P63 expression in papillary cystadenoma and mucoepidermoid carcinoma of minor salivary glands

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1 P63 expression in papillary cystadenoma and mucoepidermoid carcinoma of minor salivary glands Felipe Paiva Fonseca, DDS, a Bruno Augusto Benevenuto de Andrade, DDS, MSc, a Márcio Ajudarte Lopes, DDS, PhD, a Hélder Antônio Rebelo Pontes, DDS, PhD, b Pablo Agustin Vargas, DDS, PhD, a and Oslei Paes de Almeida, DDS, PhD a State University of Campinas, Piracicaba; and Federal University of Pará, Belém, Brazil Vol. 115 No. 1 January 2013 Objective. The aim of this study was to investigate the expression of p63 protein in mucoepidermoid carcinoma and papillary cystadenoma of the salivary glands, and to evaluate the usefulness of this protein in distinguishing these tumors. Study Design. Immunoexpression of p63 protein was studied and quantified in 9 formalin-fixed paraffin-embedded mucous retention cysts, 4 papillary cystadenomas, and 19 low-grade and 9 high-grade mucoepidermoid carcinomas. Results. All cases were positive for p63 immunoexpression; however, it was observed that p63 labeling in mucous retention cysts and papillary cystadenomas was limited to the basal layers of the cystic spaces, whereas in low-grade mucoepidermoid carcinomas, positive nuclear staining was also found diffusely in the suprabasal layers. Mucoepidermoid carcinoma presented increased immunoexpression of p63 compared with the other groups. Conclusions. P63 immunohistochemical expression pattern can be helpful in distinguishing low-grade mucoepidermoid carcinoma from papillary cystadenoma of the salivary glands. (Oral Surg Oral Med Oral Pathol Oral Radiol 2013;115:79-86) Mucoepidermoid carcinoma (MEC) is the most common malignant salivary gland tumor. In major salivary glands it usually presents as a single painless mass, whereas in minor glands it typically involves the palate, presenting as a bluish swelling. Histologically, MEC is characterized by neoplastic cells organized in solid and cystic growth patterns, and is classified into low-, intermediate-, and high-grade types. 1-4 Papillary cystadenoma of the salivary glands is a rare benign epithelial tumor characterized by multicystic growth, with focal intraluminal papillary proliferations of the epithelial lining. 5 Most cases occur in major salivary glands, particularly the parotid gland, but these can also be found in minor glands, being seen in the lip, buccal mucosa and palate. 6-8 Cystadenomas of the major glands present as asymptomatic masses, but in the oral cavity they may resemble mucoceles. 7 Histologically, cystadenomas show a close resemblance to lowgrade MEC, particularly when the latter shows a predominantly cystic papillary growth pattern. p63 protein is constitutively expressed in cell nuclei of many normal stratified epithelial tissues, including Supported by grants from the São Paulo State Research Foundation (FAPESP), process no. 2009/ a Piracicaba Dental School, State University of Campinas. b Service of Oral Pathology, João de Barros Barreto University Hospital, Federal University of Pará. Received for publication Jan 27, 2012; returned for revision Jul 11, 2012; accepted for publication Sep 11, Elsevier Inc. All rights reserved /$ - see front matter those from the skin, oral cavity, esophagus, and cervix. In addition, p63 is strongly expressed in myoepithelial cells of the breast and salivary glands In neoplastic tissues, p63 immunoreactivity has been demonstrated in transitional and squamous cell carcinomas and in several salivary gland tumors, including adenoid cystic carcinoma, polymorphous low-grade adenocarcinoma, and myoepithelial carcinoma. 12 Different myoepithelial markers have been investigated in MEC, with divergent and inconclusive results. The expression of p63 has been the subject of very few studies in this context. 9,13,14 Moreover, the expression of p63 in papillary cystadenoma has not been described previously in the literature. Therefore, the aim of the present study was to describe the immunoexpression of p63 in MEC, papillary cystadenoma, and mucous retention cysts, and evaluate if this marker could be useful for the differentiating between MEC and papillary cystadenoma. MATERIALS AND METHODS Nineteen low-grade MECs, 9 high-grade MECs, 4 papillary cystadenomas, and 9 mucous retention cysts, Statement of Clinical Relevance The distinction between cystadenoma and lowgrade mucoepidermoid carcinoma is sometimes challenging. The present study shows that p63 protein is expressed in both tumors, but with different intensity and patterns, which may be useful in distinguishing the tumors. 79

2 ORAL AND MAXILLOFACIAL PATHOLOGY OOOO 80 Fonseca et al. January 2013 derived from minor salivary glands of the oral cavity, were used to determine immunohistochemically the expression of p63 protein. All samples were fixed in 10% formalin and embedded in paraffin. The original diagnoses were reviewed by 2 independent oral pathologists, according to the World Health Organization classification. 2 For immunohistochemistry, 3- m sections were dewaxed with xylene and then hydrated in an ethanol series. For antigen retrieval, slides were immersed in 10 mmol/l monohydrated citrate buffer (ph 6.0) and underwent 3 minutes of pressure cooking. Endogenous peroxidase activity was blocked with the use of 10% hydrogen peroxide in 5 baths, each for 5 minutes. After washing in phosphate-buffered saline buffer (ph 7.4), slides were incubated overnight with the primary antibody anti-p63 clone 4A4 (Dakocytomation) diluted 1:300. All slides were subsequently exposed to Advanced Kit (Dakocytomation) and diaminobenzidin tetrahydrochloride (DAB; Sigma) and counterstained with Carazzi hematoxylin. Sections of fibrous hyperplasia were used as positive control, and the negative control was obtained by omitting the primary specific antibody. Results were considered to be positive when cells showed strong brown nuclear staining. Glass slides were subsequently scanned with the Scanscope CS Slide Scanner (Aperio Technologies). Digital images obtained were analyzed for p63 expression using the Imagescope software and its Nuclear Staining V9 algorithm (Aperio Technologies), with the purpose of quantifying the percentage of positive cells in 5 hotspot areas in each case. The software recognized positive expression as those with nuclear staining that ranged from 80 to 230 pixels of intensity. Lower values were considered to be nonspecific staining. For statistical analysis, the software Graphpad Prism 5.0 was used and a P value of.05 was considered to be statistically significant. RESULTS The clinical features are summarized in Table I. Mucous retention cysts mainly involved the floor of the mouth of female patients, with a mean age of 60.2 years (range years), and had presented as an asymptomatic swelling of unknown duration. The 28 cases of MEC corresponded to 17 women and 11 men, with a mean age of 46.3 years (range years). All papillary cystadenomas occurred in women, with a mean age of 60.5 years (range years). Most cases of MEC involved the palate, whereas 1 case of cystadenoma involved the floor of the mouth, 2 the palate, and 1 the buccal mucosa. The chief presenting complaint of MEC and cystadenomas was of an asymptomatic swelling. Histologically, the majority of the mucous retention cysts revealed a thin epithelial lining, usually composed of only 1 or 2 cell layers, (Figure 1, A). In 2 cases a prominent proliferation of the luminal cells was evident, with the presence of mucous cells in the epithelial lining (Figure 1, B). An eosinophilic fluid in the lumen of the cysts was invariably present. All 4 papillary cystadenomas presented as variably sized well-circumscribed multicystic lesions with luminal papillary growth but without an evident fibrous capsule (Figure 1, C). The epithelial lining of the cystic spaces was mainly composed of 2 layers of cuboidal or columnar cells that occasionally became thickened, forming small sheets of a more solid growth. Scattered mucous cells could be identified in all cases. In one case, oncocytic cells predominated (Figure 1, D). Eosinophilic amorphous fluid in the lumen of the cystic cavities was frequently present. Cellular atypia and mitoses were absent from all cases. Low-grade MECs were mainly composed of variably sized cystic spaces, showing papillary projections and more solid areas (Figure 2, A). Depending on the area evaluated, there was a predominance of mucous, intermediate, or squamous cells (Figure 2, B). Clear cells were present in several cases and predominated in 1 case (Figure 2, C). Mitoses and cellular atypia were rare and foci of necrosis absent. High-grade MEC presented as solid growths of intermediate and squamous cells, with mucous cells present in variable amounts, particularly lining small cystic spaces and occasionally among the squamous cells (Figure 2, D). Cytologic and nuclear pleomorphism, as well as mitoses and an infiltrative growth pattern into adjacent tissues were found in all cases. Sclerotic areas were variable, with no evident differences between MEC and papillary cystadenoma, except for 1 case of low-grade MEC that showed intense sclerosis. Normal minor salivary glands present in the surgical specimens expressed nuclear positivity for p63 in the myoepithelial/basal cells of the ductal structures and at the periphery of the acinar cells. Similarly, all mucous retention cysts and papillary cystadenomas revealed positive nuclear reactivity for p63, almost exclusively in the basal cells of the cystic structures. Occasionally, a few cells above the basal layer were positive (Figure 3). A variable staining pattern could be seen in all cases of low- and high-grade MEC. Sheets of intermediate or squamous cells were diffusely positive for p63, but in the cystic spaces the expression of p63 was not restricted to the basal cells and extended to the upper layers closer to the lumen (Figure 4, A-C). It was also noted that in several cystic spaces, the more basal cells did not show expression of p63. Finally, p63 protein expression was negative in the mucous and clear cells (Figure 4, D). With the use of

3 OOOO ORIGINAL ARTICLE Volume 115, Number 1 Fonseca et al. 81 Table I. Clinicopathologic features of mucous retention cysts, cystadenomas, and mucoepidermoid carcinomas (MECs) Case Diagnosis Age Sex Site Clinical presentation 1 Retention cyst 63 F Superior lip Asymptomatic nodule 2 Retention cyst 24 M Floor of mouth Asymptomatic nodule 3 Retention cyst 71 F Floor of mouth Asymptomatic nodule 4 Retention cyst 72 M Buccal mucosa Asymptomatic nodule 5 Retention cyst 65 F Superior lip Asymptomatic nodule 6 Retention cyst 66 F Buccal mucosa Asymptomatic nodule 7 Retention cyst 60 F Floor of mouth Asymptomatic nodule 8 Retention cyst NS F Floor of mouth Asymptomatic nodule 9 Retention cyst 60 M Floor of mouth Asymptomatic nodule 10 Cystadenoma 71 F Buccal mucosa Asymptomatic nodule 11 Cystadenoma 60 F Palate Asymptomatic nodule 12 Cystadenoma 50 F Palate Swelling 13 Cystadenoma NS F Floor of mouth Asymptomatic nodule 14 Low-grade MEC 63 F Maxilla, NOS NS 15 Low-grade MEC 38 F Buccal mucosa NS 16 Low-grade MEC 75 F Hard palate Asymptomatic nodule 17 Low-grade MEC 22 F Hard and soft palate Asymptomatic nodule 18 Low-grade MEC 44 F Mandible NS 19 Low-grade MEC 71 M Retromolar area Painful swelling 20 Low-grade MEC 41 F Palate Asymptomatic nodule 21 Low-grade MEC 31 M Hard palate Asymptomatic nodule 22 Low-grade MEC 38 F Hard palate NS 23 Low-grade MEC 42 M NS Painful swelling 24 Low-grade MEC 62 M Hard and soft palate NS 25 Low-grade MEC 30 M Soft palate Asymptomatic nodule 26 Low-grade MEC 49 F Hard palate Painful swelling 27 Low-grade MEC 28 F Soft palate Asymptomatic nodule 28 Low-grade MEC 64 F Hard and soft palate NS 29 Low-grade MEC NS M Maxilla Asymptomatic swelling 30 Low-grade MEC 35 F Hard palate Painful swelling 31 Low-grade MEC NS M Hard palate Swelling 32 Low-grade MEC NS M Hard palate Swelling 33 High-grade MEC 67 F Hard palate Asymptomatic nodule 34 High-grade MEC 16 M Soft palate Asymptomatic nodule 35 High-grade MEC 80 M Superior lip Painful swelling 36 High-grade MEC 28 F Buccal mucosa Asymptomatic nodule 37 High-grade MEC 45 F Retromolar área Asymptomatic nodule 38 High-grade MEC 17 F Hard and soft palate Painful swelling 39 High-grade MEC 42 F Hard palate NS 40 High-grade MEC 52 F Buccal mucosa Asymptomatic nodule 41 High-grade MEC 78 M Hard palate Asymptomatic nodule NS, Not specified. quantitative digital analysis, high-grade MEC cases revealed the highest index of expression of p63 (56.7%), followed by low-grade MEC (46.8%), mucous retention cysts (23.1%), and papillary cystadenomas (22.0%). Analysis of variance showed a statistically significant difference among the 4 groups studied (P.0001). In addition, with the use of Tukey post hoc test, a significant increase in positive immunohistochemical staining for p63 protein was observed in low- and high-grade MEC compared with mucous retention cyst (both P.0001) and cystadenoma (P.001 and P.0001, respectively). No significant difference was observed between mucous retention cyst and cystadenoma (P.05) or between lowand high-grade MEC (P.05; Figure 5). DISCUSSION The characteristic cystic growth pattern of low-grade MEC, with varying degrees of intracystic projections, can lead to a histologic similarity to papillary cystadenoma. The distinction between both tumors is sometimes arbitrary, leading to wrong diagnosis and to inadequate surgical treatment. The present study showed that p63 protein is expressed in both tumors, but with different intensity and patterns, and therefore could be useful in distinguishing between both tumor types. The p63 gene, mapped to 3q27-29, is the third member of the p53 family, whose transcription from 2 different promoters (TA and N) and 3 alternative

4 ORAL AND MAXILLOFACIAL PATHOLOGY OOOO 82 Fonseca et al. January 2013 Fig. 1. Histologic features of mucous retention cyst (MRC) and papillary cystadenoma. A, Cystic space of MRC is lined by a thin epithelium composed of 2 cellular layers (HE, 200). B, In 2 cases of MRC, epithelial lining was prominent with the presence of mucous cells (HE, 100). C, Papillary cystadenoma with cystic spaces lined by epithelium showing intracystic luminal growth (HE, 200). D, Papillary cystadenoma presenting predominance of oncocytic cells in the epithelial lining, which in some areas became thickened, leading to a more solid growth pattern (HE, 100). splice sites at the C-terminal region (,, and ) results in at least 6 different isoforms. 9,10 The TAp63 isoform activates p53 target genes (p21waf1/cip1, mdm2, and bax), blocks cell cycle progression, and promotes apoptosis, whereas the Np63 isoform acts in a dominant negative manner, inhibiting the transcriptional activation of the p53 gene. 9,12-15 The only commercially available antibody against p63 protein has shown to be limited in the ability to discriminate between TAp63 and Np63, because it recognizes both isoforms. In humans, p63 is expressed in many normal epithelial tissues and seems to be relevant for epithelial stem cell survival and maturation. Furthermore, p63 is a selective nuclear marker of normal myoepithelial cells in the human breast and salivary glands. 11,14-16 Although somatic mutation of the p63 gene is not a classic oncogenic event in neoplasia the p63 protein is overexpressed in various neoplasms, particularly squamous carcinomas of the aerodigestive tract, lung, skin, and uterine cervix, and benign and malignant myoepitheliomas. 13,15,17,18 In contrast, Jo and Fletcher (2011) reported that expression of p63 is practically absent in normal and malignant mesenchymal cells. 19 Salivary gland tumors have also been studied for p63 expression, and neoplasias that show proliferation of myoepithelial cells, such as pleomorphic adenoma, myoepithelioma, basal cell adenoma, adenoid cystic carcinoma, polymorphous low-grade adenocarcinoma, epithelial-myoepithelial carcinoma, myoepithelial carcinoma, and basal cell adenocarcinoma widely express p63 protein depending on their myoepithelial content. 10,12,14,20,21 MEC has classically been considered to be a neoplasm devoid of myoepithelal cells; however, the participation of these cells in the oncogenesis of MEC is now a matter of controversy. 22 According to Dardick et al. (1996), 23 MEC derives from both luminal and nonluminal cells, with a growth pattern characterized by the presence of luminal epithelial cells surrounded by intermediate cells, which were considered to be modified myoepithelial cells. Other studies have investigated the immunoexpression of myoepithelial markers in MEC, especially those relating to smooth muscle antigens such as -smooth muscle actin, caldesmon, and calponin, but the results obtained have been variable and inconclusive. 1,22,24-26

5 OOOO ORIGINAL ARTICLE Volume 115, Number 1 Fonseca et al. 83 Fig. 2. Histologic features of mucoepidermoid carcinoma (MEC). A, B, Low-grade MEC mainly composed of variably sized cystic spaces lined by mucous, intermediate, and squamous cells (HE, 100). C, Predominance of clear cells in a case of MEC (HE, 200). D, High-grade MEC showing solid growths of neoplastic cells with scarce cystic spaces (HE, 100). There are few studies describing the expression of p63 protein in MEC. García et al. (2011), 27 Bilal et al. (2003), 9 Mitani et al. (2011), 14 Weinreb et al. (2009), 28 and Seethala et al. (2005) 13 evaluated the immunoexpression of p63 in MEC, and all cases showed variable proportions of cells with nuclear positivity for p63. Similarly, Maruya et al. (2005) observed that 66.7% of MECs in their study were positive for p63 protein. 12 These results were explained by the presence of intermediate and squamous cells, which would represent modified myoepithelial cells, further supporting the ultrastructural results obtained previously that revealed the presence of myofilaments in these cells, which would have been responsible for -smooth muscle actin positive reactions. 26,29 Similarly to the the studies by García et al. 27 and by Weinreb et al. 28 which evaluated p63 immunoexpression in oncocytic MEC, in the present study it was observed that cells positive for p63 in MEC represented both intermediate and squamous cells of the solid growth areas and of the lining cystic spaces, where the immunoexpression was not limited to the basal layer, highlighting the multilayered intermediate-/ squamous-type cells of these cystic spaces. In addition, with the use of digital analysis it was observed that lowand high-grade MEC showed an increased expression of p63 compared with mucous retention cysts and papillary cystadenomas, which may be related to the predominance of intermediate and squamous cells in these tumors, as has also been reported by Maruya et al. 12 and Seethala et al. 13 It is notable that mucous cells and clear cells, which can predominate in some cases of MEC, were negative for p63. Papillary cystadenoma of the salivary gland is a well circumscribed multicystic benign epithelial neoplasm showing intracystic papillary projections of its epithelial lining. 6,8 Most cases are lined by columnar or cuboidal cells, but a predominance of oncocytic cells, resembling Warthin tumor, is not uncommon, as was the case in 1 of our 4 samples. 5 Cystadenomas of the salivary gland are rare, with few cases reported, and usually not included in large series or reviews of salivary gland tumors, being occasionally misdiagnosed as either low-grade MEC or mucous retention cysts showing multiple cavities. 8,30 There are no studies considering the immunoexpression of p63 in mucous retention cysts and in cystadenomas of salivary glands. In the present study, it was observed that cells expressing p63 were mainly located in the basal layer of the epithelial lining of the cystic spaces of the mucous retention

6 ORAL AND MAXILLOFACIAL PATHOLOGY OOOO 84 Fonseca et al. January 2013 Fig. 3. P63 protein immunoexpression in mucous retention cyst (MRC) and papillary cystadenoma. A, B, MRC showing nuclear staining in basal cells of the cystic epithelium of MRC ( 200 and 100, respectively). C, D, E, F, Positive staining in basal cells of papillary cystadenoma ( 100, 200, 400, and 400, respectively). cysts, indicating that they represent myoepithelial cells. However, in several cases positive nuclear staining was very difficult to identify, and in some areas this positivity could not be observed, which may have been due to the extremely thin epithelial lining present in these cases which prevented proper detection of positive nuclei. More interestingly, the labeling pattern observed in all cases of papillary cystadenomas was also restricted to the basal layers, suggesting that the cystic spaces present in these tumors keep a similarity to normal ducts, as described in our cases of mucous retention cysts and previously reported for necrotizing sialometaplasia. 31 This labeling pattern also indicates that the proliferative cells in cystadenomas are the luminal ones, as proposed by Dardick et al. (1996). 23 In low-grade MEC, p63 showed a distinct expression pattern, staining cells in all layers of the epithelial lining of the cystic spaces, although not uniformly in all cases. In fact, the loss of distinct and normal basal/myoepithelial cells found in some cystic spaces in MEC is a strong indication of malignancy, as characteristically occurs in breast and prostate cancer. 11,16 In conclusion, the staining pattern of p63 protein in papillary cystadenoma and low-grade MEC could be

7 OOOO ORIGINAL ARTICLE Volume 115, Number 1 Fonseca et al. 85 Fig. 4. P63 immunoexpression in mucoepidermoid carcinoma (MEC). A, B, Low-grade MEC revealed nuclear expression in intermediate and squamous cells, not only in the basal cells of the cystic spaces, but also in the luminal cells of the epithelial projections ( 100). C, P63 immunoexpression was absent in clear cells of MEC ( 200). D, High-grade tumors showing diffuse reactivity in intermediate and squamous cells ( 200). low-grade tumors, and much lower indexes were found in mucous retention cysts and papillary cystadenomas. Fig. 5. Percentage of stained cells for p63 protein in mucous retention cyst (MRC), papillary cystadenoma, low-grade mucoepidermoid carcinoma (MEC), and high-grade MEC. Analysis of variance: P a Significant difference between MRC and low- and high-grade MEC (P.0001). b Significant difference between papillary cystadenoma and low-grade MEC (P.001). c Significant difference between papillary cystadenoma and high-grade MEC (P.0001). helpful in the diagnosis in cases posing histologic difficulties. Moreover, a higher percentage of cells were positive for p63 in high-grade MEC compared with REFERENCES 1. Loyola AM, Sousa SOM, Araújo NS, Araújo VC. Study of minor salivary gland mucoepidermoid carcinoma differentiation based on immunohistochemical expression of cytokeratins, vimentin and muscle-specific actin. Oral Oncol 1998;34: Barnes L, Eveson JW, Reichart P, Sidransky. World Health Organization classification of tumors. Pathology and genetics. Head and neck tumors. Lyon: IARC; Schwarz S, Stiegler C, Müller M, Ettl T, Brockhoff G, Zenk J, Agaimy A. Salivary gland mucoepidermoid carcinoma is a clinically, morphologically and genetically heterogeneous entity: a clinicopathological study of 40 cases with emphasis on grading, histological variants and presence of the t(11;19) translocation. Histopathol 2011;58: Kwon H, Lim WB, Choi YD, Nam JH, Han CW, Kim JS, et al. High-grade oncocytic mucoepidermoid carcinoma of the minor salivary gland origin: a case report with immunohistochemical study. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2010;109:e Bezic J, Durdov MG. Salivary oncocytic cystadenoma with intraluminal crystalloids: a case report with review of the literature. Head Neck Pathol 2011;5: Guccion JG, Redman RS, Calhoun NR, Saini N. Papillary cystadenoma of the palate: a case report and ultrastructural study. J Oral Maxillofac Surg 1997;55: Auclair EG. In: AFIP atlas of tumor pathology. Series 4. Tumors

8 ORAL AND MAXILLOFACIAL PATHOLOGY OOOO 86 Fonseca et al. January 2013 of the salivary glands. Washington, DC: ARP Press: 2008; p Lim CS, Ngu I, Collins AP, McKellar GMW. Papillary cystadenoma of a minor salivary gland: report of a case involving cytological analysis and review of the literature. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2008;105:e Bilal H, Handra-Luca A, Bertrand JC, Fouret PJ. P63 is expressed in basal and myoepithelial cells of human normal and tumor salivary gland tissues. J Histochem Cytochem 2003; 51: Edwards PC, Bhuiya T, Kelsch RD. Assessment of p63 expression in the salivary gland neoplasms adenoid cystic carcinoma, polymorphous low-grade adenocarcinoma, and basal cell and canalicular adenomas. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2004;97: Crum CP, McKeon FD. p63 in epithelial survival, germ cell surveillance, and neoplasia. Annu. Rev Pathol Mech Dis 2010; 5: Maruya SI, Kies MS, Williams M, Myers JN, Weber RS, Batsakis JG, El-Naggar AK. Differential expression of p63 isotypes (DN and TA) in salivary gland neoplasms: biological and diagnostic implications. Hum Pathol 2005;36: Seethala RR, LiVolsi VA, Zhang PJ, Pasha TL, Baloch ZW. Comparison of p63 and p73 expression in benign and malignant salivary gland lesions. Head Neck 2005;27: Mitani Y, Li J, Weber RS, Lippman SL, Flores ER, Caulin C, El-Naggar AK. Expression and regulation of the N and TAp63 isoforms in salivary gland tumorigenesis. Clinical and experimental findings. Am J Pathol 2011;179: Graziano V, De Laurenzi V. Role of p63 in cancer development. Biochim Biophys Acta 2011, 1816;1816: Stefanou D, Batistatou A, Nonni A, Arkoumani E, Agnantis NJ. P63 expression in benign and malignant breast lesions. Histol Histopathol 2004;19: Weber A, Langhanki L, Schütz A, Gerstner A, Bootz F, Wittekind C, Tannapfel A. Expression profiles of p53, p63, and p73 in benign salivary gland tumors. Virchows Arch 2002;441: Boldrup L, Coates PJ, Laurell G, Nylander K. Differences in p63 expression in SCCHN tumours of different sub-sites within the oral cavity. Oral Oncol 2011;47: Jo VY, Fletcher CD. P63 immunohistochemical staining is limited in soft tissue tumors. Am J Clin Pathol 2011;136: Ramer N, Wu HS, Sabo E, Ramer Y, Emanuel P, Orta L, Burstein DE. Prognostic value of quantitative p63 immunostaining in adenoid cystic carcinoma of salivary gland assessed by computerized image analysis. Cancer 2010;116: Zhu X, Zhang J, Chen X, Feng X. Comparison of Ki-67, cyclin E, and p63 in benign and malignant human pleomorphic adenoma. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2012;113: Yook JI, Lee SA, Chun YC, Huh J, Cha IH, Kin J. The myoepithelial cell differentiation of mucoepidermoid carcinoma in a collagen gel-based coculture model. J Oral Pathol Med 2004;33: Dardick I. Color atlas/text of salivary gland tumor pathology. New York: Igaku-Shoin Medical; p Hassanin MB, Ghosh L, Das AK, Waterhouse JP. Immunohistochemical and fluorescent microscopic study of histogenesis of salivary mucoepidermoid carcinoma. J Oral Pathol Med 1989; 18: Regezi JA, Zarbo RJ, Batsakis JG. Immunoprofile of mucoepidermoid carcinomas of minor salivary glands. Oral Surg Oral Med Oral Pathol 1991;71: Foschini MP, Marucci G, Eusebi V. Low-grade mucoepidermoid carcinoma of salivary glands: characteristic immunohistochemical profile and evidence of striated duct differentiation. Virchows Arch 2002;440: García JJ, Hunt JL, Weinreb I, McHugh JB, Barnes EL, Cieply K, et al. Fluorescence in situ hybridization for detection of MAML2 rearrangements in oncocytic mucoepidermoid carcinomas: utility as a diagnostic test. Hum Pathol 2011;42: Weinreb I, Seethala RR, Perez-Ordoñez B, Chetty R, Hoschar AP, Hunt JL. Oncocytic mucoepidermoid carcinoma. Clinicopathologic description in a series of 12 cases. Am J Surg Pathol 2009;33: Dardick I, Gliniecki MR, Heathcote JG, Burford-Mason A. Comparative histogenesis and morphogenesis of mucoepidermoid carcinoma and pleomorphic adenoma. An ultrastructural study. Virchows Arch A Pathol Anat Histopathol 1990;417: Tsurumi K, Kamiya H, Yokoi M, Kameyama Y. Papillary oncocytic cystadenoma of palatal minor salivary gland: A case report. J Oral Maxillofac Surg 2003;61: Rizkalla H, Toner M. Necrotizing sialometaplasia versus invasive carcinoma of the head and neck: the use of myoepithelial markers and keratin subtypes as an adjunct to diagnosis. Histopathol 2007;51: Reprint requests: Prof. Dr. Márcio Ajudarte Lopes Departamento de Diagnóstico Oral, Semiologia Faculdade de Odontologia de Piracicaba UNICAMP Av. Limeira, 901 CEP Piracicaba São Paulo, Brazil malopes@fop.unicamp.br