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Original Articles
NUMBER 1-2 YEAR 2011
The Value of Ck19, Ki-67 and P53 Expression in the Diagnosis of Thyroid Follicular Neoplasms
1 County Hospital, Resita,
2 Department of Pathology,
3 Department of Endocrinology, Victor Babes University of Medicine and Pharmacy, Timisoara
Correspondence to:
Marioara Cornianu, Steaua Str., Bl. 15/D, Apt. 6, Timisoara, Tel. +40-748-331252.
Email: marioaracornianu@yahoo.com
REZUMAT

Introducere: Citokeratina-19, p53 si Ki-67 sunt cativa dintre markerii propusi pentru diferentierea tumorilor tiroidiene benigne de cele maligne. Material si metode: Am evaluat expresia tisulara a celor trei markeri în 60 de leziuni tiroidiene incluzând: 8 cazuri de hiperplazie nodulara (HN), 16 adenoame foliculare (AF), 26 carcinoame papilare (CP) si 10 carcinoame foliculare (CF). Sectiunile tisulare fixate în formol si incluse la parafina au fost supuse colorarii IHC (tehnica LSAB, vizualizare cu DAB) folosind anticorpi monoclonali anti-CK19 (clona RCK 108), anti-Ki67 (clona MIB-1) si anti-p53 (clona DO-7); datele au fost analizate statistic folosind testul T nepereche. Rezultate: CK19 a fost semnificativ exprimata doar în CP=20 (p<0,001). Pentru p53 si Ki-67 rezultatele analizelor statistice nu au fost semnificative pentru nici una dintre perechile comparate. Concluzii: CK19 este utila în diferentierea variantei foliculare a CP de CF si a CP de aspectele papilare din HN. Imunoreactivitatea p53 si Ki-67 nu s-au dovedit utile în diferentierea CP de HN, rezultatele fiind nesemnificative statistic.
Cuvinte cheie: tiroida, carcinom papilar, CK19

ABSTRACT

Introduction: Cytokeratin-19 (CK19), p53 and Ki-67 are some of the proposed markers for differentiating benign from malignant thyroid tumors. Material and methods: We evaluated the tissue expression of the three markers in 60 thyroid lesions including: 8 cases of nodular hyperplasia (NH), 16 follicular adenomas (FA), 26 papillary carcinomas (PC) and 10 follicular carcinomas (FC). Tissue sections fixed in formalin and embedded in paraffin were immunohistochemically (IHC) stained (LSAB technique, visualization with DAB) using monoclonal antibodies anti-CK19 (clone RCK 108), anti-Ki67 (clone MIB-1) and anti-p53 (clone DO-7); the data were statistically analyzed using the unpaired T-test. Results: CK19 was significantly expressed only in PC≥20 (p<0.001). For p53 and Ki-67 the results of statistical analysis were not significant for the compared pairs. Conclusions: CK19 is useful in differentiating the follicular variant of PC from FC and PC from the papillary aspects in NH. Immunoreactivity of p53 and Ki-67 was not proven to be useful in differentiating PC from NH, the results being statistically insignificant.
Key Words: thyroid, papillary carcinoma, CK19
INTRODUCTION

Thyroid cancer – the most common thyroid neoplasm, accounts for 1% of all the tumors of the human body, being found more frequently in women.1 The rate of incidence standardized at 100,000 people varies between 0.8-5% in men and 1.9-19.4% in women. In the last decades, an increase in the incidence of thyroid carcinomas was observed, especially of small papillary carcinomas, with a favorable prognosis.
Nodular tumors with a predominantly follicular pattern are the most common lesions of the thyroid gland. In the majority of cases, the diagnosis of these lesions can be made without difficulties, on the basis of clinical data and histological aspects. However, there are situations when some thyroid nodules have subtle or attenuated nuclear features, differentiation between benign (like circumscribed nodules with follicular architecture) and malignant lesions being difficult in the absence of invasive behavior. In the absence of vascular and/or capsular invasion criteria, follicular adenoma is not easily differentiated from follicular carcinoma.2 Among the proposed markers for differentiating benign from malignant thyroid lesions are included: galectin-3, cytokeratin-19 (CK19), p53, Ki-67 and bcl-2.3-11
CK19 is a high molecular weight cytokeratin, expressed in simple or glandular epithelium and in tumors. Some authors claim that in the thyroid gland, the expression of CK19 is limited to papillary carcinoma, finding it useful in differentiating follicular carcinoma from the follicular variant of papillary carcinoma.7 Papillary carcinomas express intense and diffuse immunoreactivity for CK7, CK18 and CK19 in 80-100% of cases. Similar but less intense staining patterns can be also observed in the follicular variant of papillary carcinoma. In poorly differentiated carcinomas, CK19 is less expressed (40%).12 Because the expression of CK19 is usually focal and less intense in follicular adenomas and carcinomas than in papillary carcinomas, this keratin is used most frequently in the investigation of thyroid lesions.13,14
P53 is a well known tumor suppressor gene, and its mutation is the most common genetic alteration found in human tumors.15 Mutations of p53 represent a late genetic event in thyroid carcinogenesis. As a result, p53 accumulation can be immunohistochemically (IHC) detected especially in anaplastic and poorly differentiated thyroid carcinomas and rarely in papillary and follicular well differentiated carcinomas, as well as in medullar carcinomas. Positive p53 immunoreactivity is an independent prognostic factor for the survival of patients with thyroid cancer.16,17
Ki-67 antigen corresponds to a nuclear nonhistone protein that is expressed by cells in the proliferative phase.18
The aim of this study is to assess the expression of CK19, Ki-67 and p53 in papillary carcinoma, follicular carcinoma, follicular adenoma and nodular hyperplasia and to establish their utility in differentiating benign from malignant thyroid lesions (derived from follicular cells).


MATERIAL AND METHOD

For testing the value of these markers in the differential diagnosis between benign and malignant thyroid lesions, we evaluated the expression of CK19, p53 protein and Ki-67 antigen on a group of 60 thyroid lesions including: nodular hyperplasias (NH) (n = 8 cases), follicular adenomas (FA) (n = 16 cases), papillary carcinomas (PC) (n = 26 cases) and follicular carcinomas (FC) (n = 10 cases).
We compared the expression of these markers according to the age of patients with papillary carcinoma: 22 cases over 20 years old (PC≥20) and 4 cases under 20 years of age (PC<20). Seven (70%) of the 10 follicular carcinomas were minimally invasive, six of the PC≥20 (23.27%) were follicular variants of papillary carcinoma and four were papillary microcarcinomas.
Tissue sections fixed in formalin and embedded in paraffin were IHC stained using the LSAB (Labeled Streptavidin Biotin) technique, visualization with DAB (3,3'–diaminobenzidine).
Tissue expression of CK19 was analyzed using the anti-CK19 antibody, clone RCK 108, IgG1 isotype – a mouse monoclonal antibody that reacts with a 40kDa protein corresponding to CK19. Prior enzymatic predigestion, incubation with diluted primary antibody (1:200) and visualization with DAB enabled the highlighting of the final reaction product, brown-colored, with membrane and cytoplasmic localization.
Anti-Ki67 mouse monoclonal antibody, clone MIB-1 (Zymed, CA, USA) recognizes a cellular proliferation nuclear antigen (a pair of proteins with molecular weights of 345 and 395 kDa, respectively) expressed in all cell cycle phases, except the G0 phase. After pretreatment by boiling 60 minutes at 90ºC in Retrieval solution, tissue sections were stained by incubation with diluted MIB-1 antibody; we obtained a nuclear immunostaining, the final reaction product having a brown color.
We analyzed the expression of p53 protein using anti-p53 mouse monoclonal antibody, clone DO-7 (DAKO, Glostrup, Denmark), that recognizes the wild and mutant form of p53 protein, a 53kDa nuclear phosphoprotein that takes part in the regulation of cell cycle, being encoded by p53 gene. We used the LSAB method, with pretreatment of sections by boiling 30 minutes at 90ºC in Retrieval solution (DAKO Target Retrieval Solution), incubation with diluted primary antibody at room temperature, visualization of reaction product with DAB and counterstain with hematoxilin.
After examining several microscopic fields and quantifying immunoreactions, the results were quantitatively expressed according to the percentage of positive tumor cells:
• 0 – positive immunoreaction in less than 5% of tumor cells; • 1(+) – immunostaining of 5-30% of tumor cells;
• 2(+) – immunostaining of more than 30% of tumor cells.
Score 0 was considered negative and scores 1(+) and 2(+) were considered positive.
According to the result of immunoreaction (positive or negative), the studied lesions were then divided into 2 groups: the "negative" group and the "positive group".
The expression of the three markers was compared between PC≥20 and follicular carcinoma, PC≥20 and follicular adenoma, PC≥20 and nodular hyperplasia, follicular carcinoma and follicular adenoma, PC≥20 and PC<20 and between follicular carcinoma and nodular hyperplasia, using the unpaired T test.

RESULTS

The results of IHC staining and statistical analysis are presented in Tables 1- 4.
CK19 expression
We evaluated the IHC expression of CK19 in tumor tissue and surrounding thyroid parenchyma. CK19 was expressed in 21 (80.7%) of the 26 PC studied (occult, intra- and extrathyroid), immunoreactivity (membrane and diffusely cytoplasmic) concerning >80% of tumor cells, with immunostaining of variable intensity from case to case. (Table 1, Figs. 1,2)
We observed a positive CK19 staining pattern, similar for papillary microcarcinomas and clinically manifested PC, with or without distant metastases. (Fig. 3) Five of nine PC with lymph node metastases expressed CK19 in the metastasis, without differences (in the intensity of staining) between primary tumors and metastases. We noted positive CK19 expression with the same staining intensity in both early-stage and advanced-stage with aggressive clinical behavior PC.
Table 1. Expression of CK19.
Table 2. Expression of p53.

Figure1. Follicular variant of papillary carcinoma (PC<20) CK19 positive immunoreactions.
Figure 2. Macrofollicular variant of papillary carcinoma (PC>20) CK19+.
Weak immunoreactivity for CK19 was observed in one of ten cases (10%) of FC, in two of 16 cases (12.5%) of FA (Fig. 4), in two of eight cases (25%) of NH and also focally in the non-neoplastic thyroid tissue surrounding areas of chronic inflammation.
The results of statistical analysis using the unpaired T test showed that CK19 was significantly expressed only in PC≥20 (p<0.001). (Table 4).
Table 3. Expression of Ki-67.
Table 4. Results of the statistical analysis.

Figure 3. Papillary microcarcinoma (PMC>20) CK19 +.
Figure 4. Classic papillary carcinoma – Ki-67 positive immunoreactions.
Figure 5. Expression of Ki-67, p53 and CK19 in the studied lesions.





P53 protein
P53 protein was expressed in two of 26 cases (7.7%) of PC, in two of ten cases (20%) of FC and it was absent in FA and NH. (Table 2) The results of statistical analysis using the unpaired T test were not significant for any of the compared pairs.
Ki-67 expression
Ki-67 antigen was expressed in 4 of 22 (18%) PC≥20, in three of ten (30%) FC, in two of 16 (12.5%) FA and it was absent in NH, the results of statistical analysis being insignificant for the compared pairs. (Fig. 4, Tables 3,4)




DISCUSSION

Cytokeratin 19, Ki-67 antigen, p53 protein, galectin-3 and bcl-2 are some of the proposed markers for the differentiation of benign from malignant thyroid nodules.
Cytokeratin 19, a high molecular weight citokeratin, is a sensitive (but not specific) marker of thyroid PC, with a high sensitivity for the classic variant and less useful in the diagnosis of follicular and oncocytic lesions with nuclear atypia.
Normal thyroid follicular epithelium is usually CK19-negative, although sometimes focal CK19 immunostaining is expressed in the normal thyroid tissue surrounding the tumor, in follicular cells from chronic lymphocytic thyroiditis and in reactive areas, often adjacent to degeneration areas from which the biopsy was taken, therefore having a reduced specificity. However, positive CK19 expression observed in "borderline" lesions helps standing for the diagnosis of PC.14
We studied the IHC expression of CK19 in 60 thyroid lesions: NH (8 cases), FA (16 cases), PC (26 cases) and FC (10 cases), to test the value of this marker in histological differentiation of benign and malignant thyroid lesions. The IHC reaction was made using the LSAB technique, with anti-CK19 mouse monoclonal antibody, clone RCK 108 (Dako, Glostrup, Denmark), visualization with DAB and counterstain with hematoxilin.
On examined sections, positive CK19 immunoreactivity was characterized by a diffuse staining pattern (membrane and cytoplasmic) with an intense immunoreaction in tumor cells, as compared to normal follicular epithelium, the number of CK-positive tumor cells and the intensity of immunostaining being variable from case to case.
We identified: (1) diffuse, moderate/intense CK19 immunostaining in 21 of the 26 (80.7%) studied PC, with cytoplasmic and membrane positive CK19 expression; (2) a similar staining pattern in microcarcinomas and clinically manifested PC, with or without involvement of cervical lymph nodes, and (3) lack of immunoreaction or weak CK19 immunostaining in one of ten FC and in the majority of studied benign lesions (NH and FA). The negative CK19 immunoreaction observed in five (19%) PC can be due to technical errors (of fixation or staining).
Yagi Y et al. noted the absence of CK19 expression differences between 11 papillary microcarcinomas (with mean diameter of 10 mm) and seven clinically manifested PC (with mean diameter of 31 mm) and they identified positive CK expression with the same staining intensity in all tumor stages.19
In their study, Beesley and McLaren point out (moderate/strong) positive CK19 immunoreactivity in all PC cases (n=26) and weak/absent immunostaining in the majority of benign lesions and in FC.7
Moon evaluated the IHC expression of cytokeratin-19, p53 and Ki-67 in PC (37 cases), FC (12 cases), FA (22 cases) and NH (23 cases).20 The results of their statistical analysis showed that CK19 is expressed only in PC, being useful in differentiating FC from the follicular variant of PC and PC from the papillary areas in NH.
Kragsterman et al. identified positive CK19 expression in all (35 cases) examined PC (occult, intra- and extrathyroid) and in 8 of 11 metastases, with variable immunoreactivity (but often intense), not observing immunostaining differences between tumors with and without metastases.21
On the basis of the results obtained, together with the positive CK immunostaining observed in FA, FC and in non-neoplastic thyroid tissue, the authors appreciate that CK19 has a limited value as a marker for the routine histopathological exam and suggest that the presence of CK19 immunoreactive cells must always be carefully examined because it arises the suspicion of a PC.
According to the results obtained – in accord with the observations of Beesley and McLaren, CK19 seems to be useful in differentiating the follicular variant of PC from FC and PC from the papillary aspects in NH. 7 The differences of CK19 expression observed in PC and FC suggest the transformation of cell type, from endocrine-type cells to nonendocrine-type cells, in PC.
P53 is a tumor suppressor gene localized on chromosome 17p13.1, with a role in cell cycle and the initiation of apoptosis as a response to DNA alteration, mutations of p53 gene being reported in over 50% of human tumors.15
Nasir et al. pointed out the presence of p53 mutations in 90% of FC with intense nuclear positive p53 expression and weak positive p53 immunostaining expressed in only 15% of FA, thus showing that IHC detection of p53 protein can be useful in differentiating FA from FC. 8 Moon et al. detected positive p53 immunoreaction in 9.1% of FA and in 16.7% of FC, with moderate to intense immunostaining, the results being statistically insignificant.20
In our study, only two of 26 PC (7.7%) and two of ten (20%) FC presented focal, mild/moderate positive p53 expression.
Ki-67 is a nuclear protein expressed by cells in proliferative phases G1,G2 M and S, being known that there is a correlation between Ki-67 immunoreaction and mitotic activity.18 In the study of Muller-Hocker and Augustynowicz, the proliferative activity appreciated through the evaluation of Ki-67 immunoreaction is significantly higher in oncocytic carcinomas than in adenomas.9,10
In our cases, four of 26 (18%) PC, three of ten (30%) FC and two of 16 (12.5%) FA presented positive Ki-67 immunoreactivity, the results of statistical analysis being insignificant.

CONCLUSIONS

The results obtained suggest that CK19 has limited value as a marker for the routine histopathological exam. CK19 seems to be useful in differentiating the follicular variant of PC from FC and PC from the papillary aspects in NH. The presence of CK19 immunoreactive cells arise the suspicion of a PC, thus requiring a careful examination. Immunostaining for p53 and Ki-67 did not prove to be significantly useful in differentiating PC from NH, the value of these markers in the differential diagnosis could be appreciated by extending the study to a larger number of cases.

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