Journal of Microscopy and Ultrastructure 1 (2013) 8–16

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Journal of Microscopy and Ultrastructure

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Original article

Osteopontin cytoplasmic immunoexpression is a predictor of poordisease-free survival in thyroid cancer

Wafaey Gomaaa,b,∗, Mahmoud Al-Ahwalc, Osman Hamourd, Jaudah Al-Maghrabia,e

a Department of Pathology, Faculty of Medicine, King Abdulaziz University, Jeddah, Saudi Arabiab Department of Pathology, Faculty of Medicine, Minia University, El Minia, Egyptc Department of Medicine, Faculty of Medicine, King Abdulaziz University, Jeddah, Saudi Arabiad Department of Surgery, King Faisal Specialist Hospital and Research Centre, Jeddah, Saudi Arabiae Department of Pathology, King Faisal Specialist Hospital and Research Centre, Jeddah, Saudi Arabia

a r t i c l e i n f o

Article history:Received 23 May 2013Accepted 4 July 2013

Keywords:ThyroidNormalThyroiditisGoitreAdenomaCarcinomaSurvivalOsteopontinImmunohistochemistry

a b s t r a c t

Background: Osteopontin (OPN) is expressed in various malignancies and may play animportant role in tumorigenesis, tumour invasion, and metastasis in various malignanciesincluding thyroid cancers. The objective of this study was to investigate the relationshipbetween OPN immunoexpression and clinicopathological characteristics in thyroid lesions.Material and methods: Paraffin blocks belonging to 160 patients with thyroid neoplasmswere retrieved from the archive of the Department of Pathology at King Abdulaziz Univer-sity, and King Faisal Specialist Hospital, Jeddah, Saudi Arabia. Immunohistochemistry wasperformed using anti-OPN antibody. Statistical tests were used to determine the relationof OPN immunoexpression to clinicopathological characteristics and survival.Results: Immunostaining results showed that OPN was localised in the cytoplasm andnucleus with higher cytoplasmic expression. Cytoplasmic and nuclear OPN was higher inthyroid cancer than other lesions. Microcarcinoma variant of papillary thyroid carcinomashowed a lower OPN cytoplasmic level than other variants. OPN cytoplasmic expressionwas not associated with most of the clinicopathological parameters tests. However, OPNcytoplasmic overexpression was associated poor survival outcome (p < 0.001).Conclusion: Upregulation of cytoplasmic OPN was associated with poor survival outcome

and was an independent predictor of margin involvement and recurrence. Nuclear OPNexpression was lower than cytoplasmic and was associated with extrathyroid extensionof thyroid cancer. OPN plays a role in thyroid carcinoma and incoming research has to befocused on the mechanistic association with invasion and metastasis.

© 2013 The Saudi Society of Microscopes. Production and hosting by Elsevier Ltd.

∗ Corresponding author at: Department of Pathology, King AbdulazizUniversity, P.O. Box 80205, Jeddah 21589, Saudi Arabia.Tel.: +966 2 6401000x21077.

E-mail address: wafgom@yahoo.com (W. Gomaa).Peer review under responsibility of Saudi Society of Microscopes.

2213-879X © 2013 The Saudi Society of Microscopes. Production andhosting by Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.jmau.2013.07.001

All rights reserved.

1. Introduction

Thyroid gland tumours are the most common neo-plasms of endocrine glands and head and neck region[1,2]. The incidence of thyroid malignancy is rapidlyincreasing [3]. Three different types of thyroid cancerhave been histologically identified: differentiated thyroidcancer (DTC) derived from thyroid follicular epithelialcells, medullary thyroid cancer (MTC); and anaplastic

thyroid cancer (ATC). DTC represent about 90% of thyroidcancers of which papillary thyroid carcinoma (PTC) isthe most frequent (75%), followed by follicular thyroidcarcinoma (FTC) (10%), Hurthle cell carcinoma (HCC)

oscopy and Ultrastructure 1 (2013) 8–16 9

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Table 1Patients’ clinicopathological characteristics.

Parameter Category n (%)

Gender (n = 160) Female 128 (80%)Male 32 (20%)

Age (n = 160, median 39 (range9–93)

<45 years 107 (66.9%)

≥45 years 53 (33.1%)Histological typing of tumours

(n = 160)Malignant 117 (73.1%)

Benign 43 (26.9%)Background thyroid (n = 108) Normal thyroid 57 (52.8%)

Goitre 34 (31.5%)Thyroiditis 17 (15.7%)

Extrathyroid extension (n = 117) Absent 95 (81.2%)Present 22 (18.8%)

Multifocality (n = 117) Absent 93 (79.5%)Present 24 (20.5%)

Lymphovascular invasion (n = 117) Absent 104 (88.9%)Present 13 (11.1%)

Capsular invasion (n = 117) Absent 92 (78.6%)Present 25 (21.4%)

Primary tumour (n = 117) Not available 1 (0.8%)pTx 5 (4.3%)pT1 45 (38.5%)pT2 19 (16.25%)pT3 28 (23.9%)pT4 19 (16.25%)

Stage (n = 117) 1 65 (55.6%)2 28 (23.9%)3 1 (0.8%)4 23 (19.7%)

Nodal metastasis (n = 117) Absent 86 (73.5%)Present 31 (26.5%)

Distant metastasis (n = 117) Absent 83 (70.9%)Present 34 (29.1%)

Margin status (n = 117) Free 76 (65%)Involved 41 (35%)

Recurrence (n = 117) Negative 105 (89.7%)Positive 12 (10.3%)

Status at end point (n = 117) Alive 83 (70.9%)

W. Gomaa et al. / Journal of Micr

5%), and poorly differentiated thyroid carcinoma (1–6%).n the other hand, MTC represents approximately 10%

hyroid tumours and ATC nearly 1% [4,5]. Tumours derivedrom thyroid follicular epithelium represent a model of

alignant transformation [6]. The transformation involveson-neoplastic lesions, benign lesions and malignantumours including PTC, FTC and ATC [7,8].

In Saudi Arabia, thyroid cancer (TC) is the fourth mostommon type of malignancy representing 6.4% of alleported cancers. PTC constitutes a large volume of thy-oid cancers followed by FTC. The median age at diagnosisas 44 years among males (range 4–86 years) and 37 years

mong females (range 8–99 years). The male to female ratioas 1:4. [9].

Osteopontin (OPN) is a secreted and calcium bindinghosphorylated glycoprotein and expressed constitutively

n a limited number of normal tissues, stress-responsivehysiological conditions, and abnormally elevated in someathological conditions [10]. OPN is considered as cytokinehat regulates cell pathways in the immune system [10,11].PN was also expressed in various malignancies and maylay important role in tumorigenesis, tumour invasion, andetastasis in various malignancies [12–22]. In addition, in

ome malignancies OPN expression was associated withoor prognosis [15]. There are few studies regarding thexpression of OPN in thyroid cancer [2,3,17,23–26]. Somef these studies reported OPN expression as a part of aulti-organ study while others focused in OPN expression

n relation to tumour characteristics.The volume of publications on the subject of OPN in

ancer diagnosis is substantially large, however the lit-rature is inconclusive. In addition, there is a growingody regarding OPN expression in thyroid carcinoma espe-ially PTC. However, its role in prognostication has noteen fully studied. To increase our understanding of OPNole in thyroid cancer, OPN immunoexpression in a sub-et of thyroid neoplasms was studied as regards relationo tumour differentiation, invasion, metastasis, recurrencend disease-free survival.

. Materials and methods

.1. Patients

The study involved 160 thyroid neoplasms (in the periodrom 1997 to 2010). Paraffin blocks and patients’ data wereetrieved from the archive of the Department of Pathol-gy at King Abdulaziz University, and King Faisal Specialistospital & Research Centre, Jeddah, Saudi Arabia. From

he surgical specimens of 160 patients, distinct samplesere obtained including; 43 benign and 117 malignant.

n 108 (67.5%) a background thyroid tissue was present.ata on tumour characteristics, prognostic factors, and

ollow up were obtained from surgical pathology reportsnd patients’ records. This data is presented in Tables 1nd 2. Staging was done according to the American Jointommittee on Cancer (AJCC) [27]. The female to male ratio

or malignant cases (117) was 3:1 and age distribution was9 (67.5%) under 45 years while 38 (32.5%) for 45 years orbove. All patient samples and related data analyses wereerformed after approval by the Research Committee of

Dead 34 (29.1%)

the Biomedical Ethics Unit, Faculty of Medicine, King Abdu-laziz University and the ethical review board of King FaisalSpecialist Hospital and Research Centre.

2.2. OPN immunostaining

Paraffin blocks of tumours were cut at 4 �m, andmounted on positive-charged slides (Leica MicrosystemsPlus Slides). Sections were deparaffinised in xylene andrehydrated in an automated immunostainer (BenchMarkXT, Ventana® Medical Systems Inc., Tucson, AZ, USA).Pre-treatment was done using CC1 (prediluted cell condi-tioning solution) for 60 min. Anti-human rabbit anti-OPNpolyclonal antibody (SpringTM Bioscience; Cat # E3281)was incubated at 37◦c for 20 min. Ventana® I-view DABdetection kit was used according to kit manufacturerinstructions. Subsequently, slides were washed, counter-stained with Mayer’s haematoxylin and mounted. Negativecontrol (substitution of primary antibody with Tris-

buffered saline) and positive control slides (normal breasttissue previously known to be positive to OPN) wereincluded.

10 W. Gomaa et al. / Journal of Microscopy and Ultrastructure 1 (2013) 8–16

Table 2Histological subtyping of thyroid neoplasms included in the study.

Category Subtype n (%)

Malignant tumours (n = 117) Papillary thyroid carcinoma (PTC) 105/117 (89.7%)Classic PTC 51/105 (48.6%)Microcarcinoma variant 23/105 (21.9%)Follicular variant (PTC-FV) 22/105 (20.95%)Microcarcinoma – follicular variant 6/105 (5.7%)Oncocytic variant 1/105 (0.95%)Hurthle cell variant 1/105 (0.95%)Insular variant 1/105 (0.95%)

Hurthle cell carcinoma 6/117 (5.1%)Anaplastic thyroid carcinoma 2/117 (1.8%)Medullary thyroid carcinoma 1/117 (0.8%)Follicular thyroid carcinoma 3/117 (2.6%)

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enoma

Benign tumours (n = 43) Follicular aden

Hurthle cell ad

2.3. Interpretation of OPN immunostaining

The expression of OPN was examined in thyroid neo-plasms and in background thyroid tissues. In order toevaluate OPN extent (%) of cytoplasmic and/or nuclearimmunostaining; cells showing cytoplasmic, or nuclearstaining were regarded as positive cells. All available cellsin each section were counted at microscope magnifica-tion 200×. Positive cells for OPN immunostaining werecounted. The mean values of positivity were calculatedand expressed as percentage in relation to total cells forboth cytoplasmic and nuclear immunostaining. Assess-ment of immunostaining intensity was performed in asemi-quantitatively. The fractions of percentage of posi-tive cells for OPN were divided as follows; (1) 0–25%, (2)26–50%, (3) 50–100%. For cytoplasmic immunostaining;3 (heavy and intense brown immunostaining), 2 (brownimmunostaining lighter than 3), 1 (brown immunostain-ing is weak), and 0 (no brown immunostaining). Nuclearstaining intensity was scored as: 3 (no blue areas of thenuclei are seen through brown staining), 2 (scarcely bluenuclear staining is seen through brown staining), 1 (blueareas of nuclei are clearly seen through brown staining),and 0 (only blue nuclear staining is seen). A 6-scale scoringsystem to categorise OPN expression was used by combi-nation of intensity and extent. For the statistical analysis,an OPN immunostaining score of 1–3 was considered aslow expression, and an OPN immunostaining score of 4–6was considered as high expression.

2.4. Statistical analysis

Differences between two groups of patients on one vari-able were tested by using Mann Whitney test. To testassociation procedure in three groups of patients on oneindependent variable the Kruskal Wallis test was used.Wilcoxon signed rank test is used to test differencesbetween two related groups of paired continuous vari-ables Binary logistic regression analysis was used to predictlymph node metastasis, recurrence, and distant metasta-

sis in relation immunoexpression of OPN. Estimated oddsratio [exponential (B)], 95% confidence interval (CI) forexp(B), and significance denoted for each analysis. For pur-pose of binary logistic analysis clinical stage and pT were

37/43 (86%)6/43 (14%)

dichotomised as low (category 1&2), and high (category3&4). The Kaplan–Meier procedure was used to calculatethe survival probabilities and the Log Rank test was usedto compare the difference between survivals. The end-point for patients was death from tumour (disease-free).Disease-free survival (DFS) was calculated as the time fromdiagnosis to the appearance of recurrent disease (or datelast seen disease-free). Statistical procedures were per-formed using SPSS® Release 16.0. Statistical significancewas determined at p-value of ≤0.05 and was 2-sided.

3. Results

3.1. OPN cytoplasmic immunoexpression

OPN immunoexpression was observed in the cytoplasmof ductal cells of normal breast (positive control). Strongpositive staining in colloid as well as macrophages andlymphocytes was seen in nodular goitre, Hashimoto’s thy-roiditis, and in neoplastic lesions. Also, strong positivestaining in Psammoma bodies of PTC. Cytoplasmic OPNimmunoexpression was observed in follicular cells normalthyroid, background nodular goitre, Hashimoto’s thyroidi-tis, and in thyroid neoplasms. Representative sections areshown in Figs. 1 and 2. OPN cytoplasmic expression wassignificantly higher than nuclear expression in all lesionsexamined (details are shown in Table 3). In malignanttumours, cytoplasmic expression was significantly higherthan in other lesions. Benign tumours showed significantlya higher expression than thyroiditis, goitre and normal thy-roid. Thyroiditis and goitre showed a significantly highercytoplasmic expression than in normal thyroid. On theother hand, there was no statistically significant differ-ence in cytoplasmic expression between thyroiditis andgoitre (Tables 3 and 4). There was no statistically signifi-cant difference between cytoplasmic expression betweenPTC and other malignant subtypes (p = 0.203). For PTCsubtypes, OPN expression in classic variant was signifi-cantly higher than in microcarcinoma variant (p = 0.05).Otherwise, there was no statistically significant differ-

ence between other subtypes. There was no statisticalsignificant difference between OPN cytoplasmic expres-sion in follicular adenoma) and Hurthle cell adenoma(p = 0.08).

W. Gomaa et al. / Journal of Microscopy and Ultrastructure 1 (2013) 8–16 11

Fig. 1. OPN immunoexpression in benign thyroid lesions. (A) OPN labelling in background normal thyroid tissue. OPN is expressed in the cytoplasm andnucleus of follicular cells [arrow]. Patchy strong staining is also observed within colloid (200×). (B) OPN labelling in background nodular goitre. Cytoplasmicand nuclear [arrow] labelling is shown in follicular cells (200×). (C) OPN labelling in background Hashimoto’s thyroiditis. Cytoplasmic labelling is observedi elling isa us sectiol omogen

3c

sTcti

TD

n

n follicular epithelium and in lymphocytes and macrophages. Nuclear labdenoma. Cytoplasmic staining is more prevalent and intense than previoabelling was done using anti-OPN antibody, diaminobenzidine as the chr

.2. Relation of OPN immunoexpression in malignantases to clinicopathological parameters

Details of the relation of OPN cytoplasmic expres-ion with clinicopathological parameters are presented in

ables 5 and 6. OPN cytoplasmic expression was not asso-iated with most of the clinicopathological parametersests. However, statistically there was a narrow signif-cant difference in some parameters; age, multifocality,

able 3istribution of scoring categories of OPN immunoexpression.

Low immunoexpres

Malignant tumours(n = 117)

C 47 (40.2%)

N 112 (94.7%)

Benign tumours(n = 43)

C 35 (81.4%)

N 42 (97.7%)

Thyroiditis (n = 17)C 17 (100%)

N 17 (100%)

Goitre (n = 34)C 34 (100%)

N 34 (100%)

Normal thyroid (n = 57)C 57 (100%)

N 57 (100%)

(%) = number of cases falling in each category; C, cytoplasmic; N, nuclear.* Cytoplasmic immunoexpression is higher than nuclear immunoexpression (W

seen also in follicular cells [arrow] (200×). (D) OPN labelling in follicularns. Nuclear labelling [arrows] is also seen (200×). Immunohistochemical, and haematoxylin as counterstain.

lymphovascular invasion, and margin status. On the otherhand, OPN cytoplasmic expression showed higher expres-sion in patients who died than those alive (p = 0.012).

Binary logistic regression analysis showed that OPNcytoplasmic expression was an independent predictor of

positivity of surgical resection margins and recurrence.However, OPN was not proven to be an independentpredictor of nodal metastasis, distant metastasis, capsu-lar invasion, or lymphovascular invasion. Kaplan–Meier

sion High immunoexpression p value

70 (59.8%)<0.001*

5 (4.3%)8 (18.6%)

<0.001*1 (2.3%)0 (0%)

<0.001*0 (0%)0 (0%)

<0.001*0 (0%)0 (0%)

<0.001*0 (0%)

ilcoxn signed rank test).

12 W. Gomaa et al. / Journal of Microscopy and Ultrastructure 1 (2013) 8–16

Fig. 2. OPN immunoexpression in malignant thyroid tumours. (A) OPN labelling in PTC. OPN is intensely and widely expressed in the cytoplasm andnucleus [arrow] of malignant follicular cells (200×). (B) OPN labelling in HCC. Cytoplasmic and nuclear [arrow] labelling is shown in malignant follicularcells (200×). (C) OPN labelling in FTC. Cytoplasmic labelling is observed in malignant follicular. Nuclear labelling is seen also in malignant follicular cells[arrow] (200×). (D) OPN labelling in ATC. Cytoplasmic and nuclear labelling [arrow] are seen in anaplastic cells (200×). Immunohistochemical labellingwas done using anti-OPN antibody, diaminobenzidine as the chromogen, and haematoxylin as counterstain.

Fig. 3. Disease-free survival curve (Kaplan–Meier). (A) OPN cytoplasmic exprrank = 0.011, p = 0.916).

ession (log rank = 11.975, p < 0.001). (B) OPN nuclear expression (log

W. Gomaa et al. / Journal of Microscopy and Ultrastructure 1 (2013) 8–16 13

Table 4Differences in OPN immunoexpression in thyroid lesions examined.

OPN cytoplasmic OPN nuclear

Malignant vs. benign <0.001a 0.021a

Malignant vs. thyroiditis <0.001a <0.001a

Malignant vs. goitre <0.001a <0.001a

Malignant vs. normal <0.001a <0.001a

Benign vs. thyroiditis <0.001b 0.834Benign vs. goitre <0.001b 0.704Benign vs. normal <0.001b 0.653Thyroiditis vs. goitre 0.785 0.686Thyroiditis vs. normal 0.005c 0.462Goitre vs. normal 0.012d 0.702

Values represent pa Expression is higher in malignant tumours (cytoplasmic and malig-

nant).b Expression is benign tumours (cytoplasmic and malignant).c Expression is higher thyroiditis.

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TDc

ni

Table 6Regression analysis for OPN cytoplasmic immunoexpression.

Variable Exp(B) 95% CI for exp(B) p-Value

Nodal metastasis 1.252 0.559–2.806 0.585Distant metastasis 0.875 0.400–1.912 0.738Multifocality 0.514 0.214–1.233 0.136Surgical resection margins 0.452 0.211–0.964 0.04Capsular invasion 0.833 0.352–1.975 0.679

d Expression is higher in goitre.

urvival analyses showed that OPN cytoplasmic immuno-xpression in malignant cases had significant associationith poor disease-free survival outcome (log rank = 11.975,

< 0.001) (Fig. 3).

able 5istribution of OPN cytoplasmic immunoexpression in relation to clini-opathological parameters of thyroid carcinoma (n = 117).

Parameter Category n (%) p-value

GenderFemale 53/91 (58.2%)

0.814aMale 17/26 (65.4%)

Age<45 years 54/79 (68.4%)

0.07a≥45 years 16/38 (42.1%)

Extrathyroidextension

Absent 54/95 (56.8%)0.751a

Present 16/22 (72.7%)

MultifocalityAbsent 59/93 (63.4%)

0.06aPresent 11/24 (45.8%)

Lymphovascularinvasion

Absent 61/104 (58.6%)0.08a

Present 9/13 (69.2%)Capsularinvasion

Absent 56/92 (60.9%)0.5a

Present 14/25 (56%)

Primary tumour

Not available 0/1 (0%)

0.217b

pTx 5/5 (100%)pT1 27/45 (60%)pT2 8/19 (42.1%)pT3 21/28 (75%)pT4 9/19 (47.4%)

Stage

1 42/65 (64.6%)

0.325b2 17/28 (60.7%)3 1/1 (100%)4 10/23 (43.5%)

Nodalmetastasis

Absent 50/86 (58.1%)0.179a

Present 20/31 (64.5%)Distantmetastasis

Absent 50/83 (60.2%)0.095a

Present 20/34 (58.8%)

Margin statusFree 51/76 (67.1%)

0.07aInvolved 19/41 (46.3%)

RecurrenceNegative 60/105 (57.1%)

0.131aPositive 10/12 (83.3%)

Status at endpoint

Alive 41/83 (49.4%)0.012a

Dead 29/34 (85.3%)

(%) = number of cases showing OPN cytoplasmic overexpression whichs defined as high immunoexpression in our study.

a Mann Whitney test.b Kruskal Wallis test.

Lymphovascular invasion 1.286 0.404–4.094 0.671Recurrence 0.127 0.016–1.022 0.05Extrathyroid extension 2.113 0.761–5.871 0.151

3.3. Osteopontin nuclear immunoexpression

Nuclear OPN expression was observed in all lesionsexamined. Representative sections are shown inFigs. 1 and 2. Expression was lower than cytoplasmicexpression. In addition, expression was higher in malig-nant cases than in benign, thyroiditis, goitre, and normalthyroid. However, there was no difference betweenother lesions (Table 4). Binary logistic regression analysisshowed that OPN nuclear expression was not proven to bean independent predictor of any of the parameters testedexpect extrathyroid extension [p = 0.035] (Table 7). Therewas no association between OPN nuclear expression anddisease free survival (Fig. 3) (log rank = 0.011, p = 0.916).

4. Discussion

The histological distinction between some benigntumours and well-differentiated malignant thyroid neo-plasms has many diagnostic difficulties. Moreover, a smallgroup of patients with well-differentiated thyroid malig-nancies are subject to develop metastasis and subsequentlya more aggressive management is required [23]. Thereis an advance in understanding the molecular eventsinvolved in thyroid cancer. However, the full understat-ing of the molecular background of thyroid cancer needs tobe unveiled for better diagnostic and therapeutic interven-tions. The efforts are being raised everywhere to discovermore molecular markers of thyroid cancer for clinical appli-cation both diagnostic and prognostic.

OPN is a secreted protein and also is found intracel-lularly. OPN is involved in different pathophysiologicalsituations. The intracellular OPN has cellular functionsdifferent from the secreted OPN as involvement in

signal transduction pathways as well as cytoskeletalrearrangement and cell motility [28–30]. OPN had beenshown to be overexpressed in several tumour types [15,31]and OPN overexpression showed a strong correlation

Table 7Regression analysis for OPN nuclear immunoexpression.

Variable Exp(B) 95% CI for exp(B) p-Value

Nodal metastasis 3.984 0.636–24.957 0.14Distant metastasis 0.563 0.016–5.211 0.612Multifocality 0.511 0.206–1.263 0.146Surgical resection margins 0.466 0.048–4.125 0.477Capsular invasion 5.812 0.919–36.768 0.061Lymphovascular invasion 2.000 0.207–19.279 0.549Recurrence 2.675 0.272–26.287 0.399Extrathyroid extension 0.136 0.021–0.871 0.035

oscopy a

14 W. Gomaa et al. / Journal of Micr

between levels of OPN protein and tumour progressionin multiple tumour types from different anatomical sitesand correlated with poor disease-free and overall patientssurvival [17,32,33].

The current study involved a group of thyroid tumours(benign and malignant) together with background thy-roid tissues to characterise OPN immunoexpression. Therewas a dual subcellular OPN immunolocalisation; cytoplas-mic and nuclear. This study showed for the first timethe nuclear localisation of OPN in thyroid gland and itsrelation to patients’ outcome in malignant lesions. In ourstudy, the cytoplasmic expression was dominant thannuclear expression in all tissues examined from normalto malignant tumours. Previously, OPN was shown to beperimembranous and cytoplasmic [34–36]. In thyroid can-cers, only cytoplasmic localisation was reported and onestudy reported a cytoplasmic and membranous localisation[23]. In the current study, there was a strong immuno-staining of OPN in colloid within normal thyroid follicles,goitre, and thyroiditis in addition to psammoma bodies.OPN immunoexpression was found also in macrophagesin all lesions examined. These findings were reported asregarding psammoma bodies and macrophages in PTC[24]. The presence of OPN immunolocalisation in colloidand in psammoma bodies supports OPN role in thyroidphysiological and pathological conditions. Our series of fol-licular adenoma and Hurthle cell adenoma is a large seriesreporting OPN expression than previous reports [3,23].OPN immunoexpression in adenoma was higher than inthyroiditis, goitre, and normal thyroid which is similarto reported previously [23]. In this study, OPN cytoplas-mic immunoexpression was significantly higher in primarythyroid carcinoma than in other benign lesions and nor-mal thyroid tissues. This stepwise increase from normalto thyroid carcinoma is consistent with previous reports[2,3,23,26]. These findings raise the possibility that OPNmay be used in differentiating benign thyroid lesions frommalignant and supports than OPN overexpression is asso-ciated with tumorigenesis.

In our study, PTC constitutes the major bulk of malig-nant cases. In a recent study by Kang et al., OPN expressionwas lower in FV-PTC than other subtypes, and subse-quently, they suggested that FV-PTC variant has betterprognosis [3]. This notion was previously reported by Guar-ino and his colleagues [26]. In the current study, OPNcytoplasmic immunoexpression was higher in classic PTCthan PTC microcarcinoma variant. PTC microcarcinoma issuggested to be an earlier stage in the papillary carci-nogenesis [37]. This variant is considered as an indolenttumour with a relatively benign course because there isprevalence up to 35% in autopsy series and is also detectedin up to 24% in total thyroidectomies for other benign con-dition [38–40]. This finding supports the relation of OPNupregulation with more aggressive variants in PTC.

In our study, the cytoplasmic expression of OPN inmalignant lesions showed no association with gender,extrathyroid extension, capsular invasion, tumour size,

stage, nodal metastasis, distant metastasis, margin status,or recurrence which was similar to that reported by Brieseet al. [23]. OPN overexpression was associated with stage,grade and progression, and poor prognosis in many cancers

nd Ultrastructure 1 (2013) 8–16

[32]. Other few studies on thyroid malignancy showeddifferent findings from ours. Sun et al., found that OPN isoverexpressed in primary tumours with nodal metastasisthan those without nodal metastasis [2,3]. Guarino et al.reported the same in addition to OPN correlation withlarger tumour size [26]. Also OPN overexpression wasreported to be prominent at the invasive front of tumouras well as necrotic areas [25]. The conflicting results maybe related using OPN expression on mRNA level, usingOPN immunostaining intensity only and in part due to toosmall sample size.

The mechanistic link between OPN and tumour growthand metastasis has been suggested and studied in differenttumours with few on PTC. OPN overexpression in tumourmetastasis is associated with more malignant phenotype[41,42]. OPN exerts its tumorigenic effects through CD44variants and/or binding to �v� integrins and promotessignalling pathways involved in tumour cell adhesion,migration, and lymph node metastasis [2,43]. In PTC celllines and tissues, OPN upregulation is characterised by thepresence of RET-RAS-MAPK rearrangements [3,26,44,45].In breast cancer, STAT3 is suggested to play an essentialrole in mediating OPN-related tumorigenesis [46]. Anti-apoptotic effect of OPN may be implicated in facilitatingtumour growth [47]. There may be a link between OPNoverexpression and induction of growth factors as thehepatocyte growth factor [48,49] and angiogenesis [50].OPN-related enhancement of tumour growth may involvematrix-degrading enzymes [51], which was supported inprostate cancer with positive correlation between matrixmetalloproteinase-9 expression and OPN [45].

Although there was no association of OPN cytoplasmicimmunoexpression and most clinicopathological features,interestingly in this study, the patients died earlier showedhigher cytoplasmic OPN immunostaining. In addition, OPNcytoplasmic overexpression is associated with poor sur-vival outcome. This finding is novel for our study andhas not been reported before. In our study, nuclear OPNexpression was significantly higher in thyroid cancer thanother lesion. This means that nuclear localisation in malig-nancy is associated with tumorigenicity. Nuclear OPN issuggested to play a role in mitosis and correlated withchromatin condensation during cell division [34]. This isthe first published work regarding the relation betweenOPN nuclear immunolocalisation and clinicopathologicalfeatures in thyroid cancer. However, there was no associa-tion of nuclear OPN expression with any of these featuresapart from extrathyroid extension.

In our perspective, the limitations of the current studywere short survival times in recently diagnosed patients.In addition, samples from nodal metastasis are better to becompared with primary tumours.

5. Conclusion

In this subset of thyroid lesions cytoplasmic OPNimmunoexpression showed a stepwise upregulation from

normal thyroid up to malignant lesions. OPN cytoplasmicexpression was not associated with most clinicopatholog-ical features; however OPN overexpression is associatedwith poor survival outcome. Nuclear OPN expression was

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W. Gomaa et al. / Journal of Micr

ess than cytoplasmic and is associated with extrathyroidxtension of thyroid cancer. The current results showedhat there may be an association between OPN overex-ression and thyroid carcinogenesis. Whether OPN plays

role or not is yet to be determined by focusing on itsechanistic association with invasion and metastasis.

onflict of interest

The authors confirm that no part of this work has beenubmitted or published elsewhere and that there are noonflicts of interest.

uthors’ contributions

WG made substantial contributions to the design ofhe study, scored osteopontin immunostaining, inter-reted data, performed statistical analysis, and drafted theanuscript. MA reviewed the clinical data and contributed

o the design of the study and revised the manuscript. OHeviewed the clinical data and contributed to the designf the study and revised the manuscript. JM performedistological examination and selection of paraffin blocks

ncluded in study, contributed to the design of the studynd revised the manuscript. The manuscript has been readnd approved by all of the authors, the requirements foruthorship have been met, and each author believes thathe manuscript represents honest work.

eferences

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