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Turkish Journal of Medical Sciences Turk J Med Sci(2013) 43: 625-630© TÜBİTAKdoi:10.3906/sag-1208-66

Diagnostic value of cytokeratin 19, HBME-1, and galectin-3 immunostaining of cell block from fine-needle aspiration specimens in papillary carcinoma of the thyroid

Hacer HALTAŞ*, Reyhan BAYRAK, Sibel YENİDÜNYA, Ümran YILDIRIMDepartment of Pathology, Faculty of Medicine, Fatih University, Ankara, Turkey

* Correspondence: hhaltas@hotmail.com

1. IntroductionThyroid nodules occur commonly among the adult population. Fine-needle aspiration (FNA) is a standard procedure in determining a treatment course for thyroid nodules. Thyroid FNA is used to triage patients for surgery, or for conservative management of nodules. Patients with FNA diagnoses that suggest malignancy and/or neoplasia are typically managed surgically, while patients with FNA results that indicate a benign lesion may be treated clinically. Papillary thyroid carcinoma (PTC) is the most common form of thyroid cancer. The cytologic criteria necessary for definitive diagnosis of papillary carcinoma from an FNA specimen include large monolayer sheets of follicular epithelial cells with enlarged nuclei containing fine and powdery chromatin, intranuclear cytoplasmic inclusions with nuclear grooves, and papillary structures (1). However, in a subset of cases that lack one or more of these defining features, accurate diagnosis of papillary carcinoma is challenging.

A number of immunocytochemical markers are available that may distinguish nonneoplastic lesions of the thyroid from papillary carcinoma and its variants (2–9). The type I intermediate filament protein cytokeratin polypeptide 19 (CK19) is the smallest known keratin and is unique in that, contrary to all other known keratins, it does not have a conjugate partner for the formation of filaments. This distinct feature of CK19 implies that regulation of CK19 expression differs from that of other keratin-encoding genes (10). CK19 is a sensitive marker for papillary carcinomas, usually apparent as diffuse cytoplasmic positive staining in these tissues (2,4,11,12). HBME-1 was originally described as a specific marker of mesothelial cells. In the literature, a number of studies have demonstrated the utility of HBME-1 membranous and cytoplasmic immunostaining in the identification of malignant follicular epithelial lesions of the thyroid (13–16). Galectin-3 is a member of a family of b-galactoside binding animal lectins. Galectin-3 is expressed by many

Aim: To evaluate the utility of immunohistochemical staining of cytokeratin 19 (CK19), HBME-1, and galectin-3 in cell block preparations of fine-needle aspiration cytology specimens as ancillary techniques for the differential diagnosis of papillary thyroid carcinoma.

Materials and methods: Immunohistochemical detection of CK19, HBME-1, and galectin-3 was completed in 45 thyroid aspiration cytology specimens fixed in paraffin-embedded blocks consisting of the following cytologic diagnoses: 1) 16 cases of papillary carcinoma, 2) 16 cases suspicious for papillary carcinoma, and 3) 13 cases involving nonneoplastic lesions.

Results: The follicular cells in the cytologically unequivocal papillary carcinoma diagnosis group expressed diffusely positive (+++) CK19, HBME-1, and galectin-3 in 12/16, 12/16, and 14/16 cases, respectively. Diagnosis of papillary carcinoma was confirmed during follow-up in all 16 cases. Positive staining for CK19 or galectin-3 was not detected among the 13 cases of cytologic and histopathologic diagnosis of nonneoplastic lesions. CK19 (9/12) and galectin-3 (9/12) were diffusely positive (+++) in the majority of papillary carcinoma samples with initial cytologic diagnostics indicating suspicion of papillary carcinoma.

Conclusion: The CK19, HBME-1, and galectin-3 immunostaining panel is a novel and informative adjunct for the diagnosis of papillary thyroid carcinoma from cell block material. Diffuse immunostaining for CK19 and galectin-3 contributes to improvements in accurate diagnosis of malignancy in cases of suspected papillary carcinoma.

Key words: Fine-needle aspiration, thyroid, papillary carcinoma, cell block, immunohistochemistry, cytokeratin 19, HBME-1, galectin-3

Received: 16.08.2012 Accepted: 18.10.2012 Published Online: 29.07.2013 Printed: 19.08.2013

Research Article

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tissues and cell types in both the nucleus and in the cytoplasm and has multiple functions including cell–cell and cell–matrix adhesion, cell growth, cell cycle regulation and apoptosis, neoplastic transformation and spread, and cell repair processes. Galectin-3 is highly up-regulated in thyroid carcinoma of follicular cell origin (17).

The objective of the current study was to examine the utility of galectin-3, HBME-1, and CK19 immunostaining in FNA-derived cell block material for the diagnosis of papillary carcinoma of the thyroid.

2. Materials and methodsFNA cytology and cell blocks were obtained from the archives of the Department of Pathology, Fatih University Faculty of Medicine, from January 2009 to July 2012. Only cases with an adequate number of cell blocks and available histologic follow-up were included in this study. The cytologic diagnoses issued for the cell blocks from Papanicolaou- and May-Grünwald Giemsa (MGG)-stained cytology smears included: 1) papillary carcinoma (16 cases), 2) suspected papillary carcinoma (16 cases), and 3) nonneoplastic lesions (13 cases).2.1. Cell block preparationFollowing smear preparations, the needles and syringes used to obtain fine-needle aspirates were rinsed in 10 mL of an alcohol–formalin mixture (a final concentration of 90% ethanol and 10% formaldehyde was used) in a specimen container. After 30 min, any residual clot or tissue in the hub of needles was carefully removed in the laboratory with the aid of another needle. The entire tissue was processed in a fashion similar to a small biopsy, with the tissue impregnated and embedded in paraffin. In each case, hematoxylin and eosin staining was performed on 4-µm-thick sections of the cell block.2.2. Immunohistochemical analysisThe 4-µm-thick sections were cut from blocks of paraffin-embedded tissue, deparaffinized, and rehydrated according to standard methods. Slides were incubated in hydrogen peroxide block for 15 min to reduce nonspecific background staining due to endogenous peroxidase. Antigen retrieval was performed by incubating the slides for 15 min with pepsin (LabVision; catalog no. AP-9007), at a concentration of 1 mg/mL, prior to immunostaining for CK19. Prior to HBME-1 and galectin-3 staining, slides were microwaved in 10 mM citric acid at pH 6.0 for 20 min. The slides were incubated for 60 min in primary antibody solution against CK19 (clone RCK108, LabVision/NeoMarkers; 1:100), HBME-1 (clone HBME-1, LabVision/NeoMarkers; 1:25), and Galectin-3 (clone 9C4, NovoCastra; 1:100) at room temperature (20–24 °C). The standard avidin–biotin–peroxidase complex technique was performed using the LabVision Secondary Detection Kit (UltraVision Detection System Anti-polyvalent, HRP),

with appropriate positive and negative controls. The chromogen used was AEC. All slides were counterstained using Mayer’s hematoxylin. 2.3. Microscopic evaluationImmunoreactivity was designated as negative, focally positive (+: less than 25% of cells reactive), positive (++: 25–50%), or diffusely positive (+++: more than 75%), according to the extent of the reaction. Reactivity in more than 25% of cells at an intensity of at least ++ was defined as positive (5,18).

Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and diagnostic accuracy were calculated for each antibody, using histologic diagnosis as the standard of comparison for each of the 3 specific immunostains.

3. ResultsClinical and immunohistochemical data relating to each case are summarized in Table 1. Figures 1A–1F illustrate the cytologic morphology of various thyroid nodules from cell blocks and FNA biopsies. Benign and malignant follicular cells were observed in both cell block preparations and FNA biopsies. However, thyroid tissue fragments were observed only in the cell block sections (Figures 1D–1F). Similar to the corresponding tissue sections (Figures 1G–1I), tissue architecture was apparent in the fragmented tissue section (Figures 1D–1F).

In the 16 cases with an unequivocal papillary carcinoma cytologic diagnosis, 12 (75%) cases demonstrated intense and diffuse (+++) cytoplasmic staining for CK19, 3 (19%) cases demonstrated moderate (++) staining, and 1 (6%) case was weakly positive for CK19. The incidence of diffuse (+++) cytoplasmic and membranous staining for HBME-1 and galectin-3 among these 16 cases was 75% and 86%, respectively.

Among the 12 cases with an initial cytologic diagnosis of suspicion of papillary carcinoma and a final diagnosis confirming papillary carcinoma, the detection rates of diffuse CK19, HBME-1, and galectin-3 positivity were 75%, 25%, and 75%, respectively.

In 4 cases with an initial cytologic diagnosis of suspicion of papillary carcinoma and a final diagnosis confirming papillary carcinoma, staining of CK19, HBME-1, and galectin-3 was not detected.

Among the 13 cases (100%) with cytologic and histologic diagnosis of nonneoplastic lesions, neither CK19 nor Galectin-3 staining was observed. HBME-1 staining was detected (+++)  in only one case of cellular microfollicular configuration.

Clearly, the intensity of galectin-3, HBME-1, and CK19 staining, as well as the number of positive cells, was enhanced in malignancy (Figures 1J–1R). The specificity, sensitivity, and PPV of HBME-1, CK19, and galectin-3 in distinguishing between PTC and nonneoplastic lesions were calculated and are presented in Table 2.

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Adenomatous nodule Papillary hyperplasia Papillary thyroid

carcinoma

FNA

Cell

block

Biopsy

CK19

HBME-1

Galectin-3

A B C

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F

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J K L

M N O

RP Q

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Figure. These photomicrographs provide a morphologic comparison of thyroid lesions on MGG-stained FNA cytology smears, cell block preparations, and tissue sections with immunostaining of CK19, HBME-1, and galectin-3 on cell blocks. The FNA cytology demonstrates benign, malign, and suspicious for papillary carcinoma follicular epithelial cells in (A) adenomatous nodule; (B) papillary hyperplasia (suspicious for papillary carcinoma); and (C) a papillary group of cancer cells, PTC. (D–F) Fragments of thyroid tissue are observed in these cell block sections. Thyroidectomy specimens revealed that the lesions were distributed. (G) An example of adenomatous nodule, (H) papillary hyperplastic lesions, and (I) classic variant papillary carcinoma. Immunodetection demonstrated weak staining for (J) CK19, (M) HBME-1, and (P) galectin-3 in adenomatous nodules. The atypical follicular epithelial cells were diffusely immunostained for CK19, HBME-1, and galectin-3 in (L, O, R) papillary carcinoma.

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Table 1. Clinicopathological and immunohistochemical features of thyroid nodules diagnosed by means of FNA cytology.

No. Sex/Age Cytologic diagnosis Final diagnosisImmunohistochemistry

CK19 HBME-1 Gal-31 F/53 PTC PTC ++ + +2 F/30 susp PTC NL - + ++3 F/61 susp PTC PTC +++ + +++4 F/45 susp PTC PTC +++ - +++5 F/43 susp PTC PTC +++ + +++6 F/55 susp PTC PTC +++ + +++7 F/48 susp PTC PTC - - - 8 F/19 NL NL + + +9 F/46 NL NL - ++ +10 F/44 NL NL - + +11 F/31 susp PTC PTC ++ ++ +12 F/32 NL NL ++ +++ +13 F/43 NL NL - - - 14 F/33 susp PTC PTC +++ +++ +++15 M/53 susp PTC NL + + +16 F/32 PTC PTC +++ +++ +++17 F/47 NL NL + - +18 M/57 PTC PTC +++ +++ +++19 F/34 susp PTC NL - - - 20 F/65 susp PTC PTC +++ +++ +++21 N/43 NL NL + - - 22 M/18 PTC PTC +++ +++ +++23 F/38 PTC PTC +++ +++ +++24 F/72 susp PTC PTC ++ - ++25 F/17 PTC PTC +++ +++ +++26 F/29 PTC PTC ++ + +++27 F/33 PTC PTC +++ +++ +++28 F/58 PTC PTC +++ ++ +++29 F/41 PTC PTC +++ +++ +++30 F/52 susp PTC PTC +++ +++ +++31 F/36 susp PTC PTC +++ + +++32 F/31 PTC PTC ++ +++ +++33 F/29 PTC PTC +++ +++ +++34 F/40 PTC PTC +++ +++ +++35 F/82 PTC PTC +++ +++ +++36 F/52 susp PTC PTC +++ ++ +++37 F/47 susp PTC NL - - ++38 F/29 PTC PTC +++ +++ +++39 F/67 PTC PTC + + +40 F/41 NL NL - - +41 F/42 NL NL - - +42 F/56 NL NL - - - 43 F/56 NL NL + + +44 M/63 NL NL - - ++45 F/62 NL NL - - +

Total N 45 45 45 45 45 45

PTC, Papillary thyroid carcinoma; susp PTC, suspicious for papillary thyroid carcinoma; NL, nonneoplastic lesion.

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4. DiscussionThe majority of PTCs are readily diagnosed using classic cytologic criteria, allowing the pathologist to differentiate benign lesions from papillary carcinomas with high precision. However, in a minority of cases, the pathologist is confronted with thyroid lesions in which the distinction between papillary carcinomas and benign lesions may be quite subtle. To aid in the diagnosis of papillary thyroid carcinoma, a variety of immunohistochemical stains have been evaluated in thyroid FNA cytology (2,6,8,18,19). Unfortunately, the immunoexpression profile of papillary carcinomas and benign lesions of the thyroid overlap significantly, and, to the best of the present authors’ knowledge, no single marker or even panel is 100% sensitive and 100% specific for differentiation of papillary carcinoma and benign thyroid lesion.

In this study, we evaluated the ability of 3 markers (HBME-1, CK19, and galectin-3) to differentiate papillary carcinoma from benign lesions of the thyroid in FNA cell blocks. The advantages of immunohistochemical staining of paraffin blocks over direct tissue smears are the ability to store paraffin blocks for long periods of time and, therefore, the number of samples that can be acquired for study. The disadvantages of direct immunostaining of tissue smears include artificially high and nonspecific staining resulting from disrupted cells and membrane fragments sticking to the slides, the lack of parallel samples of comparable cells for control or replicate testing, and increased cost due to the use of a considerable volume of antibodies to cover large areas of diffuse tissue smears. In contrast, cell blocks are found to be highly suitable for immunohistochemistry due to ease of morphological interpretation, standardized comparison with surgical pathology material, minimal background staining, and anticipated immunostaining patterns (20).

CK19 is a low-molecular-weight keratin widely expressed in epithelial cells. CK19 is a minor component of stratified epithelium, such as basal cell layers (21). The use of CK19 immunostaining has been reported in cytology smears of thyroid nodules. CK19 is a sensitive marker for papillary carcinomas, with positive immunostaining indicated by diffuse cytoplasmic reactivity (2,6). Several

studies have reported that CK19 is expressed focally in normal thyroid epithelium, Hashimoto’s thyroiditis, and in some benign tumors. Diffuse CK19 positive staining may have diagnostic value in PTC (2,4,6,11,12,22). In the present study, 75% of papillary carcinoma cases were strongly positive for CK19, and 3.57% of cases were negative or expressed CK19 weakly. In addition, focal staining was present in 5 (29.41%) of 17 benign lesions. Diffuse, strong immunostaining with CK19 was not observed in nonneoplastic lesions.

The monoclonal antibody HBME-1 binds to an unknown microvillus surface antigen that is present in normal or neoplastic mesothelial cells, as well as in other epithelial cells. Previous studies have reported that HBME-1 is a sensitive marker for PTCs with a staining pattern that is predominantly membranous with variable cytoplasmic positivity (18,22). In the present study we observed HBME-1 positivity in 64.28% of papillary carcinoma cases. There was no diffuse staining with HBME-1 in 58.82% of the nonneoplastic lesions. Only a single case of microfollicular hyperplasia in a nonneoplastic lesion showed diffuse positive staining with HBME-1.

Galectin-3 is a β-galactosyl-binding lectin protein involved in a variety of biological functions, including cell-cycle regulation, apoptosis, cell adhesion, and tumor progression. The biological function of galectin-3 expression in transformed thyrocytes is not well understood. In the published literature, many studies have demonstrated that well-differentiated thyroid carcinomas frequently express galectin-3, whereas healthy thyroid tissue and the majority of benign thyroid proliferations do not (3,8,23). These findings strongly support the use of galectin-3 immunostaining in preoperative FNA cytology as an adjunct marker for interpreting difficult pathologic cases, especially in confirming suspected malignant neoplasm (3,8,23,24). The sensitivity and specificity of galectin-3 in thyroid carcinomas were 99% and 98%, respectively, in a study by Bartolazzi et al. (3). In the present study, the observed specificity of galectin-3 in thyroid carcinomas was 82%. However, some researchers reported conflicting results with the use of the galectin-3-expression test in the diagnosis of follicular thyroid proliferations

Table 2. Sensitivity, specificity, PPV, NPV, and diagnostic accuracy of immunohistochemical markers for papillary thyroid carcinoma

HBME-1 CK19 Galectin-3

Sensitivity 64% 93% 92%Specificity 88% 94% 82%

PPV 0.90 0.96 0.89NPV 0.60 0.89 0.78

Diagnostic accuracy 73% 93% 84%

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(25,26). According to Bartolazzi et al., the controversy may result from methodological issues. According to these researchers, optimum galectin-3-expression analysis requires formalin-fixed and paraffin-embedded cytological preparations and a biotin-free immunohistocytochemical detection method. It is almost impossible to correctly perform galectin-3 immunostaining using conventional cytological smears (3).

HBME-1, CK19, and galectin-3 are functional markers for diagnosing PTC using paraffin cell block material. Diffuse CK19 and/or galectin-3 immunopositivity confirms papillary cell differentiation. The sensitivity of HBME-1 is lower than that of CK19 and galectin-3 in diagnosis of PTC. We recommend the use of this immunostaining panel in cases with features prompting suspicion of PTC.

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