p16^INK4a, and p14^ARF Expressions in Carcinogenesis of Squamous Cell Carcinoma of the Lip

Ayse Nur Akatli^1,2*, Ebru Sebnem Ayva^1,3, Onder Bozdogan^1,4

¹Department of Pathology, Kırıkkale University School of Medicine, Kırıkkale, Turkey. ²Department of Pathology, Inonu University School of Medicine, Malatya, Turkey. ³Department of Pathology, Baskent University School of Medicine, Ankara, Turkey. ⁴Department of Pathology, Ankara Gülhane Training and Research Hospital, Ankara, Turkey.

Abstract

The goal of this study was to clarify the role of p16^INK4a, p14^ARF, and p53 protein expressions in carcinogenesis in squamous cell carcinomas of the lip. The expressions of the p53, p16^INK4a, and p14^ARF proteins were examined in 46 formaline-fixed paraffin embedded tissue specimens, which included 19 cases of squamous cell carcinoma of the lip, 14 cases of actinic cheilitis, and 13 cases of normal mucosa. Immunoreactivity in the peritumoral epithelium adjacent to squamous cell carcinomas was also evaluated. p16^INK4a expression was increased in actinic cheilitis in comparison with normal mucosa (p=0.001). p14^ARF expression progressively increased from normal mucosa to actinic cheilitis (p=0.001) and was observed to decrease significantly during the process of transition from actinic cheilitis to carcinoma (p=0.003). p53 values progressively increased from normal mucosa to actinic cheilitis (p=0.001) and carcinoma (p=0.008). A significant positive correlation was found between p14^ARF and p53 in the peritumoral epithelium adjacent to carcinomas. Our findings indicated that p16^INK4a and p14^ARF immunohistochemistry does not determine whether or not actinic cheilitis has the potential to develop carcinoma. The p14^ARF /p53 pathway is activated in the peritumoral epithelium adjacent to carcinoma; however, this activation would not be adequate to prevent carcinogenesis.

Keywords: Actinic cheilitis, Carcinogenesis, Lip, p16^INK4a, p14^ARF

The patterns of p16^INK4a, p14^ARF, and p53 expression in invasive islands of SCC are summarized in (Table 3). Four cases in the SCC group demonstrated immunostaining for p16 around invasive islands. We observed focal scattered staining in four cases, a diffuse staining pattern in one case, and immunostaining in the superficial areas in four cases for p16.

Regarding p14^ARF positivity in the SCC group, 16 cases showed immunostaining at the basal aspect of the invasive islands and three cases showed a scattered staining pattern.

Regarding p53 positivity, 16 cases showed a staining pattern in the form of one to two lines in the basal layer at the periphery of the invasive islands, while three cases did not show immunoreactivity.

In the SCC group, all of the p16^INK4a negative cases demonstrated positive staining with p14^ARF and p53. The three p53 negative cases were found to show positive staining with p16 and p14^ARF.

Descriptive statistical values for the H-Scores of p16^INK4a, p14^ARF, and p53 expressions in all groups are presented in (Table 4). The distribution characteristics of the H-Score values of antibody expressions are depicted by a box-and-whisker plot (Figure 2).

The highest mean H-Score of p16^INK4a expression was higher in the AC and SCC groups compared to the C group (p=0.001, p=0.005).

*PE of SCC (Peritumoral epidermis of SCC)

The highest mean H-score of p14^ARF expression was found to be higher in the AC and SCC groups compared to the C group (p=0.001, p=0.001). The highest mean H-score of p14^ARF expression was determined to be lower in the SCC group compared to the AC group with statistical significance (p=0.003).

The highest mean H-score of p53 expression was found to be higher in the AC and SCC groups. p53 expression was determined to be higher in the SCC group than in the AC group (p=0.008).

The comparison of p16^INK4a, p14^ARF, and p53 expressions between the peritumoral epidermis and the tumor in cases with SCC revealed no significant differences concerning p16^INK4a, a decrease in p14^ARF expression in tumoral areas compared with the higher levels in peritumoral areas, and a greater expression of p53 in tumoral areas (p>0.05, p=0.007, p=0.005).

The comparisons made between the peritumoral epithelium and cases of AC without an accompanying tumor revealed no significant differences in terms of the H-Score values associated with the expressions of the three antibodies (p>0.05).

Spearman’s correlation analysis revealed no significant relationship between clinicopathological parameters and expressions of p16^INK4a, p14^ARF, and p53. No correlation was determined between the SCC, AC, and C groups in terms of p16^INK4a, p14^ARF, and p53 expressions. However, in SCC cases, a positive correlation was noted between p14^ARF and p53 expressions in the peritumoral epithelium (p=0.011).

The majority of studies conducted on head and neck cancers and oral SCCs using p16^INK4a, p14^ARF and p53 have focused on tumors in different localizations. SCCs originating from the lip were usually studied as a sub-group in these studies.^{[12, 25, 26]} The literature on SCC of the lip contains studies showing positive correlations for p53/Ki67 and CD44/VEGF in the assessment of the prognosis and progression of tumor evolution.^[27] Molecular changes such as homozygous deletion, promoter methylation and mutation have been found in INK4A-ARF genes in the tumor and tumor free margins of oral cancers.^[28] In this study, we compared the proteins produced by the genes p53, p16^INK4a, and p14^ARF in cases of AC, and investigated the changes associated with the SCC of the lip. To the best of our knowledge, this study is the first that investigates all of these three proteins in SCCs originating from the lip.

Inactivation of the p16^INK4a gene is prevalent in head-neck and oral SCCs and this inactivation is believed to be brought about by a variety of mechanisms including homozygous deletion, DNA methylation, and point mutation.^[28-31] Various ideas exist regarding the immunohistochemical evaluation of p16^INK4a gene inactivation. Sanchez-Cespedes et al. argue that immunohistochemical loss of p16^INK4a is connected to the inactivation of the p16^INK4a gene.^[32] Losses in p16^INK4a expression have also been observed in premalignant oral lesions and oral cavity tumors.^[30]

The relationship between the mutation in the p16^INK4a gene and immunohistochemical staining of protein expression continues to remain unclear. While p16^INK4a gene inactivation is the most prominent molecular change that is detected, various publications have reported different results such as an increase or a loss in p16^INK4a protein expression based on immunohistochemistry.^{[33, 34]}

In their study investigating the transition from normal skin to SCC, Hodges et al. reported that p16^INK4a expression was higher in cases of squamous cell carcinoma in situ compared to actinic keratosis, that it manifested a full-thickness staining pattern, and that expression was correlated with progression from normal skin to SCC.^[34] They showed in the same study that there was p16^INK4a staining in the lower half of the epidermis in actinic keratosis. Similar to their study, our results showed higher p16^INK4a expression in AC compared to normal mucosa. Although there are differences in the localizations when our study is compared with that mentioned above, it was determined that AC on the lips and actinic keratosis on the skin could manifest similar p16^INK4a expression.

Tokman and colleagues investigated p16^INK4a and p53 expression in oral SCC.^[20] Squamous cell carcinomas of the lip, which they investigated as a sub-group in their study, were found to manifest positive staining with p16^INK4a at a rate of 58%.^[26] Similarly, in our study, 63% of cases of SCC of the lip demonstrated positive staining.

The p16^INK4a expression patterns in cases of AC and SCC can be explained by an accumulation of inactive p16^INK4a proteins and an inability of the cells to avoid the cell cycle, or the failure or inadequacy of one of the other components of the Rb pathway, resulting in a functional overexpression of the p16^INK4a protein. p16^INK4a overexpression can support the hypothesis that UV radiation results in defective p16^INK4a tumor suppressor gene activity, in concordance with the previous studies.^[35]

There are very few studies that have investigated p14^ARF protein expression in head and neck cancers using immunohistochemistry.^{[33, 36]} Only a limited number of studies exist in the literature on AC.^{[35, 37]} Most of these studies have focused on p53 expression. In a study done by Weber et al. to determine the genetic and epigenetic changes associated with p16^INK4a and p14^ARF, it was shown that immunohistochemical expression was negative in cases of malignant and benign tumors of the head and neck area who had methylation in genes p16^INK4a and p14^ARF, whereas almost all cases without methylation manifested gene product expression at moderate-strong levels.^[36]

We observed a statistically significant increase in p14^ARF expression in transition from normal mucosa to AC and lower expression in cases of SCC than in cases of AC. While SCC cases manifested more significant expression compared to the C group, it was noticed that this expression was lower than that seen in AC cases. This increase in p14^ARF expression in the forming of the actinic cheilitis period can be considered as p14^ARF coming into play as a protective mechanism as a result of its oncogenic activation. The overexpression of p14^ARF causes MDM2-p14^ARFcomplex formation and serves to prevent it from binding to p53. The detected presence of higher p14^ARF expression levels in SCC compared to normal mucosa may suggest that p14ARF maintains its effect, however, fails to prevent the development of carcinoma.

Mutations in TP53 constitute the most commonly detected genetic disruptions in head-neck squamous cell carcinomas and studies have shown that the mutational spectrum of TP53 is different in cancers of the lip and cancers of the oral cavity.^[38]

Studies investigating p53 protein expression in AC, actinic keratosis, and SCCs have mostly used immunohistochemical markers that indicate both mutant and wild-type p53(wt-p53). Therefore, the p53 overexpression detected in these studies should not be considered entirely an indicator of mutation. While mutations in TP53 are the most commonly identified mutations in cancers, p53 mutations were not found in all skin tumors showing p53 expression.^{[38, 39]} Wild-type p53 increases after exposure to UV radiation, and since mutated p53 degrades more slowly, accumulation occurs. Therefore, the increase in p53 expression determined in studies can be either due to an increase in mutated p53 protein during tumor progression or wt-p53 expression in response to instability and DNA damaging agents in other genes. Multiple studies performed on skin cancers have determined that p53 mutation is not correlated well with immunohistochemical overexpression of the p53 protein.^{[39, 40]}

In this study, we determined the p53 protein at low levels in normal epithelial cells, in concordance with other studies.^[41] We determined a change in p53 expression that followed a pattern of increase during progression from normal mucosa to AC and SCC. This increase that we determined in p53 suggests that the wt-p53 might potentially contribute to the expression of mutant p53 as stated in other studies.^{[41, 42]} However, we believe that this argument should be supported by molecular studies detecting mutant type p53. In this study, we detected that 84.21% of our SCC cases had p53 expression and determined that p53 had increased expression in lip carcinogenesis.

Studies have determined p53 expression levels that increase in parallel with the progression from AC to SCC.^{[37, 43-45]} Moreover, they did not determine any differences between cases of AC related to, and unrelated to the tumor in terms of p53 expression.^[37] It has also been stated that some treatment modalities such as ingenol mebutate has no effect on histopathological response or p53 expressions on actinic cheilitis despite the clinical improvement.^[46] When we compared p53 expression between the peritumoral epidermis of SCC cases and cases with AC that had no relation to the tumor, no significant differences were found. This result shows that p53 immunohistochemical staining would not serve as a useful marker alone in determining the transition from AC to SCC.

The studies that have investigated protein expression in the head and neck area have reported varying results in terms of clinicopathological parameters and prognosis. In their study, Kwong et al. aimed to determine the prognostic importance of p14^ARF along with p16^INK4a, p53, p21, and E2F-1 markers in the SCC of the anterior tongue and found that the loss of p14^ARF expression affected survival negatively^[17] Immunohistochemical expression of CDKN2A/p16 revealed low values in the recurrent samples as compared to the non-recurrent ones of oral SCCs in another study.^[47] In our study; for both AC and SCC groups, the cases were older than 50 years of age, and the most frequently affected site by SCC was the lower lip (n=18) in accordance with the literature.^{[7, 8, 43, 48]} We determined no significant relationship between expressions of p53, p16^INK4a, p14^ARF and histopathological factors in cases of SCC of the lip, consistent with the study done by Cheng et al.^[45]

There are very few studies investigating the immunohistochemical features of the epithelium adjacent to SCC of the lip.^{[37, 49, 50]} We observed a positive correlation between p14^ARF and p53 protein expressions in the peritumoral epithelium of SCC cases, but no correlation was observed in AC cases without SCC. The presence of such a correlation may indicate that, during progression from AC to SCC, p14^ARF -mediated p53 overexpression comes into effect at the stage of early carcinogenesis as a preventive factor, however, proves insufficient. In addition, the fact that p14ARF and p53 are overexpressed simultaneously may suggest that the oncogenic activation of p14^ARF contributes as much as UV-induced carcinogenesis as a first step and perhaps that these mechanisms play an effective role in combination. We have determined that p14^ARF expression continues although it is reduced after the appearance of SCC, and we think that this situation preserves wt-p53 activation. Therefore, we reason that these protective mechanisms could explain the more favorable prognosis associated with the SCC of the lip compared to other oral cancers.

Rb and p53 pathways are the two main cell cycle control pathways that are frequently targeted in almost all human tumors. In our study, all cases who were p16^INK4a-negative manifested positive staining with p14^ARF and p53. The picture presented by the cases who showed negativity in this study could indicate the inactivation of p16^INK4a and therefore of the Rb pathway, the activation of p14^ARF and wt-p53, and thus explain the cause of increased p14^ARF and p53 expression in these cases.  

Conclusion

In our study, no statistically significant differences were determined in antibody expression between cases of AC and the peritumoral epithelium of SCC cases. This shows that the expressions of antibodies p16^INK4a, p14^ARF, and p53 by themselves do not constitute useful markers for determining whether AC would transform to SCC. We found a positive correlation between p14^ARF and p53 in the peritumoral epithelium of SCC. We think that, albeit inadequate, p14^ARF and p53 work in coordination to prevent early carcinogenesis and that there are other mechanisms responsible for carcinogenesis, which also simultaneously activate the ARF/p53 pathway.

Although our study is small-scale in terms of the number of cases, it suggested that p16^INK4a and p14^ARF, which are located in the same locus, were regulated by separate mechanisms in the development of AC and SCC of the lip. In this context, it represents a preliminary study showing that other studies can be conducted to investigate the relationship of the CDKN2A locus with various molecules in squamous carcinogenesis of the lip.

Acknowledgments

None.

Conflict of interest

None.

Financial support

None.

Ethics statement

This study was approved by the Ethics Committee of Kırıkkale University School of Medicine (IRB-2009/012).

1.        Gomes APN, Johann JE, Lovato GG, Ferreira AM. Comparative Analysis of the Mast Cell Density in Normal Oral Mucosa, Actinic Cheilitis, and Lip Squamous Cell Carcinoma. Braz Dent J. 2008;19(3):186-9.

2.        Warnakulasuriya S. Oral potentially malignant disorders: A comprehensive review on clinical aspects and management. Oral Oncol. 2020;102:104550.

3.        Warnakulasuriya S. Clinical features and presentation of oral potentially malignant disorders. Oral Surg Oral Med Oral Pathol Oral Radiol. 2018;125(6):582-90.

4.        Lopes MLDS, Gonzaga AKG, Mosconi C, Palomino GM, Mendonça EF, Batista AC et al. Immune response and evasion mechanisms in lip carcinogenesis: An immunohistochemical study. Arch Oral Biol. 2019;98:99-107.

5.        Rodriguez-Archilla A, Irfan-Bhatti A. Risk factors for actinic cheilitis: A meta-analysis. J Dent Res Dent Clin Dent Prospects. 2021;15(4):285-9.

6.        Türkyilmaz M, Öztürk M, Dündar S, Kavak Ergün A, Sevinç A, Tütüncü S, et al. Turkey cancer statistics 2017. 2021. Available from: https://hsgm.saglik.gov.tr/depo/birimler/kanserdb/istatistik/Turkiye_Kanser_Istatistikleri_2017.pdf

7.        Bozan N, Kocak OF, Cankaya H, Kiroglu MH, Gur AF, Erten R. Lip cancer: A 16-year retrospective epidemiological study in Eastern part of Turkey. J Pak Med Assoc. 2016;66(11):1433-5.

8.        Güneri P, Cankaya H, Yavuzer A, Güneri EA, Erişen L, Ozkul D, et al. Primary oral cancer in a Turkish population sample: association with sociodemographic features, smoking, alcohol, diet, and dentition. Oral Oncol. 2005;41(10):1005-12.

9.        Califano J, Van Der Riet P, Westra W, Nawroz H, Clayman G, Piantadosi S, et al. Genetic progression model for head and neck cancer: implications for field cancerization. Cancer Res. 1996;56(11):2488-92.

10.      Bansal R, Nayak BB, Bhardwaj S, Vanajakshi CN, Das P, Somayaji NS, et al. Cancer stem cells and field cancerization of head and neck cancer - An update. J Family Med Prim Care. 2020;9(7):3178-82.

11.      Perez-Ordoñez B, Beauchemin M, Jordan RCK. Molecular biology of squamous cell carcinoma of the head and neck. J Clin Pathol. 2006;59(5);445-53.

12.      Dragomir LP, Simionescu C, Mărgăritescu C, Stepan A, Dragomir IM, Popescu MR. P53, p16 and Ki67 immunoexpression in oral squamous carcinomas. Rom J Morphol Embryol. 2012;53(1):89-93.

13.      Berger JH, Bardeesy N. Modeling INK4/ARF tumor suppression in the mouse. Curr Mol Med. 2007;7(1):63-75.

14.      Bagazgoitia L, Cuevas J, Juarranz A. Expression of p53 and p16 in actinic keratosis, Bowenoid actinic keratosis and Bowen’s disease. J Eur Acad Dermatol Venereol. 2010;24(2):228-30.

15.      Bradley G, Irish J, Macmillan C, Mancer K, Witterick I, Hartwick W, et al. Abnormalities of the ARF-p53 pathway in oral squamous cell carcinoma. Oncogene. 2001;20(5):654-8.

16.      Gologan O, Barnes EL, Hunt JL. Potential diagnostic use of p16INK4a, a new marker that correlates with dysplasia in oral squamoproliferative lesions. Am J Surg Pathol. 2005;29(6):792-6.

17.      Kwong RA, Kalish LH, Nguyen TV, Kench JG, Bova RJ, Cole IE, et al. p14ARF Protein Expression Is a Predictor of Both Relapse and Survival in Squamous Cell Carcinoma of the Anterior Tongue. Clin Cancer Res. 2005;11(11):4107-16.

18.      Martinez A, Brethauer U, Rojas IG, Spencer M, Mucientes F, Borlando J, et al. Expression of apoptotic and cell proliferation regulatory proteins in actinic cheilitis. J Oral Pathol Med. 2005;34(5):257-62.

19.      De Freitas MCA, Ramalho LMP, Xavier FCA, Moreira ALG, Reis SRA. p53 and MDM2 protein expression in actinic cheilitis. J Appl Oral Sci. 2008;16(6):414-9.

20.      Correa GTB, Bernardes VF, de Sousa SF, Dinitz MG, Salles JMP, Souza RP, et al. Lip cancer, and pre-cancerous lesions harbor TP53 mutations, exhibit allelic loss at 9p,9q, and 17p, but no BRAFV600E mutations. Tumor Biol. 2015;36(11):9059-66.

21.      Custódio M, Pelissari C, Santana T, Trierveiler M. Expression of cancer stem cell markers CD44, ALDH1 and p75NTR in actinic cheilitis and lip cancer. Eur Arch Otorhinolaryngol. 2018;275(7):1877-83.

22.      Santana T, Matuck B, Tenório JR, Braga MM. Can immunohistochemical biomarkers distinguish epithelial dysplasia degrees in actinic cheilitis? A systematic review and meta-analysis. Med Oral Patol Oral Cir Bucal. 2020;25(1):e106-16.

23.      Akinyamoju AO, Adeyemi BF, Kolude B, Adisa AO. Histological grading of oral squamous cell carcinoma patients in Ibadan using Bryne's and Broders' grading systems--a comparative study. Afr J Med Med Sci. 2013;42(4):333-7.

24.      Ayva SK, Karabulut AA, Akatli AN, Atasoy P, Bozdogan O. Epithelial expression of extracellular matrix metalloproteinase inducer/CD147 and matrix metalloproteinase-2 in neoplasms and precursor lesions derived from cutaneous squamous cells: An immunohistochemical study. Pathol Res Pract. 2013;209(10):627-34.

25.      Cohen ER, Reis IM, Gomez C, Pereira L, Freiser ME, Hoosien G, et al. Immunohistochemistry Analysis of CD44, EGFR, and p16 in Oral Cavity and Oropharyngeal Squamous Cell Carcinoma. Otolaryngol Head Neck Surg. 2017;157(2):239-51.

26.      Tokman B, Gultekin SE, Sezer C, Alpar R. The expression of p53, p16 proteins, and prevalence of apoptosis in oral squamous cell carcinoma. Saudi Med J. 2004;25(12):1922-30.

27.      Ciurea RN, Pătraşcu V, Simionescu CE, Stepan AE, Popa DG, Ciurea ME, et al. Prognostic factors in squamous cell carcinoma of the lower lip – an immunohistochemical study. Rom J Morphol Embryol. 2017;58(1):89-97.

28.      Eljabo N, Nikolic N, Carkic J, Jelovac D, Lazarevic M, Tanic N, et al. Genetic and epigenetic alterations in the tumour, tumour margins, and normal buccal mucosa of patients with oral cancer. Int J Oral Maxillofac Surg. 2018;47(8):976-82.

29.      Khor GH, Froemming GR, Zain RB, Abraham MT, Omar E, Tan SK, et al. DNA methylation profiling revealed promoter hypermethylation-induced silencing of p16, DDAH2, and DUSP1 in primary oral squamous cell carcinoma. Int J Med Sci. 2013;10(12):1727-39.

30.      Kresty LA, Mallery SR, Knobloch TJ, Song H, Lloyd M, Casto BC, et al. Alterations of p16INK4a and p14ARF in Patients with Severe Oral Epithelial Dysplasia. Can Res. 2002;62(18):5295-300.

31.      Ishida K, Tomita H, Kanayama T, Noguchi K, Niwa A, Kawaguchi M, et al. Specific Deletion of p16INK4a with Retention of p19ARF Enhances the Development of Invasive Oral Squamous Cell Carcinoma. Am J Pathol. 2020;190(6):1332-42.

32.      Sanchez-Cespedes M, Reed AL, Buta M, Wu L, Westra WH, Herman JG, et al. Inactivation of the INK4A/ARF locus frequently coexists with TP53 mutations in non-small cell lung cancer. Oncogene. 1999;18(43):5843-9.

33.      Conscience I, Jovenin N, Coissard C, Lorenzato M, Durlach A, Grange F, et al. P16 is overexpressed in cutaneous carcinomas located on sun-exposed areas. Eur J Dermatol. 2006;16(5):518-22.

34.      Hodges A, Smoller BR. Immunohistochemical comparison of p16 expression in actinic keratoses and squamous cell carcinomas of the skin. Mod Pathol. 2002;15(11):1121-5.

35.      Salama MH, Mahmood MN, Qureshi HS, Ma C, Zarbo RJ, Ormsby AH. p16INK4a expression in actinic keratosis and Bowen’s disease. Br J Dermatol. 2003;149(5):1006-12.

36.      Weber A, Wittekind C, Tannapfe A. Genetic and epigenetic alterations of 9p21 gene products in benign and malignant tumors of the head and neck. Pathol Res Pract. 2003;199(6):391-7.

37.      Pimentel DRN, Michalany N, Alchorne M, Abreu M, Borra RC, Weckx L. Actinic cheilitis histopathology and p53. J Cutan Pathol. 2006;33(8):539-44.

38.      Ostwald C, Gogacz P, Hillmann T, Schweder J, Gundlach K, Kundt G, et al. p53 mutational spectra are different between squamous-cell carcinomas of the lip and the oral cavity. Int J Cancer. 2000;88(1):82-6.

39.      Campbell C, Quinn AG, Angus B, Rees JL. The relation between p53 mutation and p53 immunostaining in non-melanoma skin cancer. Br J Dermatol. 1993;129(3):235-41.

40.      Campbell C, Quinn AG, Ro YS, Angus B, Rees JL. p53 mutations are common and early events that precede tumor invasion in squamous cell carcinoma. J Invest Dermatol. 1993;100(6):746-8.

41.      Bukhari MH, Shahida N, Chaudhry NA. Relationship of immunohistochemistry scores of altered p53 protein expression in relation to patient’s habits and histological grades and stages of squamous cell carcinoma. J Cutan Pathol. 2009;36(3):342-9.

42.      Lopes ML, de Oliveira DH, Sarmento DJ, Queiroz LM, Miguel MC, da Silveira ÉJ. Correlation between cell cycle proteins and hMSH2 in actinic cheilitis and lip cancer. Arch Dermatol Res. 2016;308(3):165-71.

43.      Mello FW, Melo G, Modolo F, Rivero ER. Actinic cheilitis and lip squamous cell carcinoma: Literature review and new data from Brazil. J Clin Exp Dent. 2019;11(1):e62-9.

44.      Cheng TH, Hsu PK, Li AFY, Hung IC, Huang MH, Hsu HS. Correlation of p53, MDM2, and p14ARF protein expression in human esophageal squamous cell carcinoma. J Cancer Res Clin Oncol. 2009;135(11):1577-82.

45.      Lima FJ, Lopes MLDS, Barros CCDS, Nonaka CFW, Silveira ÉJDD. Modification in CLIC4 Expression is Associated with P53, TGF-β, TNF-α and Myofibroblasts in Lip Carcinogenesis. Braz Dent J. 2020;31(3):290-7.

46.      Rossini RD, Dellatorre G, Mesquita LA, Tarlé RG. Ingenol mebutate treatment for actinic cheilitis: clinical, histopathological and p53 profile of 14 cases. J Dermatolog Treat. 2021;32(8):1049-52.

47.      Padhi SS, Roy S, Kar M, Saha A, Roy S, Adhya A, et al. Role of CDKN2A/p16 expression in the prognostication of oral squamous cell carcinoma. Oral Oncol. 2017;73:27-35.

48.      Silva LVO, de Arruda JAA, Abreu LG, Ferreira RC, da Silva LP, Pelissari C, et al.  Demographic and Clinicopathologic Features of Actinic Cheilitis and Lip Squamous Cell Carcinoma: a Brazilian Multicentre Study. Head Neck Pathol. 2020;14(4):899-908.

49.      Varela-Centelles P, Gonzalez-Moles MÁ, Seoane-Romero J, Leira-Feijoo Y, Takkouche B, Seoane-Romero JM. Immunohistochemical analysis of epithelium adjacent to lip cancer: A meta-analysis. Oral Dis. 2022;28(1):57-65. doi:10.1111/odi.13643

50.      Nagata G, Santana T, Queiroz A, Caramez RH, Trierveiler M. Evaluation of epithelial dysplasia adjacent to lip squamous cell carcinoma indicates that the degree of dysplasia is not associated with the occurrence of invasive carcinoma in this site. J Cutan Pathol. 2018;45(9):647-51.