Original Research. PAX8 expression in thyroid tumors: Comparison with PAX2, TTF-1, and thyroglobulin

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1 Original Research Journal of Interdisciplinary Histopathology DOI: /jihp PAX8 expression in thyroid tumors: Comparison with PAX2, TTF-1, and thyroglobulin Ayhan Ozcan 1, Ashraf Khan 2, Steven S. Shen 3,5,6, Luan D. Truong 3,4,5,6 1 Department of Pathology, Yeni Yuzyıl University, Gaziosmanpasa Hospital, Istanbul, Turkey, 2 Department of Pathology, University of Massachusetts Medical School, Worcester, Massachusetts, 3 Department of Pathology, The Houston Methodist Hospital, Houston, Texas, 4 Department of Pathology, Baylor College of Medicine, Houston, Texas, 5 Department of Pathology, Weill Medical College of Cornell University, New York, NY, 6 Department of Pathology, The Methodist, and Research Institute, Houston, Texas Address for correspondence: Ayhan Ozcan, Department of Pathology, Yeni Yuzyıl University, Gaziosmanpasa Hospital, Gaziosmanpasa, Istanbul, Turkey. Phone: aozcan06018@ gmail.com Received: January 21, 2017 Accepted: April 11, 2017 Published: April 21, 2017 ABSTRACT Objective: Diagnostic markers for thyroid differentiation remain in development. Paired box (PAX8) is a member of a transcription factor family instrumental for fetal development and probably neoplastic transformation of the kidney, müllerian organs, and thyroid. Expression of PAX8 in thyroid tissue is evaluated and compared with traditional thyroid markers which are thyroglobulin and thyroid transcription factor-1 (TTF 1). Materials and Methods: Consecutive tissue sections of non-neoplastic thyroid tissue (n = 131), adenomatous nodule (n = 26), follicular neoplasms (n = 25), papillary carcinoma (n = 13), medullary carcinoma (n = 6), poorly differentiated carcinoma (n = 16), undifferentiated carcinoma (n = 6), and benign parathyroid tissue (n = 15) were submitted for PAX8, PAX2, TTF-1, and thyroglobulin immunostain. Staining extent (% of cells stained) and intensity (score 0-3) were evaluated. Results: PAX2 was not seen in any specimens. Strong (intensity score 3) (and diffuse 100% of cells) nuclear staining for PAX8 was noted in every case of non-neoplastic thyroid tissue and differentiated thyroid tumors. Staining for TTF-1 was similar to that of PAX8 in term of frequency, but the extent and intensity were less for some variants of papillary carcinoma or less differentiated follicular neoplasms. Thyroglobulin was noted in every case of non-neoplastic thyroid tissue and differentiated thyroid tumors, but the staining (which is cytoplasmic) was weak and focal in 66 cases of them, and this staining was often masked by strong staining of the adjacent colloid. For undifferentiated carcinoma, PAX8 was the only expressed marker, but in only 1/6 cases. For medullary carcinoma, PAX8 was not seen in any case, but TTF-1 and thyroglobulin were noted in 67% and 33% of cases, respectively. For parathyroid tissue, PAX8 was noted in 80% of cases, but the staining was weak and focal in each; TTF-1 and thyroglobulin were not seen. Conclusions: (1) PAX8 is a very sensitive marker for thyroid differentiation, regardless of diagnoses, (2) PAX8 is the only available marker, albeit of limited sensitivity, for undifferentiated thyroid carcinoma, (3) Both PAX8 and TTF-1 are sensitive markers for thyroid differentiation; with a diagnostic advantage for PAX8; and both are superior to thyroglobulin, (4) PAX8 may be the only marker needed for evaluating thyroid differentiation, and (5) In spite of an ontogenic similarity with PAX8, PAX2 is not expressed by thyroid tissue. KEY WORDS: Immunohistochemistry, paired box 2, paired box 8, thyroglobulin, thyroid cancers, thyroid transcription factor-1 INTRODUCTION Thyroid cancer is the most common malignant tumor of the endocrine system and accounts for approximately 1% of all cancers [1]. Follicular cell-derived thyroid carcinomas consist of several morphological subtypes including papillary (80%), follicular (15%), poorly differentiated (4-7%), and undifferentiated (anaplastic) carcinoma (<2%) [2-4]. The difficulties in the differential diagnosis of thyroid malignancies are due to partial or complete loss of differentiation by light microscopic and even at the immunohistochemical level, as evidenced by failure to express thyroglobulin or thyroid transcription factor-1 (TTF-1) [5-10], leading to morphologic overlap with other malignant neoplasms. Undifferentiated thyroid (anaplastic) carcinoma may be easily confused with other malignant neoplasms such as lymphoma, sarcoma, and 29

2 non-thyroidal carcinomas such as squamous cell carcinoma. This diagnosis is traditionally based on the previous history of thyroid cancer with the development of undifferentiated (anaplastic) carcinoma or undifferentiated (anaplastic) carcinoma with areas of differentiated thyroid carcinoma. Diagnostic markers may be helpful, but not be necessary to make this diagnosis. However, in the case where the available tumor tissue is limited, or it is composed entirely of the anaplastic component, a thyroid-specific marker that is retained in spite of the loss of thyroid differentiation by light microscopy would diagnostically helpful. TTF-1 is one of the three most important transcription factors for thyroid gland organogenesis. In contrast to TTF-1, the other two transcription factors, paired box (PAX8) and TTF-2 (FoxE1), have been rarely studied for diagnostic purposes [11]. Abnormal expression of these transcription factors was reported in some thyroid carcinomas [12,13]. Dedifferentiation and loss of thyroid-specific proteins are common in thyroid tumorigenesis [14,15]. PAX8 is a transcription factor expressed in normal and neoplastic thyroid follicular epithelium and only a few other normal and neoplastic tissues such as kidney and müllerian organs. This restricted expression suggests that PAX8 staining could be useful in challenging tumors with a spindle or squamoid appearance. PAX8 was recently shown to be expressed in most sarcomatoid renal cell carcinomas, indicating that for some tumor types, PAX8 expression persist as differentiated tumors undergo dedifferentiation [16-19]. In this study, we systematically evaluate the PAX8 expression and compare this with other traditional immunomarkers for thyroid follicular cell differentiation to understand its roles in thyroid tumorigenesis, to clarify the role of PAX8 in the diagnosis of thyroid tumors, and to assess its diagnostic utility in the differential diagnosis against the background of traditional thyroid immunomarkers. MATERIALS AND METHODS This retrospective study included non-neoplastic thyroid tissue (131 samples), adenomatous nodules (26), follicular neoplasms (25), papillary carcinoma (16), medullary carcinoma (6), poorly differentiated carcinoma (16), undifferentiated carcinoma (6), and benign parathyroid tissue (15). The non-neoplastic tissue samples included two categories: Benign thyroid lesions surgically excised and the non-neoplastic thyroid tissue in thyroid specimens resected for neoplasms. The first categories include 37 cases with the diagnoses of adenomatous nodules, Hashimoto thyroiditis, lymphocytic thyroiditis, or benign thyroid tissue excised during parathyroid surgery. We utilize the definitions and the diagnostic criteria, as specified by WHO (Pathology and Genetics of Tumors of Endocrine Organs, IARC Press, Lyon, France; 2004 Edition). Accordingly, poorly differentiated carcinoma is defined as follicular cell neoplasm that shows limited evidence of structural follicular cell differentiation. This tumor type is characterized by an insular, trabecular, and/or solid growth pattern, together with infiltrative growth, necrosis, and possible vascular invasion. Undifferentiated carcinoma is defined as a highly malignant tumor that histologically appears wholly or partially composed of undifferentiated epithelial cells. This tumor type is characterized by widely invasive tumor tissue composed of an admixture of spindle cells, pleomorphic cells, and epithelioid cells. For a direct comparison of PAX8, PAX2, TTF-1, and thyroglobulin expression, consecutive tissue sections of each of these cases were submitted to H&E stain and immunostain for each marker. The tissue sections were subjected to deparaffinization, hydration, and endogenous peroxidase blocking. Antigen retrieval was achieved by Dako Target Retrieval Solution, ph 6 (Dako, Carpinteria, CA) in a pressure cooker set at 95 C for 22 minutes followed by gradual cooling for 20 minutes. The stain was done by an automatic stainer from Ventana (Ventana Medical Systems, Tucson, AZ), using antibodies against: PAX8 (polyclonal, dilution 1:50; Proteintech Group, Chicago, IL), PAX2 (polyclonal, dilution 1:75; Invitrogen, Carlsbad, CA); TTF-1 (monoclonal, clone 8G7G31, prediluted, Ventana); and thyroglobulin (monoclonal, clone E1, prediluted, Ventana). Positive controls included renal carcinoma tissue and lymphoid tissue, which often also served as a built-in control since lymphoid cells are often seen in the evaluated tissue sections. Mesenchymal or epithelial cells in the evaluated tissue sections served as negative controls. For each case, the staining intensity (score 0 = no stain, score 1 = unequivocal but weak, score 2 = moderate, and score 3 = strong) and staining extent (estimated percentage of stained cells in 5% increment) were recorded. RESULTS PAX8, PAX2, TTF-1, and thyroglobulin were successfully detected in routinely processed tissue with appropriate positive and negative controls. The staining of TTF-1 was limited to the nuclei. PAX8 expression was noted in nuclei, but weak focal cytoplasmic staining was also noted, which is well recognized as diagnostically irrelevant and perhaps reflects the polyclonal nature of the utilized antibody. The staining for thyroglobulin was cytoplasmic. The staining results according to diagnosis, extent (percentage of positive cells and intensity (score 0-3) are shown in Table 1, and Figure 1a and b. Figure 2 illustrates the staining patterns according to diagnosis. Non-neoplastic Thyroid Tissue (n = 131) [Figure 2, Row a] PAX8 and TTF-1 displayed strong diffuse nuclear staining; but focal weak staining of colloid was noted for both. In contrast, staining for thyroglobulin was diffuse and strong for colloid, but weak and focal for follicular cell cytoplasm [Figure 1a and b, Table 1]. PAX2 is not identified in any specimens. 30

3 a b Figure 1: The staining results according to extent (percentage of positive tumor cell nuclei/cytoplasm) (a) and intensity (b) of tumor cells Table 1: The staining results of the thyroid tumors Diagnosis PAX8 TTF 1 Thyroglobulin Positive/Total cases (%) % of Stained cells Staining intensity Positive/Total cases (%) % of Stained cells Staining intensity Positive/Total cases (%) % of Stained cells Staining intensity Non neoplastic thyroid 131 (100) (100) (100) tissue (n=131) Adenomatous nodule (n=26) 26 (100) (100) (100) Follicular neoplasms (25) 25 (100) (100) (100) Follicular adenoma 10 (100) (100) (100) Follicular carcinoma 5 (100) (100) (100) 96 2 Hürthle cell adenoma 5 (100) (100) (100) Hürthle cell carcinoma 5 (100) (100) (100) Papillary carcinoma (n=16) 16 (100) (100) (100) 93 2 Classic variant 8 (100) (100) (100) 91 2 Follicular variant 4 (100) (100) (100) Tall cell variant 1 (100) (100) (100) 80 2 Sclerosing variant 3 (100) (100) (100) Medullary carcinoma (n=6) 0 (0) (67) (33) 15 3 Poorly differentiated 16 (100) (100) (100) carcinoma (n=16) Undifferentiated carcinoma (n=6) 1 (16) (0) (0) 0 0 Parathyroid tissue (n=15) [1] 12 (80) (0) (0) 0 0 TTF 1: Thyroid transcription factor 1; PTH: Parathyroid hormone [1] 10 70% of the cells stained for PAX 8 in positive cases Adenomatous Nodule (n = 26) The staining was virtually identical to normal thyroid tissue in term of frequency, extent, and intensity [Figure 1a and b, Table 1]. Follicular Neoplasms (n = 25) [Figure 2, Rows b-d] Overall, PAX8, TTF-1, and thyroglobulin were expressed in all cases, and PAX2 was not seen. However, significant differences were noted for the staining patterns among them. PAX8 expression was more pronounced than other markers in terms of extent (mean 100% vs % of tumor cells), and intensity (mean score 3 vs ). In follicular adenoma (row b), all positive markers have the same staining pattern in terms of frequency, extent, and intensity. In Hürthle cell adenoma (row c), PAX8 and TTF-1 displayed the same staining pattern. Staining for thyroglobulin was noted in each lesion but was rather weak and focal in some cases. In follicular and Hürthle cell carcinoma, PAX8 expression was more pronounced than other markers in terms of extent of staining (mean 100% vs % of tumor cells), and intensity (mean score 3 vs ), however, PAX8 was not expressed in undifferentiated (anaplastic) carcinoma areas of follicular carcinoma (row d) as other undifferentiated (anaplastic) thyroid carcinomas, which are only positive for PAX8 in 1/6 anaplastic cases (rest of them negative for PAX8). Although colloid displayed strong staining for thyroglobulin, focal weak staining was also noted for PAX8 and TTF-1. Papillary Carcinoma (n = 16) [Figure 2, Rows e and f] Overall, PAX8, TTF-1, and thyroglobulin were expressed in all cases, but there was no staining for PAX2. PAX8 expression was more pronounced than other positive markers in terms of extent (mean 100% vs % of tumor cells), and staining (mean score 2.6 vs ). In the classic (row e), tall cell (row f) and sclerosing variants, PAX8 and TTF-1 expressions were more pronounced than thyroglobulin in terms of extent (mean 100% vs. 91% of tumor cells), and intensity (mean score vs. 2). 31

4 Ozcan, et al.: PAX8 expression in thyroid tumors a b c d e f g h i Figure 2: The expression of PAX8, TTF-1, thyroglobulin, and PAX2 in non-neoplastic thyroid tissue, nodular goiter (a), follicular adenoma (b), Hürthle cell adenoma (c), follicular carcinoma with undifferentiated (anaplastic) carcinoma component, which is in right side (d), papillary carcinomas, classic (e) and tall cell (f) variants, medullary thyroid carcinoma (g), poorly differentiated carcinoma (h), undifferentiated (anaplastic) carcinoma (i) ( 200 for all figures in rows a, b, d-h; 400 for all figures in rows c and i) 32

5 Of note, TTF-1 was more pronounced than PAX8 in the tall cell variant, while PAX8 was more pronounced than TTF-1 in the sclerosing variant. However, in the follicular variant, all immunomarkers demonstrated the same staining pattern. Medullary Carcinoma (n = 6) [Figure 2, Row g] TTF-1 and thyroglobulin were expressed in 67% and 33% of cases, respectively; but there was no staining for PAX2 or PAX8. Poorly Differentiated Carcinoma (n = 16) [Figure 2, Row h] PAX8, TTF-1, and thyroglobulin were expressed in all cases, but there was no staining for PAX2. PAX8 expression was more pronounced than other markers in terms of extent (mean 93% vs % of tumor cells), and staining (mean score 2.9 vs. 2.1). Undifferentiated (Anaplastic) Thyroid Carcinoma (n = 6) [Figure 2, Row i] PAX8 was expressed in 16% of cases (1/6), but there was no staining for PAX2, TTF-1, or thyroglobulin. In the positive case, PAX8 expression was noted in 60% of tumor cells with moderate staining intensity (mean score 2). In each case, the undifferentiated carcinoma component was associated with a more differentiated carcinoma component, including papillary, follicular, or poorly differentiated. These latter components displayed a staining pattern as described above (Figure 2, row h). No distinctive histologic difference is noted between positive and negative tumors. Parathyroid Tissue (n = 15) PAX8 was expressed in 80% of cases; but there no staining for PAX2, TTF-1, or thyroglobulin. The staining for PAX8 was weak (score 1.5) and often focal (51% of cells). DISCUSSION To our knowledge, this is the first systematic and comprehensive study directly comparing PAX8 and PAX2, with other traditional immunomarkers such as TTF-1 or thyroglobulin in thyroid tumors, non-neoplastic thyroid, and parathyroid tissues. PAX8 and PAX2 are cell lineage-restricted transcription factors, with similar tissue expression and ontogenetic function. Both PAX2 and PAX8 are expressed in the primordial tissues of Wolffian (nephric) ducts, and Müllerian ducts [20,21]. During organogenesis, these primordial structures give rise to urogenital organs, including kidney, ureter, seminal vesicles, vas deferens, uterus, and fallopian tubes, under the partial control of both PAX2 and PAX8 [20,21]. In spite of ontogenic similarity with PAX8, PAX2 is not expressed by thyroid tissue. Three thyroid-specific transcription factors, (TTF-1, TTF-2, and PAX8), have been identified [11,13,22-24]. They play a role in organogenesis of the thyroid and are responsible for the thyroid differentiation [22-24]. They also play a role in the expression of multiple thyroid-specific proteins, such as thyroglobulin, thyroid peroxidase (TPO), sodium/iodide symporter (NIS), and thyroidstimulating hormone receptor (TSH-R) [14,15,25-31]. These thyroid-specific transcription factors bind to thyroglobulin and TPO promoters activating gene transcription [32]. In addition, TTF-1 binds to TSH-R and the NIS promoter [26,27]. The expression of these transcription factors is essential for the development of thyroid follicular cells [23,33] and C cells [9,34]. The loss of the thyroid-specific proteins and differentiation is a common process in tumorigenesis in the thyroid [14,15]. The loss of tissue-specific gene expression and differentiation in thyroid tumor may be explained with the lack of transcription factors because differentiation markers and malignant phenotype in the thyroid tumors are inversely related. Several studies demonstrated that abnormal expression of these transcription factors was detected in some thyroid tumors [12,13]. Thyroid tumors, which consist of adenomas, differentiated (papillary and follicular) carcinomas, poorly differentiated carcinomas, and undifferentiated (anaplastic) carcinomas, originating from the follicular cells. The degree of the cell differentiation of these tumors is heterogeneous. The heterogeneity in the expression of the differentiated phenotype is usually seen in tumors of the same class [14]. Expression of individual thyroid-related genes and proteins in thyroid tumors has been studied, with emphasis on mechanism of tumor genesis, rather than diagnosis. Ros et al. and Fabbro et al. suggested that TTF-1 and PAX8 were expressed in welldifferentiated adenomas and that their expression decreases in less differentiated papillary and follicular carcinomas and was lost in undifferentiated (anaplastic) carcinomas. They found parallel levels of thyroglobulin, TPO and TSH-R expression in the same tumors [12,13]. Interestingly, Ros et al. detected that TSH-R and TTF-1 gene expression in medullary thyroid carcinoma. Furthermore, they pointed out that the expression of the thyroid-specific genes and their transcription factors are lost in thyroid cells derived from follicular, papillary and undifferentiated (anaplastic) carcinomas. In these cells, they also demonstrated that thyroglobulin, TPO, and TSH-R promoter activities were absent [13]. Other studies suggested that the loss of PAX8 and TTF-1 correlated to the aggressiveness of thyroid carcinoma, and over-expression of TTF-1 and PAX8 could induce the differentiation of undifferentiated (anaplastic) thyroid carcinoma [35,36]. Zhang et al. suggested that a decrease of nuclear staining for TTF-1, TTF-2, and PAX8 from follicular adenoma to differentiated carcinoma and then to anaplastic carcinoma, which parallels the progressive dedifferentiation and increasing malignancy of thyroid tumors. As a result of these observations, they proposed that deregulation of these thyroidspecific transcription factors is a pivotal event for initiation and progression of thyroid neoplasm. They concluded that their expression could be helpful to distinguish benign and malignant thyroid tumor [11]. The diagnostic significance of thyroid-specific markers has been reported, albeit in a rather limited scope. Fabbro et al. suggested that TTF-1 was noted in all papillary (83% of positive nuclei) carcinoma, all follicular carcinoma (79.3% of positive 33

6 nuclei), all follicular adenomas (80% of positive nuclei); but there was no staining in anaplastic cancers [12]. Zhang et al. observed that intensity of TTF-1 staining decreased from anaplastic carcinoma (0.25 ± 0.46) to medullary carcinoma (1 ± 0.88), papillary carcinoma (1.44 ± 0.73) and follicular carcinoma (1.8 ± 0.41), and was highest in follicular adenoma (2.67 ± 0.49). In contrast, staining of PAX8 was positive only in 33.3% of follicular adenomas (intensity 0.67 ± 0.98), 40% of follicular carcinomas (intensity 0.67 ± 0.89), 31.2% of papillary carcinomas (intensity 0.5 ± 0.82), and negative in all anaplastic carcinoma, and medullary carcinoma samples. Our systematic comparative study corroborates and significantly expands these observations. Our results show that nuclear staining of PAX8 was positive in all cases of adenomatous nodules, follicular and Hürthle cell adenomas, follicular and Hürthle cell carcinomas, and papillary carcinomas, and poorly differentiated carcinomas. The staining is diffuse (99.4% of cells) and strong (intensity score 2-3, mean 2.9) in all these cases. Importantly, PAX8 was also unequivocally (intensity score 2, 60% of cells) noted in 16% (1/6) of undifferentiated (anaplastic thyroid) thyroid carcinomas. Review of the previous studies on the PAX8 expression on of undifferentiated (anaplastic) thyroid carcinoma shows that the frequency of positivity ranged from 0 to 100% in studies that include 2-34 cases, with most of them reporting a frequency of >75% [37]. Thus, the positive frequency in our study indeed falls to the lower range. The reasons for this discrepancy are not clear but may involve a number of wellrecognized factor including sampling, diagnostic criteria, and antibody species. In this aspect, we note that most studies, including ours, utilized a polyclonal antibody, which may be prone to batch-to-batch variation. We also note that even in positive cases, only a small portion of tumor cells are positive, again emphasizing tumor sample as a potential reason for the discrepancy of staining frequency. As expected, PAX8 is negative in all cases of medullary carcinoma. Of note, 80% of parathyroid tissue samples was positive for PAX8, albeit week and focal in each case. We also noted that TTF-1 was diffusely (94% of cells) and strongly positive (intensity score 2-3, mean 2,9) in all cases of adenomatous nodules, follicular and Hürthle cell adenomas, follicular and Hürthle cell carcinomas, papillary carcinoma, and poorly differentiated carcinomas, but negative in all undifferentiated carcinomas. Unexpectedly, significant staining of TTF-1 was noted in medullary carcinomas (in 67% [4/6] of cases; intensity score 2, and 27% of tumor cells), in contrast with a uniform negative result for PAX8 and TTF-1 in parathyroid tissue. Review of previous studies on PAX8 expression on medullary thyroid carcinoma [37] shows that the frequency of positivity ranged from 0-75% in studies that include 3-32 cases. Our study showed no staining for any of the 6 included cases. The reasons for this discrepancy are not clear, but may involve a number of well-recognized factors including sampling and antibody species. In this aspect we note that most studies, including 34 ours, utilized a polyclonal antibody, which may be prone tobatch-to-batch variation. We also note that even in positive cases, only a small portion of tumor cells are positive, again emphasizing tumor sample as a potential reason for the discrepancy of staining frequency. This study demonstrated cytoplasmic staining of thyroglobulin in all cases of adenomatous nodules, follicular and Hürthle cell adenomas, follicular and Hürthle cell carcinomas, and papillary carcinomas, poorly differentiated carcinomas, with an intensity score of 2-3, and negative in all undifferentiated carcinomas. The staining of colloid is uniformly strong, and may even mask the cytoplasmic staining of adjacent follicular cells. Unexpectedly, thyroglobulin was strongly (intensity score 3) positive in 33% (2/6) of medullary carcinomas Thyroglobulin was negative in all parathyroid tissue samples. Several differences in the expression of these markers are noted, and they may portend diagnostic significance. In spite of a similar ontogeny of PAX2 and PAX8, failure of PAX2 expression across the diagnostic borders negates its diagnostic value in the differential diagnosis of thyroid lesions. Although the expression of PAX8 and TTF-1 is comparable in general, PAX8 seems to be a better marker. As compared with TTF-1, the PAX8 expression is often stronger and with a significant more staining extent. This diagnostic advantage is particularly useful in some variants of papillary carcinoma, or less differentiated follicular neoplasms, where TTF-1 expression may be rather weak and focal. It should be emphasized that cytoplasmic staining for PAX8 may occur and parathyroid tissue may also express nuclear PAX8. Nevertheless, this aberrant staining, which is uniformly weak, and often seen against the background of strong diffuse nuclear PAX8 expression in a cell with thyroid differentiation, should not create diagnostic confusion. Of note, this aberrant staining is not seen for TTF-1. The term poorly differentiated carcinoma implies a low level of thyroid differentiation, with an expected limited expression of thyroid differentiation markers. Yet, PAX8 and TTF-1 are uniformly expressed by this tumor type. This information is not only diagnostic importance for small biopsy or metastatic tumor, but also highlights the concept of preserved lineage-specific transcription factors in spite of a concurrent lack of the corresponding proteins. In contrast to PAX8 or TTF-1, thyroglobulin may have limited diagnostic value. Although it is expressed in better-differentiated thyroid tumors, interpretational difficulty often arises due to frequent heavy background staining, even with a monoclonal antibody as utilized in this study; limited staining of target cells; and heavy staining of colloid masking cytoplasmic staining. Finally, PAX8 is the only marker, albeit of limited sensitivity, for undifferentiated carcinoma, against uniformly negative results of other markers as noted currently and in the previous reports. In conclusion, (1) PAX8 is a very sensitive marker for thyroid differentiation, regardless of diagnoses, (2) PAX8 is the only available marker, albeit of limited sensitivity, for undifferentiated thyroid carcinoma, (3) Both PAX8 and TTF-1 are sensitive markers for thyroid differentiation; with a diagnostic advantage for PAX8; and both are superior to thyroglobulin, (4) PAX8 may be the only marker needed for evaluating thyroid differentiation,

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