Introducere: Citokeratina-19, p53 i Ki-67 sunt civa dintre markerii propui pentru diferenierea

tumorilor tiroidiene benigne de cele maligne.

Material i metode: Am evaluat expresia tisular a celor trei markeri n 60 de leziuni tiroidiene
incluznd: 8 cazuri de hiperplazie nodular (HN), 16 adenoame foliculare (AF), 26 carcinoame
papilare (CP) i 10 carcinoame foliculare (CF). Seciunile tisulare fixate n formol i incluse la
parafin au fost supuse colorrii IHC (tehnica LSAB, vizualizare cu DAB) folosind anticorpi
monoclonali anti-CK19 (clona RCK 108), anti-Ki67 (clona MIB-1) i anti-p53 (clona DO-7); datele
au fost analizate statistic folosind testul T nepereche. Rezultate: CK19 a fost semnificativ
exprimat doar n CP20 (p<0,001). Pentru p53 i Ki-67 rezultatele analizelor statistice nu au
fost semnificative pentru nici una dintre perechile comparate.
Concluzii: CK19 este util n diferenierea variantei foliculare a CP de CF i a CP de
aspectele papilare din HN. Imunoreactivitatea p53 i Ki-67 nu s-au dovedit utile n
diferenierea CP de HN, rezultatele fiind nesemnificative statistic.
Biologie celular
Cancerul tiroidian cea mai frecvent form de neoplasm tiroidian, reprezint
1% din totalul tumorilor umane, fiind mai frecvent la femei. Rata incidenei
standardizate la 100.000 de locuitori variaz ntre 0,8-5% la brbai i 1,9-19,4%
la femei. n ultimele decade s-a observat o cretere a incidenei carcinoamelor
tiroidiene, mai ales a celor mici papilare, cu un prognostic favorabil.
Tumorile nodulare cu arhitectur predominant folicular sunt cele mai
frecvete leziuni ale glandei tiroide. n majoritatea cazurilor diagnosticul
acestor leziuni se face fr dificultate, pe baza datelor clinice i a aspectelor
histologice.Totui, exist situaii cnd anumii nodului tiroidieni au aspecte
nucleare subtile sau atenuate, cnd diferena ntre leziunile benigne (cum ar
fi noduluii circumscrii cu arhitectur folicular) i cele maligne este dificil
n absena comportamentului agresiv. n lipsa criteriilor de invazie vasculare
i/sau capsulare adenomul folicular este greu de difereniat de carcinomul
folicular. ntre markerii propui pentru a discerne ntre leziunile tiroidiene
benigne i cele maligne se numr: galactina-3, citokeratina-19 (CK19), p53,
Ki-67 i bcl-2.

CK19 este o citokeratin cu greutate molecular mar, prezent n epiteliile simple

sau glandulare i n tumori.Unii autori pretend c la nivelul tiroidei expresia CK19
se limiteaz la cazul carcinoamelor papilare, gsind-I utilizarea n diagnosticul
diferenial ntre carcinomul follicular i varianta folicular a carcinomului tiroidian.
Carcinoamele papilare prezint o imunoreactivitate intens i difuz pentru CK18 i
CK19 n 80-100% din cazur. imilar 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

1
hyperplasia and to establish their utility in differentiating benign from
malignant thyroid lesions (derived from follicular cells).
MATERIAL AND METHODS
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 (PC20) and 4 cases under
20 years of age (PC<20). Seven (70%) of the 10 follicular carcinomas were
minimally invasive, six of the PC20 (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 (3,3diaminobenzidine).
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 G 0

phase. After pretreatment by boiling 60 minutes at 90C 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 90C 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:

2
 0 positive immunoreaction in less than 5% of tumor cells;

3
 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 PC20 and
follicular carcinoma, PC20 and follicular adenoma, PC20 and nodular
hyperplasia, follicular carcinoma and follicular adenoma, PC20 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)

4
Figure1. Follicular variant of papillary carcinoma (PC<20) CK19 positive immunoreactions

5
Figure 2. Macrofollicular variant of papillary carcinoma (PC>20) CK19+.
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.

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Figure 3. Papillary microcarcinoma (PMC>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 PC20 (p<0.001). (Table 4)

7
8
Figure 4. Classic papillary carcinoma Ki-67 positive immunoreactions.
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.

Figure 5. Expression of Ki-67, p53 and CK19 in the studied lesions.

Ki-67 expression
Ki-67 antigen was expressed in 4 of 22 (18%) PC20, 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

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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. _____________________________ Viorel Stan et al 63

10
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

11
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._____________________________ 64 TMJ 2011,
Vol.61 , No.1 - 2

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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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thyroid tumors. Pol J Pathol 2004;55:133-41.
7. Beesley MF, McLaren KM. Cytokeratin 19 and galectin-3 immunohistochemistry in the differential
diagnosis of solitary thyroid nodules. Histopathology 2002;41:236-43.
8. Nasir A, Catalano E, Calafati S, et al. Role of p53, CD44V6 and CD57 in differentiating between
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9. Muller-Hocker J. Immunoreactivity of p53, Ki-67 and Bcl-2 in oncocytic adenomas and carcinomas of
the thyroid gland. Hum Pathol 1999;30:926-33.
10. Siironen P, Nordling S, Louhimo J, et al. Immunohistochemical expression of Bcl-2, Ki-67 and p21 in
patients with papillary thyroid cancer. Tumour Biol 2005;26:50-6.
11. Augustynowicz A, Dzieciol J, Barwijuk-Machala M, et al. Assessement of proliferative activity of
thyroid Hurthle cell tumors using PCNA, Ki-67 and AgNOR methods. Folia Histochem Cytobiol
2004;42:165-68.
12. Lam KY, Lui MC, Lo CY. Cytokeratin expression profiles in thyroid carcinomas. Eur J Surg Oncol
2001;27:631-5.
13. Cerilli LA, Mills SE, Rumpel CA, et al. Interpretation of RET immunostaining in follicular lessions of
the thyroid. Am J Clin Pathol 2002;118:186-93.
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Saunders, 2005;302-3.
16. Nishida T, Nakao K, Hamaji M, et al. Stepwise participation of p53 gene mutation during
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17. Hosal SA, Apel RL, Freeman JL, et al. Immunohistochemical localization of p53 in human thyroid
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13
 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 PC20 and
follicular carcinoma, PC20 and follicular adenoma, PC20 and nodular
hyperplasia, follicular carcinoma and follicular adenoma, PC20 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)

14