by immunostaining between FTC and PTC. Our results are in agreement with those of Boelaert et al. (17), who described pretranslational hpttg overexpression in both FTC and PTC compared with normal and hyperplastic thyroid tissue by a quantitative RT-PCR approach. Even though no significant association between the expression of hpttg mRNA and TNM status was evident, an association between hpttg mRNA and early tumor recurrence was suggested (17). In the present study the expression level of hPTTG was confirmed as an independent prognostic factor for persistent disease. The association between hPTTG overexpression and other clinically relevant outcomes, such as cancer-related death and recurrence, could not be evaluated in our study. Patients with DTC have 10-yr cancer-specific mortality rates less than 10% (2). Because of its high survival rates and prolonged clinical course, cancer-related death is an outcome only available in epidemiological studies including long-term follow-up of a large series of DTC. It has been suggested that most cases of recurrence are actually cases of persistent disease that have progressed to the point of detection (20). Current sensitive Tg assays allow for the early detection and treatment of cases of persistent disease, thus diminishing the risk of progression. It is possible that the criteria for persistent disease defined in our protocol contributed to the small rate of recurrent disease, which did not allow us to consider this a main outcome. Thus, according to our results, hPTTG expression represents a molecular marker of worse prognosis in DTC. The immunohistochemical method allows easy identification of cells overexpressing hPTTG. Tumors that expressed high levels of hPTTG showed an association with advanced TNM stages. Increased hpttg expression has been associated with vascular invasion and lymph node metastasis (14). Also, hpttg has been included in a set of genes whose expression can predict a metastastic phenotype in multiple tumor types (21). hPTTG induces the expression of vascular endothelial growth factor (22), which is considered the most important angiogenic factor involved in tumor neovascularization.
The physiopathological mechanisms leading to persistence, recurrence, and progression of thyroid cancer are largely unknown. It is thought that one mechanism relates in part to decreased sodium-iodide symporter (NIS) expression (23). NIS is an integral membrane glycoprotein that mediates active iodine transport into thyroid cells as the first step in thyroid hormone biosynthesis (24). The ability of thyroid cells to accumulate iodine has been used as an effective means for diagnostic and therapeutic doses of radioiodine to localize and destroy DTC and their metastases. Thus, prediction of a lack of responsiveness to radioiodine therapy would be extremely useful in the follow-up of these patients, an event that occurs in about one third of patients with DTC (25). hPTTG overexpression in rat FRTL5 thyroid cells and primary human thyroid cell cultures causes in vitro transformation, decreased iodine uptake, and reduced NIS expression (16). In our series, 17 of 25 patients with persistent disease after radioiodine ablation showed elevated serum Tg levels and undetectable iodine uptake; nearly all these cases showed overexpression of hPTTG in primary DTC tumors. Interestingly, aggressive clinical behavior in tumors with loss of capacity to respond to radioiodine therapy could not be predicted according to histological or TNM staging criteria. According to Heaney et al. (16), we hypothesize that hPTTG overexpression might play a role in the loss of capacity to accumulate iodine and respond to radioiodine therapy in noniodine-transporting DTC. Even though more studies are needed, analysis of immunohistochemical expression could be useful in the prediction of loss of capacity to respond to radioiodine therapy in DTC.
In DTC, numerous staging classification systems have been proposed to predict outcomes and to select individualized therapy. Recently, two consensus statements have been proposed in the follow-up of low-risk patients with DTC (26, 27). Specific molecular markers of tumor prognosis would be extremely useful in the classification of high- and low-risk DTC and could help in designing follow-up protocols. Immunohistochemical expression of hPTTG might be a powerful tool in the determination of tumor aggressiveness in DTC.
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Acknowledgments |
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Footnotes |
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The authors declare no potential conflicts of interest.
First Published Online January 17, 2006
1 C.S. and M.A.M.-B. contributed equally to this article. 
Abbreviations: BNTD, Benign nodular thyroid disease; CI, confidence interval; CT, computerized tomography; DTC, differentiated thyroid cancer; DWBS, diagnostic whole body scan; FDG-PET, [18F]deoxyglucose positron emission tomography; FTC, follicular thyroid carcinoma; MIBI, [99mTc]methoxyisobutyl-isonitrile scan; NIS, sodium-iodide symporter; PTC, papillary thyroid carcinoma; pttg, pituitary tumor-transforming gene; Tg, thyroglobulin; TgAb, Tg antibody.
Received November 21, 2005.
Accepted January 5, 2006.
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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