Original Research Article

Visfatin/pre-B cell colony-enhancing factorimmunohistochemical overexpression in oralcancers

Li-Wen Lin a, Yung-Chuan Lu b, Chao-Ping Wang c, Chia-Chang Hsu d,Li-Fen Lu e, Ming-Che Hsin f, I.-Ting Tsai g, Fu-Mei Chung c,Jer-Yiing Houng h, Yau-Jiunn Lee i, Chih-Yu Chen a,*aDepartment of Dentistry, E-Da Hospital, I-Shou University, Kaohsiung 82445, TaiwanbDivision of Endocrinologic, E-Da Hospital, I-Shou University, Kaohsiung 82445, TaiwancDivision of Cardiology, E-Da Hospital, I-Shou University, Kaohsiung 82445, TaiwandDivision of Gastroenterology and Hepatology, Department of Internal Medicine, E-Da Hospital, I-Shou University,Kaohsiung 82445, TaiwaneDivision of Cardiac Surgery, E-Da Hospital, I-Shou University, Kaohsiung 82445, TaiwanfDivision of General surgery, Department of Surgery, E-Da Hospital, I-Shou University, Kaohsiung 82445, TaiwangDepartment of Emergency, E-Da Hospital, I-Shou University, Kaohsiung 82445, TaiwanhDepartment of Medical Nutrition, Institute of Biotechnology and Chemical Engineering, I-Shou University, Kaohsiung82445, Taiwani Lee's Endocrinologic Clinic, Pingtung 90000, Taiwan

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a r t i c l e i n f o

Article history:

Received 17 June 2014

Received in revised form

13 August 2014

Accepted 18 August 2014

Available online 28 August 2014

Keywords:

Visfatin

Oral squamous cell carcinoma

Hyperplasia

Proliferating cell nuclear antigen

CD68

a b s t r a c t

Increased visfatin expression has been shown to increase gene expression, which promotes cell

survival and increases SirT1 activity thereby promoting angiogenesis. Previous studies have

shown that oral squamous cell carcinomas (OSCCs) express high levels of activated signal

transducer and activator of transcription 3 (Stat3). Since visfatin expression is increased by Stat3,

we hypothesized that visfatin protein may be highly expressed in OSCCs. Immunohistochemis-

try was the technique used to examine the expression of visfatin in 19 OSCCs and 4 hyperplastic

lesions. The results indicated that visfatin was detected in the cytoplasm and nuclei of the OSCCs

and epithelial hyperplasia as well as in the stromal tissues of patients with OSCC and oral

hyperplasia. Furthermore, co-expression of visfatin and proliferating cell nuclear antigen pro-

teins was noted in verrucous epithelial hyperplasia, and co-expression of visfatin and CD68 in the

inflammatory cells of the stromal region was noted in the OSCCs. In addition, enzyme-linked

immunosorbent assay showed that plasma visfatin concentrations were significantly increased

in the patients with OSCC and oral hyperplasia compared to those of the control subjects. In

conclusion, visfatin expression and concentrations were higher in OSCCs and oral hyperplasia,

suggesting that visfatin may play a role in the pathogenesis of oral cancers.

# 2014 Faculty of Health and Social Studies, University of South Bohemia in Ceske

Budejovice. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

* Corresponding author at: E-Da Hospital, I-Shou University, No. 1, Yi-Da Rd, Jiau-Shu Village, Yan-Chao Township, Kaohsiung 82445,Taiwan. Tel.: +886 7 615 1100x5914/5018.

E-mail addresses: chungfumei@gmail.com, orthoallen@yahoo.com.tw (C.-Y. Chen).

Available online at www.sciencedirect.com

ScienceDirect

journal homepage: http://www.elsevier.com/locate/jab

1214-021X/$ – see front matter # 2014 Faculty of Health and Social Studies, University of South Bohemia in Ceske Budejovice. Publishedby Elsevier Urban & Partner Sp. z o.o. All rights reserved.http://dx.doi.org/10.1016/j.jab.2014.08.003

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Introduction

Oral squamous cell carcinoma (OSCC) is the most commonmalignant tumor of the oral cavity and the eighth mostcommon cancer in the world (Massano et al., 2006). In Taiwan,oral cancers ranked as the sixth most prevalent cancers in bothsexes and the fourth most common cancers in males in 2006(Cancer Registry Annual Report in Taiwan Area, 2007).Therefore, it is critical to discover prognostic factors as wellas therapeutic targets for oral cancers. Visfatin, also known aspre-B cell colony-enhancing factor, catalyses the rate limitingstep of nicotinamide adenine dinucleotide (NAD+) synthesis(Garten et al., 2009). Visfatin expression promotes cell growthand survival and angiogenesis, and has been shown to behighly expressed in ovarian cancers, gastric and colorectalcarcinomas, and malignant astrocytomas/glioblastomas (Huf-ton et al., 1999; Van Beijnum et al., 2002; Yang et al., 2007;Reddy et al., 2008; Garten et al., 2009; Nakajima et al., 2009;Shackelford et al., 2010). It has also been shown that visfatinmay be induced (�1.5 to 2.0-fold above basal levels) by factorssuch as hypoxia, serum deprivation, and methylmethanesulfonate in cell cultures, and fasting in animals, via a signaltransducer and activator of the transcription 3 (Stat3)-dependent mechanism (Nowell et al., 2006; Yang et al.,2007). Stat3 is a transcription factor that has been shown toact as an oncogene in several human malignancies includingoral cancer (Bowman et al., 2000; Zhao et al., 2012). In addition,our recent study suggested that visfatin may act throughinflammatory reactions to play an important role in thepathogenesis of OSCC (Tsai et al., 2013). As biomedicine is aninterdisciplinary area connecting human medicine in everyfield, it involves the study of patho-physiological processes

Table 1 – Patients' characteristics.

Case no. Gender Age Location of primary

1 Female 61 Tongue

2 Male 46 Neck

3 Male 51 Buccal

4 Male 40 Buccal

5 Male 44 Retromolar and gum

6 Male 39 Tongue

7 Male 63 Buccal

8 Male 52 Tongue

9 Male 45 Buccal

10 Male 59 Buccal

11 Male 52 Buccal

12 Female 58 Tongue

13 Male 32 Buccal

14 Male 70 Lip

15 Male 46 Buccal

16 Male 67 Buccal

17 Male 58 Lip

18 Male 59 Tongue

19 Male 44 Buccal

20 Male 68 Palate

21 Male 53 Buccal

22 Male 53 Buccal and gum

23 Male 60 Buccal

+, score 0–2; ++, score 3–5; +++, score 6–8; ++++, score 9–12.

using the methods of modern biology and being an importantway to investigate human diseases, particularly from theperspective of devising new strategies for diagnosis andtherapy (Berger, 2011). The aim of this study was to furthersubstantiate a potential role for visfatin in oral cancer bystudying visfatin protein expression patterns in patients withoral cancer and precancerous tissue.

Proliferating cell nuclear antigen (PCNA), a nuclear non-histone antigen, is a proliferation marker that allows for theestimation of the growth fraction in a tumor (Bedavanija et al.,2003; Liu et al., 2003). Among the microenvironment compo-nents, tumor-associated macrophages (TAMs) are the majorinflammatory component of the stroma in a tumor (Manto-vani, 2005). Therefore, to gain an understanding of the role ofvisfatin in oral cancer, we also investigated the association ofvisfatin with PCNA and CD68 in patients with oral cancers.Plasma visfatin concentrations in patients with OSCC and oralhyperplasia and control subjects were also examined.

Materials and methods

Sample collection

The records of 23 patients (21 males; 2 females) with oralcancer and precancerous lesions were retrieved from E-DaHospital, I-Shou University, Taiwan (2010–2012). The mean ageof the patients was 51.5 years (range, 32–70 years). All patientshad primary lesions and had undergone total surgical excision.The detailed clinical and TNM data of these 23 patients areshown in Table 1. Thirteen cases occurred in the buccal, 5 inthe tongue, 2 in the lips, and 3 cases were found in the palate,retromolar area, and neck, respectively. Of the 19 oral cancer

TNM state Visfatin immunoscores

T1N0M0 +++T1N0M0 +++T2N0M0 ++T2N0M0 ++++T4N0M0 ++++T1N0M0 +++T4N0M0 +++T2N0M0 ++++T4N2bM1 ++++Epithelial hyperplasia ++++T2N0M0 ++++Verrucous hyperplasia ++++T4N0M0 ++++T2N0M0 ++T2N1M0 +++Squamous papilloma ++++T3N2bM0 +++T4N0M0 +++T2N0M0 ++++T2N0M0 ++++T4N0M0 +++T4N0M0 +++Verrucous hyperplasia ++++

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patients, 11 were at pathological stage T1 or T2, and 8 at stageT3 or T4. Among the 11 patients with T1-or T2-stage oralcancer, 3 had T1N0M0 tumors, 7 had T2N0M0 tumors, and 1had a T2N1M0 tumor. Further, among the 8 patients with stageT3 or T4 oral cancer, 6 had tumors classified as T4N0M0, 1 asT4N2M1, and 1 as T3N2M0. In addition, 2 had verrucoushyperplasia, 1 had squamous papilloma, and 1 had epithelialhyperplasia. The surgical specimens were fixed in 10%buffered formalin embedded in paraffin. Four micrometer-thick tissue sections were prepared for immunohistochemicalanalysis and hematoxylin and eosin staining. The histologicaldiagnoses of oral cancer and precancerous lesions wererendered in accordance with the World Health Organization(WHO) guidelines for pathologic confirmation of lesions (Wahiet al., 1966). In addition, 26 healthy volunteers (24 males; 2females) were recruited as control subjects. The controlsubjects ranged in age from 51 to 77 years, with a mean ageof 61 years. All of the patients and control subjects wereTaiwanese (single race). None of the patients or controlsubjects had a history of autoimmune diseases or hepatitis.This study was approved by the Institutional Review Board ofE-Da Hospital (98-IRB-141).

Semi-quantitative immunohistochemistry

For immunohistochemical (IHC) studies, samples from aseries of 19 consecutive patients with oral cancer and 4patients with oral precancerous lesions were used. Acommercial polymer conjugated peroxidase detection sys-tem method (Dako Corporation, Carpinteria, CA, USA) wasused for the IHC study. Four micron-thick paraffin sections ofOSCC and hyperplasia tissues were mounted on slides, driedfor 30 min in an oven (60–70 8C), and deparaffinized in xylene.The slides were pretreated with microwave heating asdescribed previously (Lai et al., 2002; Lu et al., 2012). Aftermicrowave treatment, the sections were washed in PBS, andendogenous peroxidase activity was blocked by incubation in3% H2O2 in ddH2O for 15 min, and the slides were thenincubated with protein blocker (BSB 0114, BioSB, CA, USA) for30 min to block nonspecific staining. The sections weredrained and incubated overnight at room temperature in ahumidity chamber with monoclonal antibodies directedagainst visfatin (1:250 dilution), PCNA (1:200 dilution), andCD68 (1:100 dilution), which were diluted with protein blocker(BSB 0114, BioSB, CA, USA). The primary visfatin, PCNA, andCD68 antibodies were purchased from Bioss (Wobum, MA,USA), Spring Bioscience Inc. (Pleasanton, CA, USA), and Bio SBInc. (Santa Barbara, CA, USA), respectively. The polymerdetector was applied to the sections following incubationdirectly with a primary antibody labeled sections. Stainingwas completed after incubation with DAB substrate-chrom-ogen solution, counter stained with hematoxylin, andmounted in mounting medium.

The immunostained sections were compared with thecorresponding hematoxylin and eosin-stained sections inorder to establish a topographic relationship between positive-stained areas and histopathological diagnoses. The percentage(P) of positive-stained tumor cells was determined semi-quantitatively by assessing the entire tumor section, andeach section was assigned to one of the following categories:

0 (0–4%), 1 (5–24%), 2 (25–49%), 3 (50–74%), or 4 (75–100%). Theintensity (I) of immunostaining was determined as 0 (nega-tive), 1 (light yellow), 2 (yellow-brown), or 3 (dark brown).Staining intensity was judged relative to the unstained tumorcells of the sample. Finally, an immunoscore was calculated bymultiplying the percentage of positive cells (P) by the stainingintensity score (I), as proposed by Krajewska et al. (1996). Theimmunoscores for visfatin were then evaluated and classifiedinto four groups: (+) score 0–2, (++) score 3–5, (+++) score 6–8,and (++++) score 9–12, as scored independently by the first twoauthors. When the evaluators disagreed on the scores, aconsensus was reached by discussion. Interobserver agree-ment was evaluated using kappa statistics (Landis and Koch,1977) and assessed according to the calculated kappa value: akappa value of less than 0.40 was considered to show pooragreement, 0.40–0.59 fair agreement, 0.60–0.74 good agree-ment, and 0.75–1.00 excellent agreement.

Plasma visfatin measurement

All of the blood samples were drawn after overnight fastingand plasma samples were kept at �80 8C for subsequent assay.The concentrations of plasma visfatin were determined bycommercial enzyme immunoassay kits (Phoenix Pharmaceu-ticals, Belmont, CA, USA). The intraassay coefficients ofvariation were 2.4–2.7% for visfatin. Samples were measuredin duplicate in a single experiment.

Statistical analyses

The bars represent the mean � SD. All of the statisticalanalyses were performed using the SAS software (release8.0; SAS Institute, Cary, NC, USA). The Mann-Whitney U testwas used for between-group comparisons. All statisticalanalyses were calculated at the significance level 2al-pha = 0.05.

Results

Interobserver agreement between the two observers wasexcellent for the assessment of immunoscores, with a kappavalue of 0.92. The IHC expression scores for each case of OSCCand hyperplasia are summarized in Table 1.

Visfatin was detected in the cytoplasm, nuclei, and stromaltissues of the OSCCs and epithelial hyperplasia

All oral cancer and precancerous samples showed an expres-sion of visfatin with different intensities. These proteins weremostly located in the cytoplasm and the nuclei of the OSCCsand hyperplasia cells, as well as in the stromal tissues ofpatients with OSCC and oral hyperplasia. Fig. 1 shows therepresentative IHC staining patterns of oral cavity biopsiesusing a visfatin monoclonal antibody. The results show thatnormal oral mucosa evidenced no detectable visfatin proteinimmunostaining (Fig. 1A), leukoplakia with hyperplasiashowed nuclear and cytoplasmic visfatin staining in nearlyall layers of the epithelium (Fig. 1B), and severe epithelialhyperplasia exhibited strong nuclear and cytoplasmic visfatin

Fig. 1 – Representative immunohistochemical staining patterns of oral cavity biopsies using a visfatin monoclonal antibody:(A) normal oral mucosa showing no detectable immunostaining of visfatin protein; (B) leukoplakia with hyperplasia showingstrong nuclear and cytoplasmic visfatin staining in nearly all layers of the epithelium; (C) severe epithelial hyperplasiaexhibiting strong nuclear and cytoplasmic visfatin staining in nearly all epithelial cells and stromal regions; (D) visfatinimmunostaining in verrucous hyperplasia; (E) buccal well differentiated squamous cell carcinoma (SCC) showing strongnuclear and cytoplasmic visfatin immunostaining in tumor cells; (F) buccal moderately differentiated SCC exhibiting strongnuclear and cytoplasmic visfatin staining in roughly 85% of the cells in OSCC tumor nests.

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staining in nearly all epithelial cells and stromal regions(Fig. 1C). Visfatin immunostaining in verrucous hyperplasia(Fig. 1D) and well differentiated squamous cell carcinoma(SCC) (Fig. 1E) showed strong nuclear and cytoplasmic visfatinimmunostaining in tumor cells, and moderately differentiatedSCC exhibited strong nuclear and cytoplasmic visfatin stainingin roughly 85% of the cells in OSCC tumor nests (Fig. 1F).

Co-expression of visfatin and proliferating cell nuclear antigenproteins was noted in verrucous epithelial hyperplasia

Furthermore, co-expression of visfatin and PCNA proteins wasobserved in the epithelial hyperplasia of the verrucousepithelial hyperplasia (Fig. 2A and B).

Co-expression of visfatin and CD68 in the inflammatory cellsof the stromal region was noted in the OSCCs

Moreover, co-expression of visfatin and CD68 was shown inthe inflammatory cells of the stromal regions of the OSCCs(Fig. 2C and D).

Plasma visfatin concentrations were found to increase in thepatients with OSCC and oral hyperplasia

Patients with OSCC and oral hyperplasia had statisticallysignificant higher plasma visfatin concentrations as comparedwith the control subjects (8.7 � 2.8 vs. 7.6 � 2.3 vs. 4.9 � 2.4 ng/mL, Fig. 3A), and oral cancer patients with a T classification of 3

Fig. 2 – Representative sample (case 23) of the verrucous epithelial hyperplasia showing co-expression of visfatin (A) andproliferating cell nuclear antigen (B) in the epithelial hyperplasia. Representative sample (case 11) of oral squamous cellcarcinoma showing co-expression of visfatin (C) and CD68 (D) in the stromal region.

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or 4, T classification of 1 or 2, and oral hyperplasia patients hadstatistically significant higher plasma visfatin concentrationsas compared with controls (Fig. 3B). The oral cancer patientswith clinical staging III or IV, clinical staging I or II, and oralhyperplasia patients had statistically significant higher plas-ma visfatin concentrations as compared with the controls(Fig. 3C).

Discussion

To the best of our knowledge, this is the first study to evaluatethe expression of visfatin in patients with oral cancer. Asignificant increase in visfatin expression was observed inOSCCs and hyperplasia tissues, and patients with oral cancerhad significantly higher plasma visfatin concentrationscompared with the control subjects. This is in agreementwith previous reports in which higher visfatin expressionswere observed in primary colorectal cancer than in nonneo-plastic mucosa, and that it serves as a biomarker for gastriccancer (Hufton et al., 1999; Nakajima et al., 2009).

Previous studies of other epithelial neoplasms haverevealed a general pattern of increasing visfatin expressionaccompanying malignancies, and in particular higher grademalignant neoplasms (Shackelford et al., 2010; Patel et al.,

2010). In the present study, we similarly observed high visfatinstaining in SCC, leukoplakia with hyperplasia, severe epitheli-al hyperplasia, and verrucous hyperplasia. However, weacknowledge that for these subtypes the number of tumorsexamined is too low to make any definite conclusion (Table 1).Further studies to clarify the significance of visfatin proteinexpression in these tumor types are thus warranted. Inaddition, previous studies have shown nuclear and cyto-plasmic staining for visfatin in both adrenal cells andfibroblasts, and this is thought to be dependent on the stageof the cell cycle (Kitani et al., 2003). In our study, we detectedboth cytoplasmic and nuclear staining for visfatin which is inaccordance with these findings.

PCNA is a well known marker for cell proliferation andtends to accumulate in the late G1 and S-phase of the cell cycle.The evaluation of cell proliferation using PCNA is comparableto and, under certain conditions, superior to the traditionalmethods of mitotic figure count using optical microscopy,tritiated timidin uptake and flow cytometry (Hall et al., 1990;Ogawa et al., 1993). Macrophages are involved in variousaspects of host defense mechanisms and pathophysiologicalconditions, such as chronic inflammatory disease and cancer(Adams and Hamilton, 1984). The functional competence ofmacrophages is acquired after the exposure of macrophages tostimuli in the tissue microenvironment (Hamilton et al., 1993).

ControlHyperplasiaT1+T2T3+T40

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4

6

8

10

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(B) «

«

Plas

ma

visf

atin

(ng/

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ControlHyperplasiaStage I/IIStage III/IV0

2

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10

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(C) «

«

Plas

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visf

atin

(ng/

mL)

ControlsHyperplasiaOSCC0

2

4

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«(A)

«

Plas

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visf

atin

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Fig. 3 – *Above both columns OSCC and hyperplasia hadstatistically significant higher plasma visfatinconcentrations as compared with controls (8.7 W 2.8 vs.7.6 W 2.3 vs. 4.9 W 2.4 ng/mL, A), and *oral cancer patientswith a T classification of 3 or 4, T classification of 1 or 2, andoral hyperplasia patients had statistically significanthigher plasma visfatin concentrations as compared withthe controls (B); and *oral cancer patients with a clinicalstaging of III or IV, clinical staging of I or II, and oralhyperplasia patients had statistically significant higherplasma visfatin concentrations as compared with controls(C). Bars represent the mean W SD.

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Li et al. (2002) suggested that tumor-associated macrophagespossibly play a role in angiogenesis during oral cancerprogression. Visfatin is a newly discovered insulin-mimeticadipocytokine, which directly interacts with the insulinreceptor but as the insulin-like growth factor receptor, andcan subsequently promote cancer cell proliferation (Tilg andMoschen, 2008). It is more highly expressed in primarycolorectal cancer than in non-neoplastic mucosa (Huftonet al., 1999). A basic research study revealed that visfatin maycontribute to the etiopathogenesis of breast cancer byaugmenting cell proliferation via stimulation of cell cycleprogression and by increasing the expression of genes that areimportant in angiogenesis and metastasis (Kim et al., 2010).Additionally, visfatin was originally identified as a pre-B-cellcolony-enhancing factor and was thought to play roles inimmune response and inflammation (Samal et al., 1994; Luket al., 2008; Moschen et al., 2010). Thus, there is some evidenceto suggest that visfatin activates proinflammatory cytokines inhuman monocytes (Moschen et al., 2007). In the current study,co-expression of visfatin and PCNA was noted in both thecytoplasm and nuclei of verrucous epithelial hyperplasia cells,and co-expression of visfatin and CD68 was noted in thestromal tissues of inflammatory cells of patients with oralcancer. These observations therefore suggest that there maybe a link between visfatin and cell proliferation, angiogenesis,and inflammatory responses. This might be one of themechanisms underlying the progression of OSCC.

Previous studies found that mean serum visfatin levelswere significantly higher in gastric cancer, postmenopausalbreast cancer, and colon cancer patients than in controlparticipants (Nakajima et al., 2009, 2010; Dalamaga et al., 2011).Our recent study showed that increased plasma visfatin levelswere associated with OSCC, independent of risk factors, andwere correlated with inflammatory biomarkers (Tsai et al.,2013). In the present study, we similarly observed that patientswith oral cancer had significantly higher plasma visfatinconcentrations compared with the control subjects, and thatoral cancer patients with a T classification of 3 or 4 had higherplasma visfatin concentrations compared with those with a Tclassification of 1 or 2, oral hyperplasia patients, and thecontrol subjects. Yet, the biological mechanisms involvingvisfatin in the pathogenesis of OSCCs are not yet wellunderstood. Visfatin is expressed not only in adipocytes andin total visceral adipose tissue (Fukuhara et al., 2005; Moschenet al., 2007; Sommer et al., 2008), but also in activated immunecells such as neutrophils, macrophages, and lymphocytes(Samal et al., 1994; Jia et al., 2004; Moschen et al., 2007). Visfatinactivates leukocytes and induces the production of cytokinessuch as IL-1 b, tumor necrosis factor, and IL-6 (Moschen et al.,2007), and it can therefore be regarded as a proinflammatory,immunomodulating, and apoptosis-inhibiting adipocytokine(Moschen et al., 2007). Interestingly, in our recent study,plasma visfatin levels were found to be elevated in patientswith OSCC and elevated plasma visfatin levels were associatedwith total white blood cell count and neutrophil count. Hence,we propose that visfatin could be potentially considered as amarker that participates in the process of OSCC.

Some limitations of this study need to be considered. First,our study population was relatively small, and so furtherstudies with larger populations are needed. Moreover, the

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cross-sectional design limited our ability to infer any causalrelationship between visfatin and OSCCs. A prospectivecohort study is required to completely elucidate the impor-tance of visfatin as a biomarker of OSCC and the causativeassociation between oral cancer and the changes in visfatinlevels.

In summary, the present study demonstrated that visfatinexpression and concentration are higher in OSCCs and oralhyperplasia, suggesting that visfatin may play a role in thepathogenesis of oral cancers. Further investigations arerequired to explore the detailed mechanisms of visfatinsignaling in oral cancer development.

Conflict of interest

The authors declare that they have no conflict of interests.

Acknowledgements

The authors would like to thank the E-Da Hospital of theRepublic of China, Taiwan, for financially supporting thisresearch under Contract No. EDAHP101020.

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