Loss of heterozygosity and immunoexpression of PTEN in oral epithelial dysplasia and squamous cell carcinoma

Journal Pre-proof Loss of heterozygosity and immunoexpression of PTEN in oral epithelial dysplasia and squamous cell carcinoma

Filipe Nobre Chaves, Thâmara Manoela Marinho Bezerra, Debora Chaves Moraes, Sara Ferreira dos Santos Costa, Paulo Goberlanio Barros Silva, Ana Paula Negreiros Nunes Alves, Fábio Wildson Gurgel Costa, Vanessa Fátima Bernardes, Karuza Maria Alves Pereira PII:

S0014-4800(19)30247-3

DOI:

https://doi.org/10.1016/j.yexmp.2019.104341

Reference:

YEXMP 104341

To appear in:

Experimental and Molecular Pathology

Received date:

7 April 2019

Revised date:

28 September 2019

Accepted date:

11 November 2019

Please cite this article as: F.N. Chaves, T.M.M. Bezerra, D.C. Moraes, et al., Loss of heterozygosity and immunoexpression of PTEN in oral epithelial dysplasia and squamous cell carcinoma, Experimental and Molecular Pathology(2018), https://doi.org/10.1016/ j.yexmp.2019.104341

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© 2018 Published by Elsevier.

Journal Pre-proof Tittle Page Original Article Loss of heterozygosity and immunoexpression of PTEN in oral epithelial dysplasia and squamous cell carcinoma.

Authors and name affiliations Filipe Nobre Chaves1 , Thâmara Manoela Marinho Bezerra 2 , Debora Chaves Moraes 3 , Sara Ferreira dos Santos Costa4 , Paulo Goberlanio Barros Silva 2 , Ana Paula Negreiros Nunes Alves 2 , Fábio Wildson Gurgel Costa 2 , Vanessa Fátima Bernardes 5 , Karuza Maria Alves Pereira 6* 1

School of Dentistry, Federal University of Ceará Campus Sobral, Sobral, Brazil Department of Dental Clin ic, Facu lty of Pharmacy, Dentistry and Nursing, Federal University of Ceará, Fortaleza, Brazil 3 Department of Surgery, School of Medicine, Federal University of Minas Gerais, Belo Horizonte, Brazil 4 Department of Oral Surgery and Pathology, School of Dentistry, Federal University of Minas Gerais, Belo Horizonte, Brazil 5 Department of Pathology, Biological Sciences Institute, Federal Un iversity of Minas Gerais, Belo Horizonte, Brazil 6 Departament of Morphology, School of Medicine, Federal University of Ceará, Fortaleza, Brazil

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*Correspondence author: PhD. MSc. DDS. Karuza Maria Alves Pereira Departament of Morphology, School of Medicine

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Federal University of Ceará

Rua Delmiro de Farias s/n - Rodolfo Teófilo - CEP 60416-030 Fo rtaleza CE, Brazil.

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Phone / Fax Number: +55 85 3366 8471.

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E-mail: [email protected]

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Word count: 2874 (excluding abstract and references) Conflict of interest statement: None declared. Running title: LOH of PTEN in OED and OSCC

Journal Pre-proof LOSS OF HETEROZYGOSITY AND IMMUNOEXPRESSION OF PTEN IN ORAL EPITHELIAL DYSPLASIA AND SQUAMOUS CELL CARCINOMA.

ABSTRACT Introduction: Oral epithelial dysplasia (OED) is a risk factor for developing subsequent oral squamous cell carcino ma (OSCC). Loss of heterozygosity (LOH) pro files have been validated as risk predictors of malignant transformation of OED. It is still unclear if Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) allelic loss also occurs in initial stage malignant lesions and if the allelic loss is involved as one of the mechanisms of oral carcinogenesis. Thus, this study objective investigate LOH of PTEN gene and the immunohistochemical expression of the protein in OED and OSCC samples . Material and methods: Formalin-fixed paraffin -embedded samples of 19 OEDs and 16 OSCCs were

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included to immunohistochemistry and LOH analysis. Two poly morph ic microsatellite markers

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(AFMA086W G9 and D10S1765) located in chro mosome 10 were used in this study for LOH analysis. For immunohistochemical analysis, 5 random fields with 400x magnification were evaluated quantitatively and qualitatively in epithelial and neoplastic cells .

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Results: AFMA086W G9 marker only demonstrated LOH in OEDs cases (10.5%). D10S1765 marker demonstrated LOH in 57.2% of OEDs and 50% of OSCCs. Higher nuclear immunostaining was detected

immunoexpression in OSCCs (p <0.045).

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in cases of OSCCs when co mpared to OEDs (p <0.001) and there was strong cytoplasmic

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Conclusions: We provide ev idence that the allelic loss of PTEN is present in premalignant oral lesions and OSCCs, however the LOH of PTEN does not seems to influence its protein expression.

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Immunohistochemistry.

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Keywords: Loss of heterozygosity; Oral epithelial dysplasia; Oral squamous cell carcinoma; PTEN;

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Journal Pre-proof INTRODUCTION Cancer is a of global public health problem because of its high incidence and mortality. Oral cancer has the sixth highest mortality rate among cancers , and the oral squamous cell carcino ma (OSCC) is the most frequent subtype (Dantas et al., 2016). Potentially malignant lesions (PM Ls) are associated with oral carcinogenesis. OSCC has been documented in association with or preceded by PM Ls, and their presence is a risk factor for the development of OSCC. Histopathological analysis of PMLs can show epithelial dysplasia to predict malignancy (Warnakulasuriya et al., 2008). Ho wever, progression to a malignant phenotype is not observed in most PMLs . Additionally, the reproducibility of the microscopic classification criteria for dysplastic lesions is low, which p resents significant difficulties among pathologists (Go mes et al., 2015). Thus, the identification of the mo lecular markers that may signal

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specific behaviors and the malignant transformation is very important.

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The PI3K/AKT signaling pathway is one the most dysregulated pathways in cancer; its main substrates are PIP3 (Phosphatidylinositol triphosphate) and AKT (Protein kinase B), which are important for cell growth, proliferation and survival (Wang et al., 2015; Li et al., 2014). PTEN (Phosphatase and

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tensin homologue deleted on chromosome 10) is a tumor suppressor gene that acts in this pathway through the dephosphorylation of PIP3, leading to the activation of AKT via phosphoinositide dependent

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kinase 1 (PDK 1) protein. The loss of PTEN has been reported in a variety of PM Ls and cancers, including OSCC (Sn ietura et al., 2012). Recent studies have shown that allelic loss or loss of

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heterozygosity (LOH) is involved in oral leu koplakias malignant transformat ion prediction (FonsecaSilva et al., 2016; Go mes et al., 2015), since it is involved in the tumor suppressor genes functional loss (Couto, 2011; Velickovic et al., 2002). So me studies have shown that LOH in the 10q-23.3 locus, where

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the PTEN gene is located, is present in head and neck cancers such as OSCC (Bettendorf et al., 2008; Bae

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et al., 2007; Mavros et al., 2002). However, it is still unknown whether PTEN allelic loss also occurs during the initial stage in malignant lesions or whether allelic loss is involved as one of the mechanisms

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of oral carcinogenesis.

On this basis, we perfo rmed LOH analysis on two PTEN ch ro mosomal regions (intergenic and intragenic) in oral epithelial dysplasia (OED) and OSCC samples with different histological malignancy grades, as well as PTEN immunohistochemical analysis to evaluate whether the LOH of this gene is involved in its protein expression in different OED and OSCC histological stages. This study also sought to verify whether the process of allelic loss occurs similarly between malignant lesions at different stag es of malignancy or potentially malignant disorders during the malignancy process.

MATERIAL AND METHODS

Sample selection and grading This study was approved by the Institutional Ethics Co mmittee on Research Involving Hu mans (Protocol n o 94432). Samp les of 19 OEDs and 16 OSCCs paraffin -embedded tissues were obtained from patients biopsies of the Stomatology Clinic of the Federal University of Ceará Sobral Campus. Samp les were collected fro m January 2012 to December 2015. OEDs specimens were classified by binary system (low and high risk) of grading dysplasia for prediction of malignant transformat ion (Warnakulasuriya et

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Journal Pre-proof al., 2008), and the OSCCs samples were graded and classified in lo w and h igh grade of malignancy (Silveira et al., 2007). None of the patients with OED had a previous history of oral cancer. Five samp les of normal oral mucosa were included in the study for control. These samples were specimens resulting fro m excisional biopsy mucoceles in which inflammat ion is restricted to the granulation tissue and not at mucosal lining epithelium.

DNA extraction Histological sections were reviewed and examined in order to identify the presence of epithelial tissue and lamina propria / submucosa in the OEDs, as well as tumor parenchyma and stroma in OSCCs. In OSCC, the representative areas of normal tissues (non-tumor DNA) for microdissection were the tissues of the stroma reg ion. Dysplastic epitheliu m and tu mor parenchyma, lamina propria and non-tumor tissue areas were separated by manual microdissection using an optical microscope and a surgical blade

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with series of 20-μm-thick paraffin-embedded tissue sections. From microdissected tissues, genomic DNA was extracted using QIAamp DNA FFPE Tissue Kit (Qiagen, Valenc ia, CA, USA), fo llo wing the

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manufacturer’s protocol. Loss of heterozygosity analysis

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LOH was investigated using two microsatellite markers for genetic loci flan king reg ions of chromosome 10q23.3 (AFMA 086W G9) and 10q 23.31 (D10S1765) as previously described (Fonseca-

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Silva et al., 2016). PCRs were performed in the thermocycler (Eppendorf Mastercycler gradient) under the follo wing conditions: Init ial denaturation at 95 °C for 10 minutes, followed by 45 cycles of amp lification (denaturation at 96 °C for 10 seconds, annealing at 64° C (D10S1765) or 58 °C

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(AFMA086W G9) for 30 seconds and extension at 70° C for 30 seconds followed final extension of 70° C for 30 seconds.

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All PCRs were performed for non-lesional and lesional tissues under the same parameters, using a 15 μL react ion mixture containing 4 μL of DNA temp late, 3.75 μL of deoxynucleoside triphosphate,

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0.75 μL o f magnesium chloride, 1.5 μL of commercial PCR buffer, 0.25 μL of each primer (forward and reverse) (Applied Biosystems, Foster City, CA, EUA), and 0.12 μL of Taq DNA Po limerase Reco mbinante (Thermo Fisher Scientific, Wilmington, EUA ). The amp lified PCR products were visualized in 8% polyacrylamide gel and subsequently detected using ABI PRISM 3130 (Applied Biosystems, Foster City, CA, USA) and analyzed using GeneMapper software version 4.0 (Applied Biosystems). LOH was calcu lated as the ratio between the short allele non-lesional/normal tissue (Sc) and long allele non-lesional/normal t issue (Lc) d ivided by the ratio between the short allele epitheliu m / lesion (Se) and the long allele ep itheliu m / lesion (Le) using the following formu la: (Sc:Lc)/(Se:Le). LOH was scored when an allele was decreased by more than 50% in the epitheliu m / lesion sample co mpared to the same allele in non-lesional/normal tissue. Samp le was considered non-informative (NI) when DNA fro m non-tumor tissue was homo zygous for the poly morphic marker or in cases that generated

non-

interpretable results. Samples with only one peak in the connective tissue were considered homozygous. Ho mozygous cases and those samples that even after two independent PCRs showed unclear results were considered noninformative. The frequency of allelic loss (FA L) was calcu lated for each sample by dividing the nu mber of markers that showed allelic loss by the number of informat ive markers (Go mes et al., 2015; Correa et al., 2015).

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Journal Pre-proof Immunohistochemistry Briefly, 3-µm-th ick histological sections were cut fro m paraffin-embedded tissue block p repared in glass slides. The antigenic ret rieval was performed by immersing the slides in 10 mmol/ L cit rate buffer (pH=6.0) in a Pascal water bath. Endogenous peroxidase activity was blocked for 30 minutes with 0.3% hydrogen pero xide fo llo wed by 1% protein b locking for 10 minutes. The sections were incubated with primary antibodies anti-PTEN (Rabbit polyclonal, Abcam®, [ab31392], at 1:400 d ilution, overnight at 4°C. The samp les were then incubated with the secondary antibody LSAB Kit (DA KO®, Carpentaria, CA, USA) for 30 minutes at room temperature and the reaction was revealed with a chromogen solution of 3-3’-d iaminobenzidine (Sig ma, St. Lou is, MO, USA) fo r 5 minutes in a dark chamber and Harris hematoxylin was used for counterstaining. coverslips

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samples

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slides,

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Finally,

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examined under Leica DM 2000 optical microscope. Breast carcino ma was included as positive control

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and negative control consisted in omission of primary antibody.

Evaluati on of immunohistochemistry

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Heterogeneous and homogeneous immunostaining were observed in OEDs and OSCCs, thus, due to the regional differences, the fields chosen for the photomicrography were the ones that presented

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the greatest immunostaining (hot spot). Five fields were selected, visualized and captured at 400x magnificat ion with a Leica DFC295 HD digital camera using Las software (Meyer Instruments, Houston, USA) at maximu m resolution.

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Qualitative and quantitative analyses of immunostaining were performed simultaneously on each field according to a methodology adapted from Chaves et al. using Image J software (National Institutes

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of Health, Bethesda, Maryland, USA ) (Chaves et al., 2017). Analysis consisted of counting the number of positive cells in each field and quantifying the intensity (no staining, weak, moderate or strong staining)

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and the cellu lar location (nucleus and/or cytoplasm). The data gave normal variat ion and the positive cell average was determined in every case for possible statistical analysis. Two authors carried out the analysis at separate times wh ile unaware of the clinical h istopathological data, with a concordance coefficient of 0.86 (86%).

Statistical analysis The data were statistically analy zed using the Statistical Package for the Social Science software version 20.0 (SPSS, Ch icago, IL). The Kruskal-Wallis statistical test follo wed by Dunn's post-test and the Mann-Whitney test was used for immunohistochemical analysis of this study. Both the molecu lar analysis and the association between molecular and immunohistochemical data were seen through the Fisher tes t. All results were considered statistically significant when p <0.05.

RESULTS

Immunohistochemical Analysis

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Journal Pre-proof This research aimed to perform a detailed immunohistochemical analysis of intracellu lar localization of PTEN, once this can significantly affect your cell behaviour. PTEN immunohistochemical analysis revealed nuclear and cytoplasmic immunostaining in OEDs, OSCCs and control groups (normal oral mucosa). The PTEN immunostaining pattern showed both homogeneous as heterogeneous (positive and negative areas) in samp les of OSCCs and OEDs (Figure 1 ). Breast adenocarcinoma was used as positive control which showed homogeneous immunostaining pattern. PTEN may be present in the nucleus of the cell, playing an important role in the regulation of nuclear ho meostasis and the maintenance of their mo rphology. In present study, the average number of cells with nuclear immunostaining was significantly higher in OSCCs compared to OEDs and control (p < 0.001). The average number of cells with cytoplasmic immunostaining was also significantly higher in OSCCs co mpared to OEDs and controls (p = 0.012), there was a p redominance of strong cytoplasm staining in OSCCs more than in OEDs (p =0.045) (Table 1). We speculate that a fact that may exp lain the

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high immunostaining of PTEN in OSCC samples in th is study comes fro m genetic alterat ions of this gene as a translational modifications that may suffer after PTEN gene transcription. Thus, the nuclear

anti-tumour role of this protein in this cell location.

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immunostaining of PTEN in samples of OSCCs this research does not necessarily point to an effect ive

In figure 2, we show the individual patient data on a scatter dot plot to show the inter individual

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variation.

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LOH Analysis

For the microsatellite marker AFMA086W G9, LOH was detected in 2 OED cases and was not observed in the OSCC cases. Among the OED cases, there were significantly more cases showing LOH

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for D10S1765 (57.14%) than AFMA086W G9 (10.52%) in PTEN (p = 0.003). Similar findings were also observed in OSCC cases, as LOH was only detected for D10S1765 (p = 0.007) (Table 2). Additionally,

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only one case of OED showed LOH in both markers (sample 11). The greater instability of the PTEN intergenic marker is also observed, as the examined OEDs

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and OSCCs show a significant increase in the allelic loss frequency in the D10S1765 marker co mpared to the AFMa086W G9 marker. LOH in PTEN d id not change with the h istopathological grades of OEDs and OSCCs. Ho wever, in OSCCs cases, LOH was only observed for the D10S1765 marker in 8 (57.2%) OED cases and 4 (50.0%) OSCC cases and was restricted to cases of high-grade malignancy (p = 0.011) (Table 2). The OSCC group showed a greater nu mber of cells with nuclear immuno staining than OED with LOH (p = 0.003). The Table 3 shows the number of PTEN-positive cells in the nucleus and cytoplasm for the microsatellite marker D10S1765. The allelic loss of PTEN in OEDs examined in this study was not involved in their malignant processes; in OEDs. The allelic loss appears to have occured independent of the histological grade of the lesion. Moreover, there was no association between PTEN cytoplasmic immunostaining among the groups (p = 0.503) (Table 3). DISCUSSION It has been established that LOH occurs in several ch ro mosome reg ions in tumor suppressor genes (TSGs) and is involved in the malignant transformation of PM Ls (Fonseca-Silva et al., 2016; Go mes et al., 2015; Zhang et al., 2012) via the inactivation of TSG (Couto, 2011). Thus, LOH can be an important tool for genomic characterization during the tumoral progression in many cancers (Xi and

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Journal Pre-proof Chen, 2015; Sn ietura et al., 2012), including oral cancer (Zhang et al., 2012; Correa et al., 2015; Go mes et al., 2015; Fonseca-Silva et al., 2016). Ho wever, few studies have shown whether allelic loss of PTEN also occurs in early stage malignancies or whether allelic loss is involved as one of the mechanis ms of oral carcinogenesis, which is still unknown. Additionally, it is unclear whether LOH pro files in the PTEN gene are related to its protein expression and oral carcinogenesis. Based on these results, we investigated LOH in PTEN and its immunoexp ression in OSCCs and OEDs. This study evaluated LOH in the PTEN gene by selecting microsatellite markers located near or within chro mosome 10q23-24 (Couto, 2011), including an intragenic marker (A FMA086W G9) and an intergenic marker (D10S1765); a sample with gene instability was one that presented LOH at 10q-23 in at least one microsatellite marker (Idoate et al., 2014). It has been suggested that the loss of the tumor suppressor function of PTEN occurs in the initial stages of oral carcinogenesis (Angadi and Krishnapillai, 2012). This phenomenon has been reported in

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endometrial cancers, in which PTEN mutations were observed in the init ial stages and were detected in PMLs (Mutter, 2002). Interestingly, we observed LOH in both markers in OED cases. Additionally, there was less nuclear and cytoplasmic PTEN protein expression in OEDs than in OSCCs. Th is corroborates

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the idea that functional and structural losses can occur at initial stages in the oral carcinogenesis process . This finding was confirmed in a study conducted by Miyahara et al., where PTEN allelic loss occurred in

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34.25% of o ral leukoplakia cases (Miyahara et al., 2018) and by Correa et al., where LOH occurred in 56.3% of actin ic cheilitis cases (Correa et al., 2015). The allelic loss of PTEN in the investigated OEDs

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cases appears to occur independently of the histological grade o f the lesion. Similarly, M iyahara et al. d id not observe differences in allelic loss between leukoplakia with mild OED and moderate and severe OED (Miyahara et al., 2018).

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The instability of the PTEN intergenic marker can be observed in the present study; the examined OEDs and OSCCs showed a significant increase in the allelic loss frequency in the D10S1765

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marker co mpared to AFMa086W G9 marker. The intragenic marker used in this study showed LOH in 10.5% OEDs samples, with 100% of OSCCs samples showing allelic retention. The intergenic marker

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showed LOH in 57,2% OED samples and in 50.% OSCC samples. We observed that LOH occurred more frequently in intergenic areas, and intragenic LOH was not observed in OSCCs, which indicates that this region is genetically more stable than the intergenic regions (VanHouten et al., 2000; Wang et al., 2015). Mavros et al. and Chakraborthy et al. did not find mutations in the PTEN coding region (Chakraborthy et al., 2008; Mavros et al., 2002). In the study, LOH in OSCC was only present in the intergenic regions, and in the cases of high grade of malignancy, which suggests that other genes found in the same intergenic regions may be involved in the tu morigenesis of the analyzed OSCC cases and that PTEN LOH favors tu moral progression (Papa et al., 2014). A similar result was observed by Mavros et al. in OSCCs patients (Mavros et al., 2002), Bae et al. in patients with hepatocellular carcinoma patients (Bae et al., 2007), and by Velickovic et al. in patients with clear cell renal carcino ma patients (Velickovic et al., 2002). Moreover, epigenetic regulation of PTEN may also occur, wh ich can be more significant than structural changes in the PTEN gene. This hypothesis is based on the fact that hypermethylation of the PTEN promoter region may occur in OSCCs and is an important epigenetic silencing mechanis m that may contribute to oral carcinogenesis (Sushma et al., 2016). Furthermo re, PTEN mutations may not occur in

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Journal Pre-proof head and neck cancer cases (Cohen et al., 2011; Kurasawa et al., 2008; Mavros et al., 2002; Snaddon et al., 2001; Shao et al., 1998). Interestingly, in th is study we d id not observe intragenic LOH in OSCCs and the intergenic allelic retention rate of OSCCs was higher than in OEDs. This may indicate that in the samp les of this study other carcinogenic mechanisms are present in already established malignant lesions. Another possibility is that particular loci harbor mult iple TSGs (Ry land et al., 2015), thus other genes may be involved in the OSCC carcinogenesis process. There are other ways to regulate PTEN than LOH. Genetic alterations including point mutations, large chro mosomal delet ions, and epigenetic mechanisms, such as hypermethylation, can silence PTEN (Molinari and Frattini, 2014). In addition, PTEN inactivation may occur by other non-structural alterations affect ing transcript stability, protein stability and differential subcellular co mpart mentalization. For examp le, the PTEN mRNA could be post-transcriptionally regulated by Pero xiso me Proliferat ion-Activated Receptor γ (PPARγ), Early Gro wth Response protein 1

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(EGR1), p53, PTEN-targeting microRNAs (such as miR19 and miR21) and also PTEN pseudogene (PTENP1) (Song et al., 2012). These mult iple forms of regulation may be difficult to assess in a single signaling pathway, which is a limitation of present study. In addition, the low sample quantity as well as

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the absence of pair-wise LOH co mparisons of OED and OSCC samples may also have affected this result.

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The allelic retention in OSCC cases and the apparent lack of association between LOH and oral carcinogenesis may occur due to a broad variety of signaling abnormalit ies in different tissues in the same

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oral cavity microenviron ment. In our study, we analyzed oral cavity cases in different locations. One the limitat ion of the present study might be that pairwise LOH co mparisons of OED and OSCC samp les were not performed.

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We performed a detailed immunohistochemical analysis of the intracellular location of PTEN because this factor may significantly affect its cellular ro les (Liu et al., 2007). We observed that OSCC

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samples showed a significant increase in nuclear immunostaining co mpared with OED and control samples. The nuclear location of PTEN has been associated with cell growth inh ibition , maintenance of

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cell chro mosome stability, regulation of nucleolar ho meostasis and maintenance of nucleolar mo rphology (Wang et al., 2015; Li et al., 2014; Liu et al., 2007). In the nucleus, PTEN exh ib its activities independent of AKT, wh ich in turn interferes with the suppressive activity of cell gro wth (Wang et al., 2015; Liu et al., 2014). In our previous study (Chaves et al., 2017), we observed higher AKT expression in OSCCs, and in the present study, we observed nuclear PTEN expression in OSCC. Regarding the increase in nuclear PTEN expression, as can be observed for the OSCCs samples, this increase may be explained as an attempt to control the elevated expression of AKT, as has been suggested by Miyahara et al. in dysplastic lesions (Miyahara et al., 2018), because this proportionally increases with the malignancy of oral lesions (Chaves et al., 2017). Nuclear PTEN is required fo r cell cycle arrest (Liu et al., 2007), suggesting an attempt to exert its tumor-suppressing functions in established malignant lesions with high AKT activity. Moreover, nuclear PTEN was more conspicuous in the OSCC samples with LOH than in OED. Thus, LOH might affect PTEN cellu lar patterns, showing that there is a clear tumor suppressor role for PTEN in the establishment of malignant lesions and changes in the allelic balance. It seems that the PTEN gene only loses its tumor suppressor function in the more advanced stages of oral carcinogenesis

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Journal Pre-proof In conclusion, the present study provided evidence that allelic loss of PTEN occurred in OED and OSCC cases. LOH of PTEN appears to occur in the init ial stages of oral carcinogenesis development , and the allelic retention in intragenic regions might be responsible for maintaining a positive PTEN regulation. Additionally, the heterogeneity of PTEN protein exp ression in the analyzed OED and OSCC samples, regardless of their histological grade, was demonstrated, and PTEN localization may be crit ical to its function.

ACKNOWLEDGEMENTS The authors would like to thank Prof. Dr. Lu iz Armando Cunha de Marco fro m the Laboratory of Molecular Medicine of the Faculty of Medicine of the Federal University o f M inas Gerais, Brazil, for the contribution in physical structure of his Laboratory.

The authors declare that they have no conflict of interest. Ethical approval

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Conflict of interest

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This article does not contain any studies with human participants or animals performed by any of the authors.

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Informed consent

For this type of study, formal consent is not required.

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Funding

This work was supported by grants fro m Conselho Nacional Desenvolvimento Científico e Tecnológico

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Mavros, A., et al., 2002. Infrequent genetic alterations of the tumor suppressor gene PTEN/MMAC1 in squamous cell carcinoma of the oral cavity. J Oral Pathol Med. 31, 270-276. https://doi.org/10.1034/j.1600-0714.2002.310504.x Miyahara, L.A.N., et al., 2018. PTEN allelic loss is an important mechanism in the late stage of development of oral leukoplakia into oral squamous cell carcinoma. Histopathology. 72, 330-8. https://doi.org/10.1111/his.13381. Molinari, F., Frattini, M., 2014. Functions and Regulation of the PTEN Gene in Colorectal Cancer. Front Oncol. 16, 326. https://doi.org/10.3389/fonc.2013.00326. Mutter, G.L., 2002. Diagnosis of premalignant endometrial disease. J Clin Pathol. 55, 326-331. Papa, A., et al., 2014. Cancer-associated PTEN mutants act in a dominant-negative manner to suppress PTEN protein function. Cell. 157, 595-610. https://doi.org/10.1016/j.cell.2014.03.027. Ryland, G.L., et al., 2015. Loss of heterozygosity: what is it good for? BMC Med Genomics. 1, 45. https://doi.org/10.1186/s12920-015-0123-z. Shao, X., et al., 1998. Mutational analysis of the PTEN gene in head and neck squamous c ell carcinoma. Int J Cancer. 77, 684–688. https://doi.org/10.1002/(SICI)1097-0215(19980831)77:5<684::AID-

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Journal Pre-proof IJC4>3.0.CO; 2-R. Silveira, E.J., et al., 2007. Correlation of clinical, histological, and cytokeratin profiles of squamous cell carcinoma of the oral tongue with prognosis. Int J Surg Pathol. 15,376-383. https://doi.org/10.1177/1066896907304992. Snaddon, J., et al., 2001. Detection of functional PTEN lipid phosphatase protein and enzyme activity in squamous cell carcinomas of the head and neck, despite loss of heterozygosity at this locus. Br J Cancer. 84, 1630–1634. https://doi.org/10.1054/bjoc.2001.1848. Snietura, M., et al., 2012. PTEN as a prognostic and predictive marker in postoperative radiotherapy for squamous cell cancer of the head and neck. PLoS One. 7, :e33396. https://doi.org/10.1371/journal.pone.0033396. Song, M.S., Salmena, L., Pandolfi. P.P., 2012. The functions and regulation of the PTEN tumour suppressor. Nat Rev Mol Cell Biol. 13, 283-296. https://doi.org/10.1038/nrm3330.

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Sushma, P.S., et al., 2016. PTEN and p16 genes as epigenetic biomarkers in oral squamous cell carcinoma (OSCC): a study on south Indian population. Tumor Biol. 37, 7625–7632. https://doi.org/10.1007/s13277-015-4648-8.

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VanHouten, V.M., et al., 2000. Molecular assays for the diagnosis of minimal residual head-and-neck cancer: methods, reliability, pitfalls, and solutions. ClinCancer Res. 6, 3803-16.

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Velickovic, M., et al., 2002. Intragenic PTEN/MMAC1 loss of heterozygosity in conventional (clear-cell) renal cell carcinoma is associated with poor patient prognosis. Modern Pathol. 15,479-485.

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https://doi.org/10.1038/modpathol.3880551

Wang, X., Huang, H., Young, K.H., 2015. The PTEN tumor suppressor gene and its role in lymphoma pathogenesis. Aging (Albany NY). 7, 1032-1049. https://doi.org/10.18632/aging.100855

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Warnakulasuriya, S., et al., 2008. Oral epithelial dysplasia classification systems: predictiv e value, utility, weaknesses and scope for improvement. J Oral Pathol Med. 37, 127-133.

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https://doi.org/10.1111/j.1600-0714.2007.00584.x. Xi, Y., Chen, Y., 2015. Oncogenic and therapeutic targeting of PTEN loss in bone malignancies. J Cell

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Biochem. 116, 1837-1847. https://doi.org/10.1002/jcb.25159. Zhang, L., et al., 2012. Loss of heterozygosity (LOH) profiles —validated risk predictors for progression to oral cancer. Cancer Prev Res (Phila). 5, 1081-1089. https://doi.org/10.1158/1940-6207.CAPR12-0173

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Journal Pre-proof TABLES Table 1: Mean percentage of immunopositive cells in cytoplasm and nucleus of oral epithelial dysplasia and oral squamous cell carcinoma. Samples / Group Control

OED

OSCC

p-Value

9.56 ± 13.11

1.25 ± 2,48

17.08 ± 10.42* †

PTEN / Staining Nuclear Cytoplasm

93.77 ± 9.44

93.39 ± 13,56

98.58 ± 3.01*

† †

70.80 ± 30.74

57.43 ± 16,17

69.37 ± 13.04

Cytoplasm (weak)

22.97 ± 22.87

36.04 ± 10,02

29.21 ± 11.51

a

0.012

a

0.045 a

0.054

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OED: oral epithelial dysplasia; OSCC: oral squamous cell carcinoma Control tissues (n=5); OED tissues (n=19); OSCC tissues (n=16). *p<0,05 versus Control, †p<0,05 versus OED; a Kruskal-Wallis/Dunn test (mean + SD).

<0.001

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Cytoplasm (strong)

a

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Journal Pre-proof Table 2: Loss of heterozygosity in OED and OSCC according to histopathological grading AFMA086WG9

D10S1795

Allelic retention

LOH

Allelic retention

LOH

OED

17 (89.5 %)

2 (10.5 %)

6 (42.8 %)

8 (57.2 %)

OSCC

16 (100 %)

0 (0.0 %)

4 (50 %)

4 (50 %)

c

p-Value

c

0.498

1 (9.1%)

High risk

7 (87.5 %)

1 (12.5%)

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OSCCb grading

0.007*

4 (50.0%)

4 (50.0 %)

c

0.111

2 (33.3%)

4 (66.7 %)

c

0.090

c

1.000

c

0.287

0 (0.0 %)

1 (100.0 %)

0 (0.0 %)

11 (100.0 %)

0 (0.0 %)

3 (42.8 %)

4 (57.2 %)

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5 (100.0 %)

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p-Value

c

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1.000

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c

p-Value

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10 (90.9 %)

High grade

0.003*

1.000

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Low risk

Low grade

c

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OEDa grading

p-Value

c

1.000

c

c

0.011*

1.000

OED: oral epithelial dysplasia; OSCC: oral squamous cell carcinoma *p<0,05, cFisher. a According by Warnakulasuriya et al. b According by Silveira et al. Data expressed as absolute and percentage frequency

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Journal Pre-proof Table 3: Loss of heterozygosity in OED and OSCC for marker D10S1765 in relation to nuclear and cytoplasmic immunoexpression of PTEN. Allelic retention PTEN / Staining (% ) Nuclear Cytoplasm

LOH

OED

OSCC

p-Value

2.69 ± 4.03

10.25 ± 9.11

a

86.85 ± 23.70

97.29 ± 3.13

a

OED

OSCC

p-Value

0.262

0.33 ± 0.68

15.71 ± 6.75

a

0.381

97.50 ± 1.87

97.07 ± 5.44

a

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OED: oral epithelial dysplasia; OSCC: oral squamous cell carcinoma *p<0,05, aMann-Whitney test (mean + SD).

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0.003

0.503

Journal Pre-proof FIGURES Figure 1: (A-E) Hemato xylin and eosin staining (200x) and immunostaining of PTEN (F-J) in OED, OSCC and normal mucosa. (F) Ho mogeneous immunostaining is observed in the normal mucosa sample (200x). (G and H) Heterogeneous cytoplasmic and nuclear immunostaining in OSCC low and high grade (400x). Heterogeneous areas of PTEN immunoexpression in OED: (I) Strong nuclear labeling in low-risk OED (400x). (J) Absence of nuclear labeling in high-risk OED (400x).

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Figure 2: Individual patient data on a scatter dot plot showing the inter individual variation.

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Journal Pre-proof Highlights (bullet points) -

Loss of heterozygosity of PTEN is involved in the OLs malignant transformation prediction.

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The cell location of the PTEN proteins has involved in its function in the OED and OSCC.

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Observed presence of heterogeneity in protein expression of PTEN in the samples evaluated.

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LOH of PTEN seems not to influence its protein expression

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Figure 1

Figure 2