Urias Barreras, Cynthia Marina¹; Ramírez Camou, Jessica Guadalupe²; Ríos Burgueño, Efrén Rafael³

Language: English/Spanish [Versión en español]
References: 24
Page: 215-223
PDF size: 491.08 Kb.

ABSTRACT

Introduction: P63 belongs to the p53 gene family, and encodes different isoforms. TAp63 transactivates the p53 target gene that induces apoptosis. Np63 inhibits the activity of TAp63 and p53, promoting cell proliferation and growth. P63 antibody recognizes the TAp63 and Np63 isoforms, while p40 antibody only recognizes the Np63 isoform. Objectives: To analyze the p63/p40 immunoprofile properties to discriminate aggressiveness in benign odontogenic tumors and cysts. Material and methods: Five odontogenic tumors (3 ameloblastomas, 1 primordial odontogenic tumor, and 1 ameloblastic fibroma) and twenty-five odontogenic cysts (12 odontogenic keratocysts, 6 radicular cysts, 5 dentigerous cysts, 1 glandular odontogenic cyst, and 1 case with epithelial nests in the cyst wall) were evaluated with p63 and p40 antibodies. Results: Ameloblastomas and ameloblastic fibroma presented p63+/p40+ in all epithelial neoplastic cells. The primordial odontogenic tumor showed positivity in the epithelial component, except in superficial layer areas. Odontogenic keratocysts showed p63+/p40+ in all epithelial layers, except in the parakeratinized superficial layer. Dentigerous cysts were positive in all epithelial cells, some of them with less intensity in the superficial layer. It was not possible to evaluate the glandular odontogenic cyst since it lost the epithelial component during the process. Radicular cysts showed p63+ in all epithelial strata, except for one case with intense focal positivity. P40 showed positivity in some cases very strong, in others moderate or focal. On the other hand, p63+/p40+ was observed in odontogenic epithelial nests in the cyst wall. Conclusions: P63/p40 immunophenotype does not discriminate aggressiveness in the diagnosis of benign odontogenic tumors and cysts.

INTRODUCTION

P63 transcription factor belongs to the p53 gene family that is involved in epithelial development and maintenance, stem cell biology, carcinogenesis, and proliferation of limb and craniofacial structures. P63 is normally expressed in the stratified epithelium basal layer, in myoepithelial cells and in neoplasms derived from these epithelia. Stem cells lacking p63 undergo a premature proliferative rundown. P63 encodes different isoforms, three with the transactivation domain (TA) and three lacking the N-terminal domain (N) which exert opposite biological properties. TAp63 transactivates the p53 target gene that induces apoptosis. On the other hand, Np63 inhibits TAp63 and p53 activity, promoting cell growth and proliferation. Different p63 isoforms have been shown to play roles in various cells and tissues, such as the epidermis, oocytes, muscles, and cochlea. P63 antibody recognizes the TAp63 and Np63 isoforms of the p63 protein, while p40 antibody only recognizes the Np63 isoform, the more specific squamous form.^1-11

ΔNp63 isoform acts as an enhancer of cell proliferation in human tumors, suggesting that it may play an oncogenic role through its regulation of metabolic reprogramming in tumors. In recent studies, the p63/p40 combination has been proposed as an effective diagnostic method in salivary gland neoplasms, sinonasal tract tumors, and lung carcinomas. ΔNp63 expression in benign odontogenic tumors has been associated with a high risk of recurrence, more than in non-aggressive benign odontogenic tumors with a low risk of recurrence. In addition, p63 is expressed in more than 50% of malignant odontogenic tumors compared to non-aggressive benign odontogenic tumors.^11-13

Furthermore, increased angiogenesis and p53, p63, p73 expression may contribute to the locally aggressive and invasive behaviors of odontogenic keratocysts (OKC), previously called odontogenic keratocystic tumors).¹⁴ On the other hand, Alsaegh et al.¹⁵ reported that ΔNp63 correlates with the proliferative capacity of the odontogenic epithelium in dentigerous cyst (DC) and odontogenic keratocyst (OKC), but not in ameloblastoma (AME). This diversity could reflect a different role and pathways of ΔNp63 in odontogenic tumor than in odontogenic cyst.

This paper aims to analyze the expression of the combined p63/p40 immunoprofile in benign odontogenic tumors and cysts to determine its property to discriminate aggressiveness in these lesions.

MATERIAL AND METHODS

Benign odontogenic tumors and odontogenic cysts were selected from the pathology department (Departamento de Patología del Centro de Investigación y Docencia en Ciencias de la Salud de la Universidad Autónoma de Sinaloa) diagnosed from January 2014 to April 2019. Processed specimens stained with hematoxylin and eosin were reviewed to confirm the diagnosis. The group of benign odontogenic tumors is made up of 3 ameloblastomas (AME), 1 primordial odontogenic tumor (POT) and 1 ameloblastic fibroma (AF). The group of odontogenic cysts includes 12 odontogenic keratocysts (OKC), 6 radicular cysts (RC), 5 dentigerous cysts (DC), and 1 glandular odontogenic cyst (GOC). In addition, odontogenic epithelial nests (OEN) present in a cyst wall were analyzed. Clinical data on the age and sex of the patients, as well as the location of the lesion, were collected (Table 1).

For the immunohistochemical study, levels were made at 1.5 microns of each block. Each section was deparaffinized at 80 ^oC for 20 minutes and rehydrated with xylol-alcohol according to the standard protocol of the immunohistochemical process. Subsequently, antigenic unmasking was performed for 18 minutes at a temperature of 105 ^oC with RHP Bio & SB. Each slide was subjected to a capillarity process by cover plate with PVC at pH 7.4 to an internal block ADE peroxidase with 3% hydrogen peroxide and subsequent PVC rinses. P63 and p40 primary antibodies were placed at 1:200 and 1:50 dilutions according to the manufacturer's specifications (Bio & SB and Diagnocell) for 30 minutes. Bio & Sb HRP secondary antibody was used for 10 minutes and rinsed with PVC. Finally, it was labeled with a Bio & SB complex for a biotinylated system for 10 minutes. After rinsing it was revealed with diamino-benzidine and counterstained with hematoxylin and eosin for further interpretation.

A semi-quantitative assessment of p63 and p40 expression was performed by assigning cases to one of the following three categories: (I) score 0, when the stained cells were from 0 to < 5%; (II) score 1, when the stained cells were from > 5 to < 50%; (III) score 2, when the stained cells were from > 50% of the total cell population.^14,16

RESULTS

In AME and AF p63 and p40 were positive in all neoplastic epithelial cells (score 2); only one case of AME showed positivity with a weak intensity. POT showed p63+/p40+ in the epithelial component (score 2) except some superficial layer areas (Figure 1). In OKC, positivity for p63/p40 was observed in all stratified epithelia, except for the parakeratinized layer (score 2). In DC cases, p63+/p40+ was observed in all stratified epithelia except for some superficial layer cells (score 2). However, one case showed positivity with moderate intensity. It was not possible to evaluate the GOC, since the epithelial component was lost during the process. On the other hand, p63+/p40+ was observed in the epithelial nests present in a cyst wall (Figure 2). RC showed p63+ in all stratified epithelial (score 2) except for one case with intense focal positivity (score 0). P40 expression was variable: three cases showed very strong positivity (score 2), two cases moderate positivity (score 1) and one case, focal positivity (score 0) (Figure 3).

DISCUSSION

In this study it was observed that p63/p40 in AME was positive (with variable intensity) in all neoplastic epithelial cells, as reported by Argyris et al,¹ who also detailed that AME showed intense positivity for p63 in central and peripheral cells. Atarbashi et al.¹⁷ and Martínez et al.¹⁸ obtained different results in which AME was strongly positive only in peripheral cells. On the other hand, Lo Muzio et al.¹⁶ observed p63 reactivity in odontogenic tumors such as AME, ameloblastic carcinoma, adenomatoid odontogenic tumor, primary intraosseous carcinoma, and clear cell odontogenic carcinoma. Furthermore, p63 expression in malignant odontogenic tumor was detected in > 50% of cells in all cases and was significantly higher than in non-aggressive benign odontogenic tumors with low risk of recurrence. Adittionaly, p63 expression did not differ significantly between locally aggressive benign odontogenic tumors with high risk of recurrence and malignant odontogenic tumors. In our study, only benign odontogenic tumors were included, and no differences in p63 expression were observed between locally aggressive benign odontogenic tumors (AME and AF) and with low risk of recurrence (POT).

Gratzinger et al.¹⁹ found that p63 was strongly expressed in the basal half of the oral epithelium and was generally not expressed in mature superficial layers. Consistent p63 expression was the norm in AME and calcifying epithelial odontogenic tumors (CEOT). P63 polarity expression was maintained in keratocystic odontogenic tumors (KOT) and GOC. Similarly, in our study, p63 expression was uniform in AME, AF, and most of the epithelial component of POT, and p63 was not expressed in the mature superficial layer of OKC, formerly KOT. Likewise, de Brito et al.²⁰ reported that P63 immunoexpression in epithelial component of the KOT was positive in basal and suprabasal layers of all the cases analyzed. Argyris et al.¹ reported that OKC, DC, and RC showed a strong and diffuse p63+/p40+ expression throughout the thickness of the epithelial lining, except in the superficial keratin layer in the OKC and in the orthokeratinized cyst. Similarly, in our study, KOCs were positive for p63/p40 in all stratified epithelial but in the parakeratinized layer. Regarding to DCs, these were p63+/p40+. However, moderate positivity was identified in some epithelial cells of the superficial layer, as well as in basal and parabasal layers. RCs showed p63+ in all stratified epithelial, except for one case with intense focal positivity. P40 positivity was much more variable.

We observed that DC and OKC had a p63 score greater than 50% on the semi-quantitative scale. The RC had a score higher than 50%, except for one case that had a score lower than 5% (Table 1). In a different way, Lo Muzio et al.¹¹ reported that in most follicular cysts (FC) the stained cells were from 0 to < 5% of the total and in OKC 64% of the cases showed stained cells from > 5 to < 50%. Approximately 1/3 of the RC presented stained cells from 0 to < 5% and almost 2/3 from > 5 to < 50%. Moreover, Atarbashi et al.¹⁷ reported that immunostaining for p63 in DC and RC was found mainly in basal and parabasal layers. In our study, in 4 of 5 DC cases, intense reactivity was also observed in the upper layers. Likewise, Lo Muzio et al.¹¹ reported that in almost all FC, immunostaining for p63 was limited to basal and parabasal layers of the epithelium. Only 2 cases out of 30 showed positivity of the intermediate layer. Three RC presented negative epithelium for p63 immunostaining, whereas residual cases showed positivity not only in basal and parabasal layers but more than half, also in the intermediate layer.

As previously Mascitti et al.²¹ reported, p63 positivity was higher in superficial layer of DC and RC than OKC. On the other hand, Seyedmajidi et al.²² found that KOT revealed the highest p63 expression between dentigerous cyst and radicular cyst. Gurgel et al.²³ found no significant differences in Ki-67, p53, and p63 expression between primary and recurrent KOT or between nevoid basal cell carcinoma syndrome-associated KOT. Foschini et al.²⁴ reported that in recurrent OKC, ΔNp63 antibody staining was found in 60 to 90% of cells and localized to all layers. In non-recurrent lesions, it was found in less than 70% of cells, and it was limited to basal and parabasal layers. They suggested that lesions with high p40 expression are more likely to recur. Interestingly, in our study p40 expression was detected in all layers, but the parakeratinized layer of all OKC, which had been described as non-recurrent primary lesions up to that time.

CONCLUSION

P63/p40 immunophenotype in benign odontogenic tumors and cysts do not discriminate aggressiveness in the diagnosis of these lesions.

REFERENCES

1. Argyris PP, Malz C, Taleb R, Koutlas IG. Benign and malignant odontogenic neoplasms of the jaws show a concordant nondiscriminatory p63/p40 positive immunophenotype. Oral Surg Oral Med Oral Pathol Oral Radiol. 2018; 126 (6): 506-512.

2. Soares E, Zhou H. Master regulatory role of p63 in epidermal development and disease. Cell Mol Life Sci. 2018; 75 (7): 1179-1190.

3. Jaafari-Ashkavandi Z, Geramizadeh B, Ranjbar MA. P63 and Ki-67 expression in dentigerous cyst and ameloblastomas. J Dent. 2015; 16 (4): 323-328.

4. Tilson MP, Bishop JA. Utility of p40 in the differential diagnosis of small round blue cell tumors of the sinonasal tract. Head Neck Pathol. 2014; 8 (2): 141-145.

5. Senoo M, Pinto F, Crum CP, McKeon F. p63 is essential for the proliferative potential of stem cells in stratified epithelia. Cell J. 2007; 129 (3): 523-536.

6. Mills AA. p53: link to the past, bridge to the future. Genes Dev. 2005; 19 (18): 2091-2099.

7. Kumamoto H, Izutsu T, Ohki K, Takahashi N, Ooya K. p53 gene status and expression of p53, MDM2, and p14 proteins in ameloblastomas. J Oral Pathol Med. 2004; 33 (5): 292-299.

8. Tsujita-Kyutoku M, Kiuchi K, Danbara N, Yuri T, Senzaki H, Tsubura A. p63 expression in normal human epidermis and epidermal appendages and their tumors. J Cutan Pathol. 2003; 30 (1): 11-17.

9. Yang A, Kaghad M, Caput D, McKeon F. On the shoulders of giants: p63, p73 and the rise of p53. Trends Genet. 2002; 18 (2): 90-95.

10. Kaelin WG. The p53 gene family. Oncogene.1999; 18 (53): 7701-7705.

11. Lo Muzio L, Santarelli A, Caltabiano R, Rubini C, Pieramici T, Fior A et al. p63 expression in odontogenic cysts. Int J Oral Maxillofac Surg. 2005; 34 (6): 668-673.

12. Argyris PP, Wetzel SL, Greipp P, Wehrs RN, Knutson DL, Kloft-Nelson SM et al. Clinical utility of myb rearrangement detection and p63/p40 immunophenotyping in the diagnosis of adenoid cystic carcinoma of minor salivary glands: a pilot study. Oral Surg Oral Med Oral Pathol Oral Radiol. 2016; 121 (3): 282-289.

13. Rooper L, Sharma R, Bishop JA. Polymorphous low grade adenocarcinoma has a consistent p63+/p40- immunophenotype that helps distinguish it from adenoid cystic carcinoma and cellular pleomorphic adenoma. Head Neck Pathol. 2015; 9 (1): 79-84.

14. Chandrangsu S, Sappayatosok K. p53, p63 and p73 expression and angiogenesis in keratocystic odontogenic tumors. J Clin Exp Dent. 2016; 8 (5): 505-511.

15. Alsaegh MA, Altaie AM, Zhu S. p63 Expression and its relation to epithelial cells proliferation in dentigerous cyst, odontogenic keratocyst, and ameloblastoma. Pathol Oncol Res. 2020; 26 (2): 1175-1182.

16. Lo Muzio L, Santarelli A, Caltabiano R, Rubini C, Pieramici T, Giannone N et al. p63 expression correlates with pathological features and biological behaviour of odontogenic tumours. Histopathology. 2006; 49 (2): 211-214.

17. Atarbashi Moghadam S, Atarbashi Moghadam F, Mokhtari S, Eini E. Immunohistochemical analysis of P63 expression in odontogenic lesions. Biomed Res Int. 2013; 2013: 624176.

18. Martínez-Martínez M, Mosqueda-Taylor A, Carlos-Bregni R, Pires F-R, Delgado-Azanero W, Neves-Silva R et al. Comparative histological and immunohistochemical study of ameloblastomas and ameloblastic carcinomas. Med Oral Patol Oral Cir Bucal. 2017; 22 (3): 324-332.

19. Gratzinger D, Salama ME, Poh CF, Rouse R V. Ameloblastoma, calcifying epithelial odontogenic tumor, and glandular odontogenic cyst show a distinctive immunophenotype with some myoepithelial antigen expression. J Oral Pathol Med. 2008; 37 (3): 177-184.

20. de Brito Monteiro BV, Cavalcante RB, Maia Nogueira RL, da Costa Miguel MC, Weege Nonaka CF, da Silveira ÉJD. Participation of hMLH1, p63, and MDM2 proteins in the pathogenesis of syndromic and nonsyndromic keratocystic odontogenic tumors. Oral Surg Oral Med Oral Pathol Oral Radiol. 2015; 120 (1): 52-57.

21. Mascitti M, Togni L, Balercia A, Balercia P, Rubini C, Santarelli A. p53-family proteins in odontogenic cysts. Appl Immunohistochem Mol Morphol. 2020; 28 (5): 369-375.

22. Seyedmajidi M, Shafaee S, Shafigh E, Bijani A, Hamidi H. p63 expression in randomized odontogenic cysts. Saudi Med J. 2011; 32 (5): 463-466.

23. Gurgel CAS, Ramos EAG, Azevedo RA, Sarmento VA, da Silva Carvalho AM, dos Santos JN. Expression of Ki-67, p53 and p63 proteins in keratocyst odontogenic tumours: an immunohistochemical study. J Mol Histol. 2008; 39 (3): 311-316.

24. Foschini MP, Cocchi R, Marucci G, Pennesi MG, Magrini E, Ligorio C et al. High DeltaN p63 isoform expression favours recurrences in odontogenic keratocyst--odontogenic keratocystic tumour. Int J Oral Maxillofac Surg. 2006; 35 (7): 673-675.

AFFILIATIONS

¹ Patóloga bucal. Profesora de la Facultad de Odontología. Profesora del Departamento de Patología en el Centro de Investigación y Docencia en Ciencias. Universidad Autónoma de Sinaloa, Sinaloa, México.

² Pasante de Servicio Social en el Instituto Mexicano del Seguro Social Unidad Médica Familiar No. 37. Estudiante de la Facultad de Odontología. Universidad Autónoma de Sinaloa, Sinaloa, México.

³ Médico Patólogo, Jefe del Departamento de Patología del Centro de Investigación y Docencia en Ciencias de la Salud. Universidad Autónoma de Sinaloa, Sinaloa, México.

CORRESPONDENCE

Cynthia Marina Urias Barreras. E-mail: cynthia.urias@uas.edu.mx

Received: Febrero 2021. Accepted: Abril 2021.