Immunoexpression of DNA base excision repair and nucleotide excision repair proteins in ameloblastomas, syndromic and non-syndromic odontogenic keratocysts and dentigerous cysts

Journal Pre-proof Immunoexpression of DNA base excision repair and nucleotide excision repair proteins in ameloblastomas, syndromic and non-syndromic odontogenic keratocysts and dentigerous cysts Hellen Bandeira de Pontes Santos, Everton Freitas de Morais, Roberta Barroso Cavalcante, Renato Luiz Maia Nogueira, Cassiano ´ Batista de Souza, Roseana de Francisco Weege Nonaka, Lelia Almeida Freitas

PII:

S0003-9969(19)30818-0

DOI:

https://doi.org/10.1016/j.archoralbio.2019.104627

Reference:

AOB 104627

To appear in:

Archives of Oral Biology

Received Date:

13 August 2019

Revised Date:

1 December 2019

Accepted Date:

2 December 2019

Please cite this article as: de Pontes Santos HB, de Morais EF, Barroso Cavalcante R, Maia Nogueira RL, Weege Nonaka CF, de Souza LB, de Almeida Freitas R, Immunoexpression of DNA base excision repair and nucleotide excision repair proteins in ameloblastomas, syndromic and non-syndromic odontogenic keratocysts and dentigerous cysts, Archives of Oral Biology (2019), doi: https://doi.org/10.1016/j.archoralbio.2019.104627

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IMMUNOEXPRESSION OF DNA BASE EXCISION REPAIR AND NUCLEOTIDE EXCISION REPAIR PROTEINS IN AMELOBLASTOMAS, SYNDROMIC AND NONSYNDROMIC ODONTOGENIC KERATOCYSTS AND DENTIGEROUS CYSTS

Short title: DNA repair proteins in odontogenic lesions

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Hellen Bandeira de Pontes Santos1, Everton Freitas de Morais1, Roberta Barroso Cavalcante2, Renato Luiz Maia Nogueira3, Cassiano Francisco Weege Nonaka4, Lélia Batista de Souza1,

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Roseana de Almeida Freitas1* [email protected]

Department of Dentistry, Federal University of Rio Grande do Norte, Natal, RN, Brazil

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Department of Oral Pathology, University of Fortaleza, Fortaleza, Ceará, Brazil.

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Department of Oral Surgery, Federal University of Ceará, Fortaleza, Ceará, Brazil.

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Department of Dentistry, State University of Paraíba, Campina Grande, Paraíba, Brazil.

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Corresponding author: Roseana de Almeida Freitas, Departamento de Odontologia,

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Universidade Federal do Rio Grande do Norte. , Av. Senador Salgado Filho, 1787, Lagoa

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Nova, CEP 59056-000 Natal, RN, Brasil. Phone/Fax: +55843215-4138.

Highlights

This is the first study about DNA BER and NER proteins in odontogenic lesions. Overexpression of APE-1, XRCC-1 and XPF was found in solid AMEs, NSOKCs, and SOKCs.

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APE-1, XRCC-1 and XPF may regulate events related to a more aggressive behavior. APE-1 expression may be synergistic with XRCC-1 and XPF in all studied lesions.

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These proteins may be involved in the development of odontogenic lesions.

Abstract

Objective: To evaluate the immunoexpression of DNA base excision repair (BER)

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[apurinic/apyrimidinic endonuclease 1 (APE-1), X-ray repair cross complementing 1 (XRCC1)] and nucleotide excision repair (NER) [xeroderma pigmentosum complementation group

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(XPF)] proteins in benign epithelial odontogenic lesions with different biological behaviors. Design: Thirty solid ameloblastomas, 30 non-syndromic odontogenic keratocysts (NSOKCs),

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29 syndromic odontogenic keratocysts (SKOCs), 30 dentigerous cysts (DCs) and 20 dental follicles (DFs) were evaluated quantitatively for APE-1, XRCC-1 and XPF through

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immunohistochemistry.

Results: Nuclear expression of APE-1 was significantly higher in NSOKCs, SOKCs, and

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ameloblastomas in comparison to DCs (p<0.001). Nuclear expression of XRCC-1 was higher

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in NSOKCs and SOKCs than in DCs (p<0.05). At the nuclear level, XPF expression was higher in NSOKCs and SOKCs than in DCs and ameloblastomas (p<0.05). A statistically significant higher expression of APE-1 (nuclear), XRCC-1 (nuclear), and XPF (nuclear and cytoplasmic) was found in all odontogenic lesion samples as compared to DFs (p<0.05). For all lesions, there was a positive correlation between nuclear expression of APE-1 and XRCC1 or XPF (p<0.05). Conclusions: Our results suggest a potential involvement of APE-1, XRCC-1 and XPF

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proteins in the pathogenesis of benign epithelial odontogenic lesions, especially in those with more aggressive biological behavior, such as ameloblastomas, NSOKCs, and SOKCs. We also showed that the expression of APE-1 was positively correlated with the nuclear expression of XRCC-1 and XPF, which may suggest an interaction between the BER and NER pathways in all odontogenic lesions studied herein.

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Keywords: Odontogenic tumour; odontogenic cyst; DNA repair; immunohistochemistry.

Introduction

Odontogenic lesions represent a heterogeneous group of cysts, tumours and

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hamartomatous processes, with complex clinicopathological features and biological behavior.

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Ameloblastoma is the most frequently encountered and clinically significant benign odontogenic tumour of the jaws, which is characterized by an aggressive biological behavior

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and a high recurrence potential (Brown et al., 2014; Johnson, Gannon, Savage, & Batstone, 2014; Kurppa et al., 2014). Odontogenic keratocyst is one of the most frequent odontogenic

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cysts in gnathic bones, representing a distinct form of odontogenic developmental cyst, which deserves special attention due to its aggressive clinical behavior, high recurrence rate and

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possible association with Gorlin Syndrome (Mendes, Carvalho, & van der Waal, 2010; ElNaggar, Chan, Grandis, Takata, & Slootweg, 2017; Gomes, Guimarães, Diniz, & Gomez,

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2017). In contrast, dentigerous cyst (DC) presents an excellent prognosis exhibiting very low recurrence rates when correctly treated (Allison & Garlington, 2017; El-Naggar et al., 2017). Nevertheless, the mechanisms underlying the pathogenesis of these lesions and the reasons that justify different biological behaviors are not yet fully elucidated, which might be due to the little information available on the molecular characteristics of the lesions.

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The DNA repair pathways act on specific types of damage to the genetic material, thereby regulating several cellular processes (Kansikas, Nyström, & Peltomäki, 2017). Among the main DNA repair pathways, the most notable are the base excision repair (BER) and the nucleotide excision repair (NER) (Dalhus, Laerdahl, Backe, & Bjørås, 2009; Tell, Quadrifoglio, Tiribelli, & Kelley, 2009; Nesic, Wakefield, Kondrashova, Scott, & McNeish, 2018). Key proteins such as apurinic/apyrimidinic endonuclease 1 (APE-1) and X-

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ray repair cross-complementing 1 (XRCC-1) are required for viability and an efficient repair of DNA damage in the BER pathway (Whitaker, Schaich, Smit, Flynn, & Freudenthal, 2017). APE-1, also known as redox effector factor 1 (Ref-1), is a multifunctional enzyme that plays an essential role in the BER pathway as an apurinic/apyrimidinic-site endonuclease, and it is

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also involved in the redox regulation of important transcription factors, such as nuclear factor-

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κB and hypoxia inducible factor-1α (Whitaker et al., 2017). In addition, the inhibition of APE-1 redox activity promotes odontogenic and osteogenic differentiation of dental papilla

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cells (Chen et al., 2015). XRCC-1 recognizes and binds to the DNA breaks and facilitates BER pathway by acting as a scaffold protein to recruit and physically interact with several

Rotte, 2014).

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components of the repair machinery (Abdel-Fatah et al., 2013; Bhandaru, Martinka, Li, &

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As an NER protein, xeroderma pigmentosum complementation group F (XPF), also known as the excision repair cross-complementing group 4, is one of the most important

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DNA repair proteins. It enables the enzymatic activity of XPF-excision repair crosscomplementing group 1 (ERCC1) heterodimer, an endonuclease that incises at the 5’ side of various DNA lesions (Qiu et al., 2014; Faridounnia, Folkers, & Boelens, 2018). Thus, this protein is critically involved in the NER pathway and has also an important role in recombination repair, mismatch repair and, possibly, immunoglobulin class switching (Qiu et al., 2014; Faridounnia et al., 2018). Evidence has indicated that APE-1, XRCC-1 and XPF are

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dysregulated and sometimes highly expressed in some malignancies, contributing to tumour development and progression (Li, 2011; Seiwert et al., 2014; Thakur et al., 2014; Brown, & Betz, 2015; Shah et al., 2017; Whitaker et al., 2017; Azambuja et al., 2018). While investigations have demonstrated the participation of growth factors, matrix metalloproteinases, tumour suppressor genes and oncogenes in the biological behavior of benign epithelial odontogenic lesions (Kurppa et al., 2014; Li., 2011; Sweeney et al., 2014;

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Brown, & Betz, 2015; Gomes et al., 2017), only a few studies have evaluated the possible involvement of DNA repair proteins in these lesions. Among the various proteins involved in DNA repair pathways, studies involving benign odontogenic lesions have explored only

proteins from the mismatch repair system (Castrilli et al., 2001; de Brito Monteiro et al.,

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2015; Amaral-Silva et al., 2018; Bologna-Molina et al., 2018) as well as the possible

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involvement of the polymorphism in XRCC-1 and the risk of ameloblastoma (Yanatatsaneejit, Boonsuwan, Mutirangura, & Kitkumthorn, 2013).

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Considering the heterogeneous biological behavior of odontogenic lesions and the absence of studies that evaluated the immunoexpression of DNA repair proteins of the BER

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and NER pathways in benign epithelial odontogenic lesions (Pubmed Database, Scopus, Web of Science, LILACS, SIGLE), we evaluated the immunoexpression of BER proteins APE-1

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and XRCC-1 and NER protein XPF in solid ameloblastomas, non-syndromic odontogenic keratocysts (NSOKCs), syndromic odontogenic keratocysts (SOKCs) (associated with Gorlin

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syndrome) and dentigerous cysts (DCs) in order to provide insights for a better understanding of the role of these proteins in the varying biological behavior between the lesions.

Material and Methods Sample

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This was cross-sectional study previously approved by the Research Ethics Committee of the Federal University of Rio Grande do Norte (UFRN, Natal, Brazil) (Approval number: 2.535.458/ 9 March 2018). A total of 139 formalin-fixed paraffin-embedded tissue samples were retrieved from the files of the Oral Pathology Service at the UFRN, comprising 30 solid ameloblastomas, 30 NSOKCs, 29 SOKCs, 30 DCs and 20 dental follicles (DFs) obtained from healthy patients submitted to impacted third molar surgery. DFs were used as a control

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group, representing healthy adult odontogenic tissues. Microscopic aspects of all lesions were reviewed by two oral pathologists to confirm their diagnoses following current World Health Organization guidelines (El-Naggar et al., 2017). Ameloblastoma samples were comprised of multiple histopathological types. The plexiform type prevailed in 18 cases, whereas the

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follicular type was more prevalent in the other 12 cases, with 6 cases demonstrating

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expressive additional areas of acanthomatous or granular pattern. Patients with Gorlin syndrome had been diagnosed according to the criteria proposed by Evans et al. (1993) and

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presented multiple odontogenic keratocysts. Patients with NSOKCs presented single lesions and had been submitted to clinical and radiographical evaluation to exclude the presence of

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other manifestations of Gorlin syndrome. All odontogenic lesions submitted to the marsupialization technique prior to biopsy and those that presented secondary inflammation

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based on histopathological analysis were not included. Clinical data (sex, age and anatomical location of the lesions) were collected from the patient’s medical records and biopsy request

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forms. The clinical characterization of the patients is summarized in Table 1. All procedures were conducted in full accordance with the World Medical Association Declaration of Helsinki.

Immunohistochemistry

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Histological sections with 3-μm-thickness were obtained from the formalin-fixed paraffin-embedded material and mounted on glass slides previously prepared with organosilane (3-aminopropyltriethoxysilane, Sigma Chemical Co., St Louis, United States of America) as adhesive. The sections were deparaffinized, rehydrated and submitted to antigen retrieval with Trilogy (1:100, Cell-Marque, United States of America) in a Pascal pressure cooker (Dako, Carpinteria, United States of America) for 30 minutes. Next, the tissue sections

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were immersed in 10 volumes of hydrogen peroxide solution to block endogenous peroxidase and then incubated with protein block (Thermo Scientific, Runcorn, United Kingdom) for 5 minutes. Subsequently, the tissue sections were incubated with the following monoclonal primary antibodies: anti-APE-1 (clone C4; sc-17774; Santa Cruz Biotechnology, United

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States of America; 1:3000; 60 minutes), anti-XRCC-1 (clone 33-2-5; Thermo Scientific,

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Barrington, United States of America; 1:1500; overnight) and anti-XPF (clone 219; Thermo Scientific, Barrington, United States of America; 1:800; overnight). Sections were then

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washed twice in phosphate-buffered saline and incubated in the HiDef visualization system (HiDef Detection™ HRP Polymer System, Cell-Marque, United States of America) at room

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temperature. The reactions were revealed with 3.3’-diaminobenzidine (Liquid DAB + Substrate; Dako, Carpinteria, United States of America), resulting in a brown reaction

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product. Finally, tissue sections were counterstained with Mayer's hematoxylin and coverslipped. Human ovarian carcinoma specimens were used as positive controls for the

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APE-1 and XRCC-1 reactions and human tonsil fragments were used for XPF. Negative controls were performed by omitting the primary antibody in the protocol described above.

Immunostaining assessment All slides were scanned into high-resolution images using a digital slide scanner system (Pannoramic MIDI II, 3DHISTECH, Budapest, Hungary), and the images were

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visualized by the Panoramic Viewer 1.15.2 software (3DHISTECH Kft.29-33, Budapest, Hungary). The tissue sections were analyzed blindly by one previously trained examiner. APE-1, XRCC-1 and XPF immunoexpression was analyzed quantitatively in the parenchymal cells of ameloblastomas and epithelial lining of odontogenic keratocysts, DCs and DFs. Nuclear and cytoplasmic reactivity was analyzed separately for APE-1 and XPF while only nuclear immunoexpression was evaluated for XRCC-1. Adapting the method from Zhang et

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al. (2018), the five areas of highest anti-APE-1, anti-XRCC-1 and anti-XPF immunoreactivity were selected along the epithelial component. These five fields were digitally photographed at a magnification of 400×, each field corresponding to an area of 0.0998mm2, and the images were transferred to the ImageJ® software (Image Processing and Analysis in Java, National

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Institute of Mental Health, Bethesda, Maryland, United States of America). Immunostained

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and negative cells were counted in each photographed field, and the percentage of positive

Statistical analysis

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cells relative to the total number of counted cells was established for each antibody.

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The results were analyzed using the IBM Statistical Package for the Social Sciences (SPSS) Statistics 20.0 program (IBM Corp., Armonk, United States of America). Descriptive

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statistics was used for characterization of the sample. Immunopositivity percentages were submitted to distribution analysis by the Kolmogorov-Smirnov test, which revealed a non-

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normal data distribution. Thus, the non-parametric Kruskal-Wallis (KW) and Mann-Whitney (U) tests were used to compare the median percentages of immunoreactivity between the groups of lesions. Correlations between the immunoexpression of APE-1, XRCC-1 and XPF were evaluated by the Spearman’s correlation test. All statistical tests considered a 5% significance level (p ≤ 0.05).

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Results

Characterization of APE-1, XRCC-1 and XPF immunoexpression Analysis of the expression of APE-1, XRCC-1 and XPF in ameloblastomas revealed a higher immunoexpression of these proteins at the peripheral/ ameloblastic layer of plexiform and follicular cases and in solid areas of plexiform cases (Figure 1A-B). Solid areas consisted

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of central areas in the anastomosing strands and cords epithelium that presented a large number of epithelial cells in the plexiform ameloblastomas as well as in the central areas of the epithelial islands of follicular ameloblastomas.

Protein expression varied considerably, with no particular pattern of distribution in the

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different cell layers of the epithelial lining of NSOKCs, SOKCs, and DCs (Figure 1D-L). In

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DFs, a discrete nuclear staining was observed in some cases (Figure 1M-O).

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Nuclear APE-1 and XRCC-1 are overexpressed in NSOKCs, SOKCs and ameloblastomas All NSOKC and SOKC samples showed nuclear APE-1 immunoexpression in the

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epithelial lining, while 29 ameloblastomas (96.66%), 24 DC (80.0%), and 7 DF (35.0%) samples exhibited nuclear expression of APE-1 in the epithelial component (Figure 1A, 1D,

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1G, 1J, 1M). Among all groups, only 4 cases of SOKCs (13.79%) and 6 cases of NSOKCs (20.0%) exhibited cytoplasmic immunoreactivity for APE-1 (Figure 1D and 1G). XRCC-1

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nuclear immunoexpression was observed in 27 ameloblastomas (90.0%), 28 NSOKC (93.33%), 28 SOKC (96.55%), 24 DC (80.0%) samples and in only 4 DF (20.0%) samples (Figure 1B, 1E, 1H, 1K, 1N). No case exhibited cytoplasmic immunoreactivity for XRCC-1 in any of the groups. There was a higher nuclear expression of APE-1 in NSOKCs when compared to DCs (p < 0.0001) and ameloblastomas (p = 0.001). Similarly, SOKCs demonstrated higher nuclear

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expression of this protein compared to DCs (p < 0.0001) and AMEs (p = 0.009). A significantly higher nuclear expression of APE-1 was found in ameloblastoma samples when compared to DC ones (p = 0.014). However, no statistically significant differences in APE-1 expression were observed between NSOKCs and SOKCs at the nuclear (p = 0.301) and cytoplasmic (p = 0.366) levels (Figure 2A). Nuclear expression of XRCC-1 was significantly higher in NSOKCs (p = 0.016) and

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SOKCs (p < 0.0001) as compared to DCs, with no statistically significant differences between NSOKCs and SOKCs (p = 0.114) and between NSKOCs and ameloblastomas (p = 0.667). In addition, nuclear expression of XRCC-1 was greater, albeit non-significant, in ameloblastoma samples in comparison to DCs (p = 0.066) (Figure 2B). No significant difference was found

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between APE-1 and XRCC-1 expression and the predominant histological subtype of

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ameloblastoma (p > 0.05).

Interestingly, all odontogenic lesions exhibited significantly higher nuclear expression

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of APE-1 and XRCC-1 when compared to DFs (p < 0.0001) (Figure 2A-B).

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Nuclear XPF is overexpressed in NSOKCs, SOKCs and ameloblastomas Nuclear XPF immunoreactivity was observed in the epithelial cells of 27 AMEs

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(90.0%), all NSOKCs (100.0%), 28 SOKCs (96.55%), 25 DCs (83.33%) and 4 DFs (20.0%). Cytoplasmic XPF expression was noted in the epithelial component of 15 AMEs (50.0%), 18

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NSOKCs (60.0%), 9 SOKCs (31.03%) and 15 DCs (50.0%) (Figure 1C, 1F, 1I, 1L). No case of DF presented XPF cytoplasmic expression (Figure 1O). A significantly higher nuclear XPF expression was observed in NSOKCs compared to

DCs (p < 0.0001) and ameloblastomas (p = 0.025) by Mann-Whitney test. Likewise, SOKCs also demonstrated higher nuclear expression of this protein compared to DCs (p = 0.001) and ameloblastomas (p = 0.043). However, no statistically significant differences in nuclear XPF

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expression were observed between NSOKCs and SOKCs (p = 0.724). Although nonsignificant, nuclear XPF immunoreactivity was higher in ameloblastomas compared to DCs (p = 0.156) (Figure 2C). Expression of XPF at the cytoplasmic level was increased in NSOKCs compared to SOKCs (p = 0.039) and in DCs compared to SOKCs (p = 0.04), but no statistical difference in cytoplasmic XPF reactivity was detected between NSOKCs and DCs (p = 0.864) and between

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ameloblastomas and NSOKCs (p = 0.072), SOKCs (p = 0.845) and DCs (p = 0.094) (Figure 2D).

No significant difference was found between nuclear and cytoplasmic expression of XPF and the predominant histological subtype of ameloblastoma (p>0.05). Importantly, all

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epithelial odontogenic lesions showed a greater number of XPF immunopositive cells when

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compared to DFs in both the nucleus (p < 0.0001) and cytoplasm (p < 0.01) (Figure 2C-D).

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Correlation between APE-1, XRCC-1 and XPF expression Possible correlations between APE-1, XRCC-1 and XPF expression were analyzed in

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the five study groups. In ameloblastomas, nuclear expression of APE-1 correlated positively with XRCC-1 (r = 0.850; p < 0.0001) and XPF (r = 0.600; p < 0.001) expression. Similarly,

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there was a positive correlation between nuclear expression of XPF and XRCC-1 (r = 0.566; p = 0.001) and between nuclear XPF and cytoplasmic XPF (r = 0.597; p < 0.0001) (Table 2).

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In NSOKCs, nuclear APE-1 was positively correlated with nuclear XPF (r = 0.571; p

= 0.001); cytoplasmic XPF was positively correlated with both nuclear XPF (r = 0.459; p = 0.011) and nuclear XRCC-1 (r = 0.465; p = 0.01). Furthermore, cytoplasmic expression of APE-1 was positively correlated with nuclear APE (r = 0.388; p = 0.034) and cytoplasmic XPF (r = 0.429; p = 0.018) (Table 2).

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In SOKCs, nuclear expression of APE-1 was positively correlated with both nuclear XRCC-1 (r = 0.762; p < 0.0001) and nuclear XPF (r = 0.470; p = 0.01). Likewise, XPF expression was correlated positively with XRCC-1 expression (r = 0.381; p = 0.041) at the nuclear level. Cytoplasmic expression of APE-1 and XPF was not significantly related to the expression of the other proteins screened in SOKC samples (p > 0.05) (Table 2). In DCs, nuclear expression of APE-1 correlated positively with both nuclear XRCC-1

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(r= 0.631; p < 0.0001), nuclear XPF (r = 0.622; p < 0.0001) and cytoplasmic XPF (r = 0.635; p < 0.0001). XPF expression in the cytoplasm was positively correlated to expression of

nuclear XRCC-1 (r = 0.560; p = 0.001) and nuclear XPF (r = 0.760; p < 0.0001) (Table 2). Discussion

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Evolutionarily, cells have conserved DNA repair and maintenance mechanisms to

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cope with threats to their genetic material. Hence, misregulation or defects in DNA repair pathways might result in the development and progression of several diseases, including

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neoplasms (Moraes, Neto, & Menck, 2012; Zou, & Maitra, 2018). Nevertheless, little is known about the role of DNA repair proteins in odontogenic cysts and tumours, and no

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previous investigation has determined the expression levels of the BER proteins APE-1 and XRCC-1 and the NER protein XPF in these lesions. In this study, we found a nuclear

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overexpression of these proteins in ameloblastoma, NSOKC and SOKC lesions, which commonly show a locally aggressive behavior and a tendency to relapse, which suggests their

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involvement in the regulation of DNA repair and maintenance mechanisms. The BER is the most used pathway to handle with simple modifications (alkylation

and oxidation) of single bases. The BER pathway is also involved in repairing DNA singlestrand breaks (SSB) induced by free radical agents (Whitaker et al., 2017). One of the key enzymes of the BER pathway in mammals is APE-1. APE-1 is a multifunctional protein involved both in the BER pathway of DNA lesions, acting as the major apurinic/apyrimidinic

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endonuclease, and in transcriptional regulation of gene expression (Tell, Damante, Caldwell, & Kelley, 2005; Thakur et al., 2014; Whitaker et al., 2017). This effect is obtained as a redox co-activator of different transcription factors (Tell et al., 2005; Thakur et al., 2014; Whitaker et al., 2017). In malignancies, transcription factors downstream of APE-1 promote growth, migration, and survival in tumour cells as well as angiogenesis and inflammation in the tumour microenvironment (Shah et al., 2017). In this study, we found a significantly higher

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nuclear expression of APE-1 in ameloblastomas, NSOKCs and SOKCs when compared to DCs and DFs. Taken together, these findings suggest that the BER pathway is probably

upregulated due to greater genomic instability and also that APE-1 may trigger a higher transcriptional activity of genes related to proliferation and migration in solid

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ameloblastomas, NSOKCs and SOKCs. Of note, these lesions present a higher proliferation

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index, a more aggressive behavior, as well as the presence of important molecular genetic changes (Kurppa et al., 2014; Gomes et al., 2017; Brown, & Betz, 2015).

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Importantly, the compound APE1/Ref-1 redox inhibitor (APX3330) has been extensively characterized as a direct, highly selective inhibitor of Ref- 1/APE-1 redox activity

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that does not affect the protein endonuclease activity in tumours. Treatment with APX3330 slowed tumour growth and progression, with limited toxicity, in both in vitro and in vivo

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models (Shah et al., 2017; Zou, & Maitra, 2018; Fishel et al., 2011; Cardoso et al., 2012) and is entering into the clinical phase against various types of cancer and other diseases (Shah et

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al., 2017). Thus, it would be valid to evaluate the possible functional effects of APX3330 on ameloblastomas and odontogenic keratocysts in order to discover future therapeutic targets that could provide a personalized treatment approach for these lesions. The subcellular distribution of APE-1 in different mammalian cell types occurs mainly in the cell nucleus due to the predominant DNA repair and co-transcriptional activity of this protein, which controls cell proliferation and other processes (Tell et al., 2005; Shah et al.,

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2017). In most cell types, APE-1 occurs only in the nucleus, whereas in other cells it may be present in the cytoplasm or both in the nucleus and cytoplasm (Tell et al., 2005; Shah et al., 2017). Such a complex distribution pattern suggests that localization is not random but, on the contrary, is controlled by a strictly regulated process. It has been shown that a shift to greater cytoplasmic localization is present in some aggressive malignant tumours with poor prognoses (Sheng et al., 2012; Shah et al., 2017). This expression pattern is very often

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associated with lesions with high metabolic or proliferative rates in a cell cycle-dependent fashion (Tell et al., 2005; Tell et al., 2009; Sheng et al., 2012). Possible explanatory

hypotheses for the cytoplasmic expression of APE-1 may come from repair activities in mitochondrial DNA (Tell et al., 2009) or by the need to maintain newly synthesized

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transcription factors in a reduced state during translocation to the nucleus (Tell et al., 2009).

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Interestingly, in all groups evaluated here, cytoplasmic expression of APE-1 was found only in NSKOCs and SKOCs. This is probably attributed to the high proliferative capacity and

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aggressive biological behavior of these cysts (Gomes et al., 2017). Since this is the first study to report cytoplasmic APE-1 expression in OKCs and other odontongenic lesions, further

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investigation with a larger sample size, clinical parameters, and longer follow-up needs to be performed to establish the biological and clinical influence of extranuclear expression of

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APE-1 in these lesions.

Another key BER molecule is XRCC-1, which functions as a scaffold protein and is

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intimately involved in the coordination of DNA repair by interacting with several components of the BER pathway (Abdel-Fatah et al, 2013; Bhandaru et al., 2014). Cells with mutated and non-functional XRCC-1 are reportedly susceptible to chromosomal aberrations and deletions (Ladiges, 2006; Bhandaru et al., 2014). It has also been shown that the expression of XRCC-1 in malignancies is related to better prognosis and disease-specific survival of patients with melanoma (Bhandaru et al., 2014) or a worse prognosis in ovarian cancer (Abdel-Fatah et al,

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2013) and colorectal cancer (Azambuja et al., 2018). Since these malignancies have different pathogenesis, XRCC-1 may be influenced by extra and intracellular factors and act as an oncogene or tumour suppressor gene depending on the scenario and tumour type. In our study, we found a greater expression of XRCC-1 in the most aggressive odontogenic lesions analyzed (SOKCs, NSOKCs and ameloblastomas) when compared to DCs and DFs. These findings suggest a high activity of the BER pathway components in these lesions and that

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XRCC-1 may drive events related to a more aggressive biological behavior. NER pathway proteins perform a highly coordinated excision of the damage as a

single-stranded oligonucleotide and restoration of the original DNA sequence using the nondamaged strand as a template (Faridounnia et al., 2018). Among several molecules, XPF is an

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essential protein in this pathway, forming a heterodimer with ERCC1 and being involved in a

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multitude of mechanisms, including inter-strand crosslink repair and homologous recombination (Faridounnia et al., 2018). In our study, we found a higher expression of

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nuclear XPF in NSOKCs and SOKCs in comparison to DCs and DFs (p < 0.0001) and, although non-significant, nuclear expression of this protein was higher in ameloblastomas

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than in DCs. These findings show that XPF is overexpressed in benign epithelial odontogenic lesions with a more aggressive behavior. Consistent with this, it has been suggested that high

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nuclear expression of XPF is linked to a poor prognosis in head and neck squamous cell carcinoma (Vaezi et al., 2011) and to the onset, development and poor prognosis of gastric

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cancer (Li, & Ma, 2018). On the other hand, Qiu et al. (2014) found that bladder cancer patients with low levels of XPF had a higher recurrence rate than those with high levels of XPF, suggesting that this protein exerts a protective role in the development of bladder cancer. Altogether, it seems that XPF may be upregulated in NSOKCs, SOKCs and ameloblastomas due to the necessity to deal with DNA damage-related activities. Further

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research is warranted to thoroughly elucidate the biological mechanism of such interactions and differences. Interestingly, while some cases with all the lesions studied herein presented XPF cytoplasmic staining, no DF sample showed XPF immunopositivity. Ahmad et al. (2010) showed that cytoplasmic XPF expression is common in XPF mutant cells and in Xeroderma pigmentosum patients. These authors demonstrated that at least part of the DNA repair defect

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and symptoms associated with mutations in XPF are due to mislocalization of XPF-ERCC1 within the cell cytoplasm, likely due to protein misfolding (Ahmad et al. 2010). Thus, it is possible that odontogenic lesions may be associated with XPF mutations and thereby display a pathological impact on the cell function, but this remains to be explored in future studies.

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Besides the previous hypotheses, it is also possible that the BER proteins APE-1 and

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XRCC-1 and the NER protein XPF are overexpressed in ameloblastomas and odontogenic keratocysts due to the necessity to deal with mutations and other genetic alterations that are

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highly frequent in these lesions. Some examples include activation of mutations in important genes of the mitogen-activated protein kinase (MAPK) pathway, especially the oncogenic

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BRAFV600E mutation, present in approximately 80% of human solid ameloblastomas (Brown et al., 2014; Kurppa et al., 2014; Brown, & Betz, 2015; Pereira et al., 2016) and in the

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Hedgehog signaling pathway genes, especially the Patched 1 (PTCH1) mutation, commonly present in odontogenic keratocysts (Qu et al., 2015; Gomes et al., 2017). Nuclear expression

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of these three proteins was low in DCs and only few cases of DFs were positive. It is important to highlight that an analysis performed with qPCR assay and Sanger sequencing revealed that DCs do not carry BRAF V600E mutations (Pereira et al., 2016) and although the loss of heterozygosity of the PTCH1 has been related in some of these cyst cases (Levanat, Pavelić, Crnić, Oresković, & Manojlović, 2000; Pavelić et al., 2001) it still needs confirmation, as sequencing data on these lesions has not been presented yet. Thus, we

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reasoned that the genetic mutations frequently present in ameloblastomas, NSOKCs, and SOKCs may induce activation of the DNA repair machinery. These findings are supported by an in vitro study by Sheu et al. (2012), which shows that transfection of BRAFV600E, but not the wild-type BRAF, into epithelial cells directly induced DNA strand breaks and DNA damage response, leading to DNA repair activation. In line with these findings, Diniz, Gomes, de Sousa, Xavier, & Gomez et al. (2017) found higher levels of endonuclease III-like N-

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glysosylase 1 (NTHL1), a gene involved in DNA damage response, that is responsible for repairing oxidative lesions in double-stranded DNA and also in the BER pathway,

BRAFV600E-solid ameloblastomas when compared to unicystic ameloblastomas and normal oral mucosa. Taken together, it seems that misregulation of these genes in odontogenic

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epithelial lesions, such as ameloblastomas, NSOKCs and SOKCs, may be associated with

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oncogenic activation by mutant BRAF in ameloblastomas or probably mutant PTCH1 in odontogenic keratocysts. Whether BER and NER repair activities influence genetic alterations

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in the pathobiology of benign epithelial odontogenic lesions remains to be determined. Previous studies have investigated possible differences in the immunoexpression

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pattern between NSOKCs and SOKCs (de Brito Monteiro et al., 2015; Mendes et al., 2017). In agreement with the present study, nuclear APE-1, XRCC-1 and XPF were not differentially

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expressed in these groups, and such DNA repair proteins are not likely to explain the different biological behavior of NSOKCs and SOKCs. In line with our findings, De Brito Monteiro et

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al. (2015) found no significant difference in the mismatch frequency of the repair protein hMLH1 between NSOKCs and SOKCs. It is possible that a larger number of cases could provide enough sensitivity to distinguish between these groups, as other authors have found significant differences between them (Leonardi et al., 2015; Hoyos Cadavid et al., 2019). A recent study established cell lines from SOKC and NSOKC cases and reported similar protein expression levels between them (Noguchi et al., 2017). The authors suggested that common

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mechanisms underlie the development of both types of odontogenic keratocysts (Noguchi et al., 2017). In our study, all odontogenic lesions presented a significantly higher expression of APE-1, XRCC-1 and XPF when compared to DFs. These findings suggest that such proteins are potentially altered in the odontogenic lesions analyzed. Interestingly, Spearman’s correlation test showed a positive correlation in all groups between nuclear expression of

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APE-1 and XRCC-1 or XPF. In addition, cytoplasmic APE-1 was positively correlated with nuclear XPF and nuclear APE-1 in NSOKCs. These findings suggest that the BER and NER pathways can interact to deal with the common genomic stress that occurs in such

odontogenic lesions. Moreover, there was a positive correlation between cytoplasmic XPF

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and nuclear XPF in AMEs, NSOKCs and DCs. Although Ahmad et al. (2010) proposed that

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cytoplasmic XPF-ERCC1 prevents this nuclease complex from participating DNA repair in fibroblasts from XFE progeroid patients, it is possible that, in the studied odontogenic lesions,

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which present different pathogenesis and molecular alterations, extranuclear XPF localization may not directly influence DNA repair mechanisms.

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In conclusion, the high nuclear expression of the DNA repair proteins APE-1, XRCC1 and XPF in solid ameloblastomas, NSOKCs, and SOKCs, suggests their participation in

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biological events related to a more aggressive behavior and peculiar molecular alterations. In addition, overexpression of these proteins may probably be related to a high activity of the

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BER and NER pathways in these lesions. Thus, differences in the biological behavior of the studied lesions may be related to expression of these proteins. We further showed that expression of APE-1 may be synergistic with expression of nuclear XRCC-1 and nuclear XPF, which may suggest an interaction between the BER and NER pathways in all studied odontogenic lesions. The absence of clinical and follow-up information of our sample represents a limiting factor. While this study presents novel aspects related to DNA BER and

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NER proteins in epithelial odontogenic lesions, further research that includes clinical data and longer follow-up is required to shed light on their functional role in pathogenesis of benign epithelial odontogenic lesions. Our results may guide future studies to elucidate the differences in the biological behavior of these lesions.

Authors’ Contributions

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H. B. P. Santos contributed to the study’s conception and design, immunohistochemistry analysis, data analysis and manuscript writing; E. F. Morais, R. C. Cavalcante and R. L. M. Nogueira contributed to acquisition of data and drafting of the article; C. F. W. Nonaka and L.B Souza contributed to the study design and manuscript writing; and R. A. Freitas contributed to the study’s conception and design and manuscript writing. All authors gave final approval and agree to be accountable for all aspects of the work.

Conflicts of interest

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obtained in this study.

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The authors state that they have no potential conflict of interest that could bias the results

Acknowledgements

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This study was supported by the National Council for Scientific and Technological Development (CNPq) and Coordination of Higher Education Personnel (CAPES) - Finance Code 001. RAF and CFWN are CNPq research fellows.

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Figure Captions

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Figure 1. APE-1, XRCC-1 and XPF immunoexpression in epithelial cells of benign epithelial odontogenic lesions and normal epithelial odontogenic tissue - dental follicle (DF). A-C, High expression of APE-1, XRCC-1 and XPF in ameloblastoma cases (scale – 100 µm). D-I, A strong expression of APE-1 (nucleus/ cytoplasm), XRCC-1 (nucleus) and XPF (nucleus/ cytoplasm) was identified in non-syndromic odontogenic keratocysts (NSOKCs) and syndromic odontogenic keratocysts (SOKCs) (scale – 50 µm). J-L, weak positive staining of

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APE-1, XRCC-1 and XPF in dentigerous cysts (DCs) (scale – 50 µm). M-O, Dental follicles (DFs) weakly positive for APE-1, XRCC-1 and XPF proteins (M,O, scale – 50 µm) (N, scale

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– 100 µm) (Hidef; DAB staining).

Figure 2. Increased expression of APE-1, XRCC-1 and XPF are observed in samples of ameloblastomas (AME), odontogenic keratocysts (NSOKCs) and syndrome odontogenic keratocysts (SOKCs). Median of the percentages for APE-1, XRCC-1 and XPF expression in epithelial cells + interquartile range (Kruskal Wallis test). There was a higher nuclear expression of APE-1 (A), XRCC-1 (B) and XPF (C) in NSOKCs, SOKCs and AMEs when compared to dentigerous cysts (DCs). The graph shows a significantly higher expression of

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nuclear APE-1 (A), nuclear XRCC-1 (B) and nuclear/ cytoplasmic XPF (C-D) in all epithelial odontogenic lesions in comparison to dental follicles (DFs). *p < 0.05; **p < 0.01; ***p <

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0.001; ****p < 0.0001.

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Tables

Table 1. Distribution of cases of benign epithelial odontogenic lesions according to clinical data. NSOKC

SOKC

DC

34.07 ±16.67

34.38 ±16.73

18.31 ±4.26

34.69 ±17.34

Female

19 (63.3%)

15 (50.0%)

16 (55.2%)

17 (56.7%)

Male

11 (36.7)

15 (50.0%)

13 (44.8%)

13 (43.3%)

Mandible

27 (90.0%)

23 (76.7%)

17 (58.6%)

21 (70.0%)

Maxilla

2 (10.0%)

7 (23.3%)

12 (41.4%)

9 (30.0%)

Age (in years)

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AME Sex

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Location

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AME, ameloblastoma. NSOKC, non-syndromic odontogenic keratocyst. SOKC, syndromic odontogenic keratocyst. DC, dentigerous cyst.

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Table 2. Sample size, Spearman’s correlation coefficient (r) and statistical significance (p) of the immunoexpression for APE-1, XRCC-1 and XPF in the epithelial component of AMEs, NSOKCs, SOKCs, DCs and DFs.

Pairwise comparison

NSOKC (n = 30)

pr

AME (n = 30) p

r

APE-1 (nucleus) × XRCC-1 (nucleus) APE-1 (nucleus) × XPF (nucleus) XPF (nucleus) × XRCC-1 (nucleus)

0.800 0.600 0.566

< 0.0001 < 0.0001 0.001

XPF (cytoplasm) × APE-1 (nucleus) XPF (cytoplasm) × XRCC-1 (nucleus) XPF (cytoplasm) × XPF (nucleus)

0.256 0.267 0.597

e-

r

Pr

0.239 0.571 0.319

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0.176 0.154 < 0.0001

0.356 0.465 0.459

SOKC (n = 29)

DC (n = 30)

DF (n = 20)

p

R

p

r

p

r

p

0.204 0.001 0.086

0.762 0.470 0.383

< 0.0001 0.01 0.041

0.631 0.622 0.318

< 0.0001 < 0.0001 0.086

0.188 0.498 -0.042

0.428 0.025 0.859

0.053 0.01 0.011

0.187 0.012 0.282

0.33 0.951 0.138

0.635 0.560 0.760

< 0.0001 0.001 < 0.0001

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APE-1 (cytoplasm) × XPF (nucleus) 0.206 0.274 0.223 0.244 APE-1 (cytoplasm) × XRCC-1 0.260 0.166 0.102 0.599 (nucleus) APE-1 (cytoplasm) × XPF (cytoplasm) 0.429 0.235 0.220 0.018 APE-1 (cytoplasm) × APE-1 (nucleus) 0.388 0.199 0.300 0.034 AME, ameloblastoma. NSOKC, non-syndromic odontogenic keratocyst. SOKC, syndromic odontogenic keratocyst. DC, dentigerous cyst. Significant differences are highlighted in bold.

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