Journal List > J Korean Med Sci > v.26(9) > 1021451

J Korean Med Sci. 2011 Sep;26(9):1191-1195. English.
Published online September 01, 2011.  https://doi.org/10.3346/jkms.2011.26.9.1191
© 2011 The Korean Academy of Medical Sciences.
Expression of X-linked Inhibitor of Apoptosis Protein in Neoplastic Thyroid Disorder
Ji Hye Yim,1 Jong Ho Yoon,2 Sun A Kim,3 Won Gu Kim,1 Min Ji Jeon,1 Ji Min Han,1 Tae Yon Sung,2 Tae Yong Kim,1 Won Bae Kim,1 Suck Joon Hong,2 Young Kee Shong,1 and Gyungyub Gong3
1Department of Internal Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea.
2Department of Surgery, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea.
3Department of Pathology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea.

Address for Correspondence: Jong Ho Yoon, MD. Department of Surgery, Asan Medical Center, University of Ulsan College of Medicine, 88 Olymphic-ro 43-gil, Songpa-gu, Seoul 138-736, Korea. Tel: +82.2-3010-3931, Fax: +82.2-3010-6962, Email: jjong126@paran.com
Received March 16, 2011; Accepted July 28, 2011.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.


Abstract

X-linked inhibitor of apoptosis protein (XIAP) is associated with tumor genesis, growth, progression and metastasis, and acts by blocking caspase-mediated apoptosis. In the present study, we sought to evaluate the expression patterns of XIAP in various neoplastic thyroid disorders and determine the association between XIAP expression and clinicopathologic factors. Expression of XIAP was evaluated with immunohistochemical staining using monoclonal anti-XIAP in 164 specimens of conventional papillary thyroid carcinoma (PTC) and 53 specimens of other malignant or benign thyroid tumors. XIAP positivity was observed in 128 (78%) of the 164 conventional PTC specimens. Positive rates of XIAP expression in follicular variant PTC, follicular, medullary, poorly differentiated, and anaplastic thyroid carcinoma specimens were 20%, 25%, 38%, 67%, and 38%, respectively. Six nodular hyperplasia specimens were negative and 1 of 7 follicular adenomas (8%) was positive for XIAP. Lateral neck lymph node metastases were more frequent in patients negative for XIAP expression (P = 0.01). Immunohistochemical staining for XIAP as a novel molecular marker may thus be helpful in the differential diagnosis of thyroid cancer. Moreover, high XIAP expression in conventional PTC is strongly associated with reduced risk of lateral neck lymph node metastasis.

Keywords: X-linked Inhibitor of Apoptosis Protein; Papillary Thyroid Carcinoma; Thyroid Neoplasms; Lymph Nodes; Metastasis

INTRODUCTION

X-linked inhibitor of apoptosis protein (XIAP) is a member of the Inhibitor of Apoptosis (IAP) protein family that selectively binds and inhibits caspases-3, -7, and -9. XIAP blocks the downstream portion of the apoptosis pathway and inhibits cell death in response to multiple stimuli (1-3). All cells have a finite lifespan, and cell death occurs mainly as a result of passive necrotic processes and/or active apoptosis (programmed cell death) (4, 5). Apoptosis is the most common mechanism by which the body eliminates damaged or unnecessary cells without local inflammation from leakage of cell contents (6, 7). This process relies on a tightly balanced signaling pathway involving both pro- and anti-apoptotic proteins. Defective apoptosis represents a major causative factor in the development and progression of cancer (8). The IAP protein family plays critical roles in maintaining tight apoptogenic regularities.

XIAP expression is increased in a number of human malignancies, including colorectal, esophageal, ovarian, renal cell, hepatocellular, and prostate cancer (2). Several reports have confirmed higher XIAP expression in thyroid cancer, compared to benign thyroid disorder (9). Moreover, a recent study showed the utility of XIAP expression as a marker to predict the aggressiveness of papillary thyroid carcinoma (PTC) (10).

In the present study, we evaluated the expression patterns of XIAP in various neoplastic thyroid disorders. We additionally aimed to delineate possible associations between XIAP expression and clinicopathologic parameters in conventional PTCs, the most common type of thyroid cancer.

MATERIALS AND METHODS

Thyroid samples

We analyzed 217 formalin-fixed, paraffin-embedded thyroid tissue specimens obtained with thyroidectomy at the Asan Medical Center between 1996 and 2003. The study specimens comprised 164 conventional PTCs and 40 other malignant thyroid tumors, including 10 follicular variant PTCs, 8 follicular thyroid carcinomas (FTC), 8 medullary thyroid carcinomas, 8 poorly differentiated thyroid carcinomas and 8 anaplastic thyroid carcinomas. The remaining 13 specimens were benign thyroid tumors, including 6 nodular hyperplasia and 7 follicular thyroid adenomas.

All thyroid specimens were stained using hematoxylin and eosin, and reviewed by two pathologists. Diagnosis was confirmed according to the World Health Organization histologic classification of thyroid tumors.

Immunohistochemical analysis of XIAP

Sections of formalin-fixed, paraffin-embedded tumor tissue specimens were deparaffinized, treated with 3% hydrogen peroxide to block endogenous peroxidase activity, and microwaved in 0.01 M/L citric acid (pH 6.0) for 6 min at 100℃, followed by slow cooling for antigen retrieval. Slides were incubated for 72 hr at 4℃ with a monoclonal anti-XIAP antibody (BD Biosciences, Franklin Lakes, NJ, USA), stained using Envision-Plus reagents (DAKO, Carpinteria, CA, USA) and diaminobenzidine as chromogen, and counterstained with hematoxylin.

The extent of staining was graded semiquantitatively as follows: no foci (0), rare-few foci (1+), multiple foci (2+), and regional-diffuse staining (3+). Staining intensity was graded semiquantitatively as follows: negative (0), weak (1+), moderate (2+), and strong (3+). Specimens were classified as XIAP-positive when the sum of the extent score plus intensity score was equal to more than 4 points (Fig. 1).


Fig. 1
XIAP expression in PTC specimens. (A) Weak-rare foci positive XIAP staining; (B) Moderate multifocal positive XIAP staining; (C) Strong diffuse positive XIAP staining.
Click for larger image

Clinicopathologic features of conventional papillary thyroid carcinoma

Patients diagnosed with conventional PTC and subjected to thyroid surgery at Asan Medical Center between 1996 and 1998 were retrospectively analyzed. Demographic data, including age and gender, and pathologic data, such as tumor size, multifocality, extrathyroidal invasion and AJCC TNM 2002 staging, were reviewed.

Statistics

Categorical variables are presented as numbers and percentages, and continuous variables as means ± standard deviations or median values with a range. Comparison of clinicopathologic parameters according to positivity of XIAP expression was performed using the Student's t test for continuous data and Fisher's exact test for categorical data. All P values were two-sided. Data were considered statistically significant at P < 0.05. R version 2.11.1 and R libraries car and Cairo were used to analyze data (R Foundation for Statistical Computing, Vienna, Austria, http://www.R-project.org) (11).

Ethics statement

Formalin-fixed, paraffin-embedded tumor specimens were obtained from the hospital research material pool after approval from the institutional review board of the Asan Medical Center (2010-0477). Informed consent was exempted by the board.

RESULTS

XIAP expression in malignant and benign thyroid tumors

Among the 164 conventional PTC specimens, 128 (78%) stained positive for XIAP (Table 1). In terms of staining extent, 130 (82%) specimens scored 2+ or 3+. Intensity scores of 1+, 2+, and 3+ were conferred to 31 (19%), 81 (49%), and 27 (16%) specimens, respectively.


Table 1
Summary of XIAP immunostaining results for thyroid neoplasms and disorders
Click for larger image

Positive XIAP staining was found in 2 (20%) of 10 follicular variant PTCs, 2 (25%) of 8 FTC, 3 (38%) of 8 medullary thyroid carcinomas, 4 (67%) of 6 poorly differentiated thyroid carcinomas, and 3 (38%) of 8 anaplastic thyroid carcinomas.

Among the benign thyroid nodule specimens, none of the 6 nodular hyperplasias and only one of 7 (14%) follicular thyroid adenomas displayed XIAP-positive staining.

Clinicopathological factors of classic PTC according to positivity for XIAP expression

Among the 128 patients in the XIAP-positive group, 35 (27%) were in the N0 stage, 80 (63%) in the N1a stage and 11 in the N1b stage (Table 2). Among the 34 patients in the XIAP-negative group, 5 (14%) patients were in the N0 stage, 20 (56%) in the N1a stage and 11 (33%) in the N1b stage. The incidence of metastasis to lateral neck lymph node was higher in patients with negative XIAP expression than those positive for XIAP (P = 0.01).


Table 2
Clinicopathological features of conventional-type papillary thyroid carcinoma according to XIAP expression
Click for larger image

No significant differences in age, sex, tumor diameter, multifocality, lymphovascular invasion, extrathyroidal extension, lymph node metastasis, and AJCC TNM 2002 stage were observed between the two groups.

DISCUSSION

The major histologic thyroid cancer types are composed of differentiated thyroid carcinomas, such as PTC and FTC, and undifferentiated thyroid carcinomas, such as medullary thyroid cancer or anaplastic thyroid cancer. PTC is the predominant thyroid cancer type in most parts of the world. Thyroid fine-needle aspiration cytology (FNAC) is a standard diagnostic tool for thyroid neoplasms (12). However, diagnostic challenges of inadequate specimens and indeterminate cytology categories that fail to meet the criteria for definitive diagnosis of cancer present significant obstacles to clinicians (13, 14). Data from the present study support the potential of XIAP as a molecular marker in thyroid cancer diagnosis.

More recently, molecular diagnoses, such as galectin-3, HBME-1, cytokeratin-19 or B-type raf (BRAF) gene mutations have been introduced, but their clinical value is yet to be established (15-17). Some groups have shown that immunohistochemical testing for a combination of 2 or more markers improves the accuracy of diagnosis (18-21). In our experiments, most specimens with benign pathology stained negative for XIAP. Thus, XIAP immunostaining from FNAC specimens, in combination with other potential markers, may be promising for the differential diagnosis of thyroid neoplastic disorders.

The incidence of positive XIAP expression was higher overall in thyroid cancer, particularly in cases of conventional PTC, compared with benign thyroid nodules. These findings are consistent with other studies suggesting that XIAP contributes to tumor cell survival as an apoptosis inhibitor (2). However, XIAP immunostaining is inappropriate to differentiate between other histologic types of thyroid cancer and benign nodules. In particular, FTC and follicular adenoma, which are difficult to diagnose using cytology, showed similar patterns of XIAP expression in this study. However, the number of FTC and follicular adenoma samples analyzed was limited, and thus, a follow-up study on a larger scale is needed to evaluate the diagnostic value of XIAP expression in these diseases.

Interestingly, follicular variant PTCs, such as FTC, showed a lower incidence of positive XIAP expression than conventional PTC. Although PTC as a group constitutes tumors with favorable biologic behavior that share characteristic microscopic features, increasing evidence suggests that they represent a heterogeneous group of tumors, and specific genetic alterations influence histologic variants and tumor behavior (22). Adeniran et al. (23) reported that the molecular profile of the follicular variant PTC is close to that of FTC, with high prevalence of RAS and a very low BRAF mutation rate. Riveral et al. (24) reported that the mutational patterns of encapsulated follicular variant PTC and FTC are similar. Infiltrative follicular variant PTC displays a BRAF and RAS genotype in between FTC and conventional PTC. Encapsulated follicular variant PTC has behavioral profile similar to that of FTC, while infiltrative follicular variant PTC has an invasive and behavioral profile similar to that of conventional PTC. XIAP appears to be associated with histologic variant and clinical behavior, similar to BRAF and RAS.

We further evaluated the potential association between XIAP expression and clinicopathological factors in patients with conventional PTC. XIAP expression was negatively associated with risk of lateral neck lymph node metastasis in our study. This result was inconsistent with a recent report from China (10). In the earlier study, XIAP expression was positively associated with the presence of lymph node metastasis in PTC. Several Korean groups have shown a high prevalence of the BRAF V600E mutation (around 70%-80%) in Korea (15-17), compared with other parts of the world. Additionally, Gu et al. (25) detected the BRAF V600E mutation in 34.1% of classical PTC samples. This regional difference in the prevalence of the BRAF mutation in PTC between Korea and China could explain the discrepancy regarding the association of XIAP with neck node metastasis. Further studies are required to explore the potential interactions between BRAF and XIAP.

Moreover, controversial results have been reported with regard to the association of XIAP expression with clinical outcomes in various cancer types other than thyroid cancer. A number of studies have shown that increased XIAP is correlated with decreased survival in acute myeloid leukemia, diffuse large B-cell lymphoma, and renal cell carcinoma (26). In another study, high levels of XIAP were correlated with favorable outcomes in prostate cancer and early-stage non-small cell lung cancer (27). In the current investigation, XIAP expression was negatively associated with risk of lateral neck lymph node metastasis. Lateral neck lymph node metastasis is one of the important prognostic factors for recurrence and disease-specific mortality in thyroid cancer patients (28). Eventually, XIAP expression may be used as a surrogate marker to predict prognosis in thyroid cancer.

The possible mechanisms linking XIAP expression to clinical outcomes have been explored. The anti-apoptotic activity of XIAP is regulated by Smac/DIABLO. This protein is normally localized to mitochondria and released into the cytosol during the early stages of apoptosis, where it promotes caspase activity by inhibiting XIAP. Recent studies have reported that the relative proportion of XIAP, compared with Smac/DIABLO, rather than absolute value of XIAP expression, may determine tumor cell survival (27, 29, 30). However, the present investigation focused solely on XIAP expression, and follow-up studies are required to establish the roles of XIAP and Smac/DIABLO in tumorigenesis and metastasis of thyroid cancer.

In conclusion, immunohistochemical staining for XIAP as a potential novel molecular marker may aid in differential diagnosis of thyroid cancer. Furthermore, high XIAP expression in conventional PTC is strongly associated with reduced risk of lateral neck lymph node metastasis.

Notes

This study was supported by a grant (2010-475) from the Asan Institute for Life Sciences, Seoul, Korea.

AUTHOR SUMMARY

Expression of X-linked Inhibitor of Apoptosis Protein in Neoplastic Thyroid Disorder

Ji Hye Yim, Jong Ho Yoon, Sun A Kim, Won Gu Kim, Min Ji Jeon, Ji Min Han, Tae Yon Sung, Tae Yong Kim, Won Bae Kim, Suck Joon Hong, Young Kee Shong and Gyungyub Gong

X-linked inhibitor of apoptosis protein (XIAP) is associated with tumor genesis, growth, progression and metastasis by blocking caspase-mediated apopotosis. We evaluated the expression of XIAP in thyroid tumors. XIAP was positive in 78% of conventional papillary thyroid cancer (PTC). Lateral neck lymph node metastases were more frequent in patients with negative XIAP. XIAP expression might be helpful in differential diagnosis of thyroid cancer.

References
1. Hunter AM, LaCasse EC, Korneluk RG. The inhibitors of apoptosis (IAPs) as cancer targets. Apoptosis 2007;12:1543–1568.
2. Holcik M, Korneluk RG. XIAP, the guardian angel. Nat Rev Mol Cell Biol 2001;2:550–556.
3. Holcik M, Gibson H, Korneluk RG. XIAP: apoptotic brake and promising therapeutic target. Apoptosis 2001;6:253–261.
4. Bree RT, Stenson-Cox C, Grealy M, Byrnes L, Gorman AM, Samali A. Cellular longevity: role of apoptosis and replicative senescence. Biogerontology 2002;3:195–206.
5. Fulda S, Debatin KM. Apoptosis signaling in tumor therapy. Ann N Y Acad Sci 2004;1028:150–156.
6. Fadeel B, Orrenius S. Apoptosis: a basic biological phenomenon with wide-ranging implications in human disease. J Intern Med 2005;258:479–517.
7. Kasibhatla S, Tseng B. Why target apoptosis in cancer treatment? Mol Cancer Ther 2003;2:573–580.
8. Lowe SW, Lin AW. Apoptosis in cancer. Carcinogenesis 2000;21:485–495.
9. Xiao GQ, Unger PD, Burstein DE. Immunohistochemical detection of X-linked inhibitor of apoptosis (XIAP) in neoplastic and other thyroid disorders. Ann Diagn Pathol 2007;11:235–240.
10. Gu LQ, Li FY, Zhao L, Liu Y, Chu Q, Zang XX, Liu JM, Ning G, Zhao YJ. Association of XIAP and P2X7 receptor expression with lymph node metastasis in papillary thyroid carcinoma. Endocrine 2010;38:276–282.
11. CoreTeam D. A language and environment for statistical computing. [accessed on Dec 5 2007].
12. Baloch ZW, Sack MJ, Yu GH, Livolsi VA, Gupta PK. Fine-needle aspiration of thyroid: an institutional experience. Thyroid 1998;8:565–569.
13. Elliott DD, Pitman MB, Bloom L, Faquin WC. Fine-needle aspiration biopsy of Hurthle cell lesions of the thyroid gland: a cytomorphologic study of 139 cases with statistical analysis. Cancer 2006;108:102–109.
14. Jemal A, Siegel R, Ward E, Hao Y, Xu J, Murray T, Thun MJ. Cancer statistics, 2008. CA Cancer J Clin 2008;58:71–96.
15. Kim TY, Kim WB, Rhee YS, Song JY, Kim JM, Gong G, Lee S, Kim SY, Kim SC, Hong SJ, Shong YK. The BRAF mutation is useful for prediction of clinical recurrence in low-risk patients with conventional papillary thyroid carcinoma. Clin Endocrinol (Oxf) 2006;65:364–368.
16. Kim SW, Lee JI, Kim JW, Ki CS, Oh YL, Choi YL, Shin JH, Kim HK, Jang HW, Chung JH. BRAFV600E mutation analysis in fine-needle aspiration cytology specimens for evaluation of thyroid nodule: a large series in a BRAFV600E-prevalent population. J Clin Endocrinol Metab 2010;95:3693–3700.
17. Jo YS, Li S, Song JH, Kwon KH, Lee JC, Rha SY, Lee HJ, Sul JY, Kweon GR, Ro HK, Kim JM, Shong M. Influence of the BRAF V600E mutation on expression of vascular endothelial growth factor in papillary thyroid cancer. J Clin Endocrinol Metab 2006;91:3667–3670.
18. Wiseman SM, Melck A, Masoudi H, Ghaidi F, Goldstein L, Gown A, Jones SJ, Griffith OL. Molecular phenotyping of thyroid tumors identifies a marker panel for differentiated thyroid cancer diagnosis. Ann Surg Oncol 2008;15:2811–2826.
19. Rossi ED, Raffaelli M, Mule' A, Miraglia A, Lombardi CP, Vecchio FM, Fadda G. Simultaneous immunohistochemical expression of HBME-1 and galectin-3 differentiates papillary carcinomas from hyperfunctioning lesions of the thyroid. Histopathology 2006;48:795–800.
20. Scognamiglio T, Hyjek E, Kao J, Chen YT. Diagnostic usefulness of HBME1, galectin-3, CK19, and CITED1 and evaluation of their expression in encapsulated lesions with questionable features of papillary thyroid carcinoma. Am J Clin Pathol 2006;126:700–708.
21. Nga ME, Lim GS, Soh CH, Kumarasinghe MP. HBME-1 and CK19 are highly discriminatory in the cytological diagnosis of papillary thyroid carcinoma. Diagn Cytopathol 2008;36:550–556.
22. Kondo T, Ezzat S, Asa SL. Pathogenetic mechanisms in thyroid follicular-cell neoplasia. Nat Rev Cancer 2006;6:292–306.
23. Adeniran AJ, Zhu Z, Gandhi M, Steward DL, Fidler JP, Giordano TJ, Biddinger PW, Nikiforov YE. Correlation between genetic alterations and microscopic features, clinical manifestations, and prognostic characteristics of thyroid papillary carcinomas. Am J Surg Pathol 2006;30:216–222.
24. Rivera M, Ricarte-Filho J, Knauf J, Shaha A, Tuttle M, Fagin JA, Ghossein RA. Molecular genotyping of papillary thyroid carcinoma follicular variant according to its histological subtypes (encapsulated vs infiltrative) reveals distinct BRAF and RAS mutation patterns. Mod Pathol 2010;23:1191–1200.
25. Gu LQ, Li FY, Zhao L, Liu Y, Zang XX, Wang TX, Chen HP, Ning G, Zhao YJ. BRAFV600E mutation and X-linked inhibitor of apoptosis expression in papillary thyroid carcinoma. Thyroid 2009;19:347–354.
26. Xiang G, Wen X, Wang H, Chen K, Liu H. Expression of X-linked inhibitor of apoptosis protein in human colorectal cancer and its correlation with prognosis. J Surg Oncol 2009;100:708–712.
27. Ferreira CG, van der Valk P, Span SW, Ludwig I, Smit EF, Kruyt FA, Pinedo HM, van Tinteren H, Giaccone G. Expression of X-linked inhibitor of apoptosis as a novel prognostic marker in radically resected non-small cell lung cancer patients. Clin Cancer Res 2001;7:2468–2474.
28. Ito Y, Miyauchi A. Prognostic factors and therapeutic strategies for differentiated carcinomas of the thyroid. Endocr J 2009;56:177–192.
29. Du C, Fang M, Li Y, Li L, Wang X. Smac, a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating IAP inhibition. Cell 2000;102:33–42.
30. Verhagen AM, Ekert PG, Pakusch M, Silke J, Connolly LM, Reid GE, Moritz RL, Simpson RJ, Vaux DL. Identification of DIABLO, a mammalian protein that promotes apoptosis by binding to and antagonizing IAP proteins. Cell 2000;102:43–53.