Histopathology 2010, 57, 410–417. DOI: 10.1111/j.1365-2559.2010.03645.x
Human salivary gland morphogenesis: myoepithelial cell maturation assessed by immunohistochemical markers Renata Fraga Ianez,1 Marcilei E Buim,1 Claudia M Coutinho-Camillo,1 Regina Schultz,2 Fernando A Soares,1,3 & Silvia Vanessa Lourenc¸o3 1
Department of Surgical Pathology, Hospital A.C. Camargo,2Department of Surgical Pathology, Hospital das Clı´nicas, Medical School, and 3Department of General Pathology, Dental School, University of Sa˜o Paulo, Sa˜o Paulo, Brazil
Date of submission 29 October 2009 Accepted for publication 14 December 2009
Ianez R F, Buim M E, Coutinho-Camillo C M, Schultz R, Soares F A & Lourenc¸o S V (2010) Histopathology 57, 410–417
Human salivary gland morphogenesis: myoepithelial cell maturation assessed by immunohistochemical markers Aims: Myoepithelial cells are important components of salivary gland structure, aiding the expulsion of saliva from acinar lobules. The aim was to evaluate the expression of smooth muscle actin (SMA), calponin, caldesmon, CD10, CD29, S100 protein, glial fibrillary acidic protein (GFAP) and p63 in myoepithelial cells during salivary gland morphogenesis to understand the maturation process of these cells and their possible use in the diagnosis of salivary gland lesions. Methods and results: Major and minor human salivary glands at various stages of development, derived from fetuses at 8–26 weeks of gestation, were studied immunohistochemically. Fully developed salivary
glands were used as controls. The protein p63 was present in all stages of salivary gland morphogenesis from initial bud to terminal bud stage. CD29, S100 and calponin were detected increasingly as salivary gland structure matured and in fully developed salivary gland. Proteins GFAP, CD10 and caldesmon were not observed in myoepithelial cells of salivary glands. Conclusions: The proteins SMA, calponin, CD29, S100 and p63, which are present from the earliest stages of salivary gland maturation, are valuable myoepithelial markers but, although very specific, are not exclusive markers for this cell type.
Keywords: human, morphogenesis, myoepithelial cells, salivary gland Abbreviations: GFAP, glial fibrillary acidic protein; SMA, smooth muscle actin
Introduction Similar to other organs, the formation of the salivary gland involves coordination of morphogenetic mechanisms, including regulated changes in cell shape and gene expression, and directed cell migration leading to a fully developed gland with important secretory functions. Morphologically, all salivary glands develop similarly, starting with the proliferation of a solid cord of cells from the epithelium of the stomatodeum into the underlying ectomesenchyme. This cord of cells Address for correspondence: Dr S V Lourenc¸o, Faculdade de Odontologia da Universidade de Sa˜o Paulo, Av. Prof. Lineu Prestes, 2227, Sa˜o Paulo-SP, CEP: 05508-000, Brazil. e-mail:
[email protected] 2010 Blackwell Publishing Limited.
extends deeply into the ectomesenchyme and branches extensively, forming channels with secretory end pieces.1,2 In this glandular structure, myoepithelial cells are important components, though their participation during the stages of development is still largely unknown. Myoepithelial cells are closely related to the secretory units and proximal ducts, being located between the basal lamina and the acinar or ductal cells. The distribution of myoepithelial cells varies considerably between types of glands and even in the same gland during the course of development. The morphology of these cells may vary and their role includes contraction when the secretory function of the gland is stimulated, compressing and strengthening the cells of the glandular parenchyma and assisting the expulsion of saliva.
Salivary gland myoepithelial maturation
Their origin is not yet fully elucidated but they are believed to derive from stem cells.3,4 Given the importance of myoepithelial cells in salivary gland physiology and tumour aetiopathogenesis, the objective of this study was to evaluate the expression of smooth muscle actin (SMA), calponin, caldesmon, CD10, CD29, S100 protein, glial fibrillary acidic protein (GFAP) and p63 in myoepithelial cells during salivary gland morphogenesis, in order to understand the maturation process of these cells and explore the possible use of these markers in the diagnosis of salivary gland lesions.
Material and methods Specimens from the oral cavity of 30 human fetuses at 8–26 weeks of gestation were obtained from the Medical School of University of Sa˜o Paulo, in accordance with the authorization of the Ethics Committee of this Institution. The specimens were collected from different oral sites, including the buccal mucosa, tongue, hard palate, lips, submandibular and parotid gland. All specimens were fixed in buffered formalin and embedded in paraffin. Sections of the specimens were stained with haematoxylin and eosin to confirm the presence of salivary glands, to study their morphology and to determine their developmental phase. Adult salivary glands were used as positive controls in accordance with the specifications of each antibody.
411
retrieval methods as well as primary antibody clones, source and title are given in Table 1. The sections were then incubated in 3% aqueous hydrogen peroxide for 15 min to quench endogenous peroxidase activity and with protein block serum-free (DakoCytomation, Carpinteria, CA, USA) for 20 min at room temperature to suppress non-specific binding of subsequent reagents. The reaction was followed by incubation with the primary antibody in two steps for double labelling. The first primary antibody was incubated overnight and the second for 2 h. All information about the primary antibody used is depicted in Table 1. The sections were rinsed in phosphate-buffered saline for 5 min between each step of the protocol. The immunolabelling of the first primary antibody was developed using Novolink Polymer (Dako Cytomation) and with 3,3¢-diaminobenzidine (Dako Cytomation) as the chromogen. Immunolabelling of the second primary antibody was developed with En Vision phosphatase (Dako Cytomation) for 30 min with Permanent red (Dako Cytomation) as the chromogen. The slides were then rinsed in distilled water and counterstained with Carazzi’s haematoxylin. Finally, the sections were air dried and mounted in aqueous mountant and a glass cover slip. Negative controls were performed by incubation of the sections with non-immune serum. Positive controls were employed according to the antibody manufacturers’ instructions.
Results immunohistochemistry: double staining Serial sections (4 lm) of the developing salivary gland samples were deparaffinized, re-hydrated and then submitted to antigen retrieval. Details of antigen Table 1. Primary serum, clones, manufacturer, title and antigen retrieval
The specimens studied were of the major and minor human salivary gland at various stages of development. These stages were classified according to Tucker5: initial bud, pseudoglandular stage, canalicular
Primary serum
Clone
Manufacturer
Title
Antigen retrieval
p63
4A4
1:100
Santa Cruz, Santa Cruz, CA, USA
Citrate, pH 6.0
Smooth muscle actin
1A4
1:600
Dako, Carpinteria, CA, USA
Citrate, pH 6.0
Caldesmon
H-CD
1:100
Dako
Citrate, pH 6.0
Calponin
CALP
1:100
Dako
Citrate, pH 6.0
S100
DAK-S100
1:100
Dako
Citrate, pH 6.0
CD10
SS2 ⁄ 36
1:80
Dako
Citrate, pH 6.0
CD29
HIP2
1:100
Santa Cruz
Citrate, pH 6.0
GFAP
6F2
1:1200
Dako
Citrate, pH 6.0
2010 Blackwell Publishing Ltd, Histopathology, 57, 410–417.
412
R F Ianez et al.
A
C
B
Figure 1. Stages of salivary gland morphogenesis. A, Initial bud: solid cluster of epithelial cells derived from the oral surface epithelium (H&E). B, Pseudoglandular: initial phase of a ductal system and beginning of lumen formation. Some clusters of branches and buds are visible at this stage (H&E). C, Canalicular: formation of complex architecture of ductal branches producing a multi-lobed gland (H&E). D, Terminal bud: differentiation of terminal buds into acinar lobules (H&E).
D
stage and terminal bud stage (cytodifferentiation) (Figure 1). i mm u no h i sto c h em i c a l an al y si s The protein p63 was present at all stages of salivary gland morphogenesis from initial bud to terminal bud stage. Initially, p63 could be observed in the basaloid cells of the solid bud (and was the only marker present at this stage), and as long as morphogenesis advanced, basal cells and myoepithelial cells along the ductal system and around acinic lobules were positive for the protein p63. At pseudoglandular and canalicular stages of salivary gland morphogenesis, when canalization was more evident, there was co-location of protein p63 with SMA, calponin, CD29 and S100 protein in myoepithelial cells around the rudimentary terminal sacs. A few cells localized along the ductal network were also positive for these markers. Additionally, p63 positivity was observed in the nuclei of basal cells of the entire developing gland system. During the terminal bud stage, when cytodifferentiation of acinar lobules was observed, a greater number of myoepithelial cells could be detected with the markers employed in the study. The protein p63, besides being present in myoepithelial cells, was also
seen in basal cells along the entire structure of the ducts of the salivary glands. The presence of GFAP, CD10 and caldesmon was not observed in any phase of gland morphogenesis, only in nerve bundles, stromal cells and vessel walls, respectively. Finally, adult normal salivary glands, used as control for the reactions, showed expression of SMA, calponin, CD29, S100 protein and p63 in myoepithelial cells around the acinar lobules and in the proximal portions of salivary ducts (intercalated ducts). The protein p63 was also detected in basal cells of the entire ductal system including the excretory ducts that merge with the covering epithelium of the oral mucosa. A summary of immunohistochemical markers of myoepithelial cells in developing and fully developed salivary glands is given in Table 2. The results are also illustrated in Figures 2 and 3.
Discussion The results of morphological analysis of the salivary glands of fetuses between 8 and 26 weeks of gestation have shown that the development of salivary gland occurs at different times in different sites studied, and in the same glandular structures, different stages of differentiation can be observed. Following the classification of Tucker,7 we were able to identify salivary 2010 Blackwell Publishing Ltd, Histopathology, 57, 410–417.
Salivary gland myoepithelial maturation
413
Table 2. Myoepithelial markers in developing and fully developed human salivary glands SMA
Calponin
S100
CD29
P63
GFAP
Caldesmon
CD10
)
)
)
)
+
)
)
)
Pseudoglandular
+
)
)
)
+
)
)
)
Canalicular
++
+
+
+
++
)
)
)
Terminal bud
++
++
+
+
++
)
)
)
++
++
++
++
++
)
)
)
Developing human salivary gland Initial bud
Fully developed salivary gland
), negative; +, few positive cells; ++, positivity in the majority of myoepithelial cells.
gland structures in their full range of morphological developmental stages, i.e. initial bud, pseudoglandular, canalicular and terminal bud stages. These stages are classically used to examine murine submandibular glands, which have been used as a classic laboratory model to investigate both the development of and the fully developed salivary glands. Allied to laboratory models of salivary glands, the use of other experimental methods to study the differentiation of cellular components of the salivary glands and tumours that affect these glands has progressed considerably in recent years. Immunohistochemical techniques that enable in situ analysis of these components have provided great advances. However, several studies investigating myoepithelial cells have been performed in animal models, and these models can harbour important physiological differences from human tissues, as we have previously demonstrated in studies on odontogenesis.6 There are several proteins that characterize and distinguish the various types of epithelial and myoepithelial cells of salivary glands in animal models and human samples of normal and neoplastic salivary tissue. These proteins are described in original articles and in various reviews.7–12 Some proteins described as myoepithelial markers in these studies are employed not only in research, but also for diagnostic purposes.10,12 Others still need further investigation to standardize their use. The contractile proteins SMA and calponin are among the classical markers of myoepithelial cells. These proteins were found in an increasing number of cells as salivary gland morphogenesis progressed. At the pseudoglandular stage of morphogenesis, SMA+ cells were located at the terminal buds, but at more advanced stages of salivary gland morphogenesis they were present around the acinic lobule up to the end of the intercalated ducts. The indentification of calponin occured almost simultaneously with 2010 Blackwell Publishing Ltd, Histopathology, 57, 410–417.
the start of differentiation of acinar lobules. In this context, maturation of myoepithelial cells will aid expulsion of saliva stored in acinar cells. Our results are in accordance with studies that describe SMA+ myoepithelial cells in developing salivary gland especially after initial maturation of acinar cells.13,14 Furthermore, Ogawa et al.15 consider calponin to be a useful tool for the identification of myoepithelial cells of salivary glands during their differentiation. Both these markers are considered useful for the identification of myoepithelial cells in tumours.16,17 S100 protein is also considered to be a classical marker for myoepithelial cells by various authors.18,19 In our study, the presence of S100 protein was observed from the pseudoglandular stage of salivary gland morphogenesis, in myoepithelial cells around the terminal sacs. In both later stages of salivary gland morphogenesis and in fully developed salivary glands, this protein was detected in myoepithelial cells around acinar lobules and intercalated ducts. Other workers have produced diverse data on the expression of S100 in myoepithelial cells. Grandi et al.20 reported expression of S100 in myoepithelial cells of the breast, bronchial and sweat glands, but not salivary glands. Jolicoeur et al.21 in their investigation of myoepithelial cells of developing and adult breast suggest that expression of the protein S100 might be a transient phenomenon during the early differentiation process of mammary glands, and describe a ‘lack of sensitivity’ of S100 protein as a marker of differentiated mammary myoepithelial cells. These differences probably reflect peculiarities in research tissue as well as subtle distinct roles of myoepithelial cells in different systems. Additionally, important work by Dardick et al.22 has shown that S100 protein is not expressed by normal salivary gland myoepithelium. In this work, the authors demonstrated that in parotid tissue, S100 is only expressed by the rich network of unmyelinated
414
R F Ianez et al.
A
(p63 + SMA)
B
(p63 + SMA)
C
(p63 + SMA)
D
(S-100)
E
(S-100)
F
(S-100)
G
(p63 + calponin)
H
(p63 + calponin)
I
(p63 + calponin)
J
(p63 + CD29)
K
(p63 + CD29)
L
(p63 + CD29)
2010 Blackwell Publishing Ltd, Histopathology, 57, 410–417.
Salivary gland myoepithelial maturation
415
A
(p63)
B
(SMA)
C
(S100)
D
(calponin)
E
(CD29)
F
(caldesmon)
Figure 3. Myoepithelial markers in fully developed salivary glands. A, p63 nuclear expression in basal cells of excretory duct and in basal cells of oral surface epithelium. B–E, Expression of smooth muscle actin, S100, calponin and CD29 in myoepithelial cells around acinar lobules. F, The protein caldesmon is only positive in blood vessels walls.
nerves in the gland. These thin nerve fibres may erroneously be interpreted as myoepithelium, as the authors found no combined expression of S100 protein or other markers of myoepithelial cells such as musclespecific actin in striated ducts, for example. In our study, we found some myoepithelial cells positive for calponin, but negative for S100. This mosaic expression pattern may indicate that the population of developing myoepithelial cells is heterogeneous and some of them may be representative of a pluripotent
(stem cell) population. This needs to be confirmed in further investigations. Supporting this evidence, Zarbo et al.23 have reported that S100 protein immunoreactivity in normal salivary glands is variable. Myoepithelial cells are observed along the epithelial– mesenchymal interface of human organs such as breast, sweat glands and salivary glands.24 Therefore, b1-integrin (CD29), a cell ⁄ extracellular matrix ligand, has been regarded as a marker for myoepithelial cells.21 In our study, expression of b1-integrin (CD29) was
Figure 2. Myoepithelial markers during salivary gland branching morphogenesis. A, B, C, In A (pseudoglandular stage), p63 (brown) is present in basal cells of initial ductal system; rare cells are positive for smooth muscle actin (SMA) (red) (myoepithelial cells). In canalicular phase (B) an increasing number of cells are positive for SMA and p63+ cells (brown) are seen in the basal layer of the branched ductal system and co-localize with SMA+ cells. In terminal bud phase (C), SMA+ cells (red) (myoepithelial cells) are seen around the acinar lobules and intercalated ducts and co-localize with p63 nuclear stain (brown). Basal cells of the entire ductal system are positive for p63 (nuclear stain). Blood vessel positive for SMA (arrow). D, E, F, In D (pseudoglandular stage), only rare cells and a nerve bundle (arrow) are positive for S100. In canalicular phase (E) an increasing number of cells are positive for S100 around the ductal system terminal buds. F, S100+ cells (myoepithelial cells) are seen around the acinar lobules and intercalated ducts. G, H, I, In G (pseudoglandular stage), p63 (brown) is present in basal cells of initial ductal system; only a few cells are mildly positive for calponin (red). In canalicular phase (H) an increasing number of cells are positive for calponin (red), around the final extremity of the ductal system; p63+ cells (brown) are seen in the basal layer of the branched ductal system. In terminal bud phase (I) calponin-positive cells (red) (myoepithelial cells) are seen around the acinar lobules and intercalated ducts (positivity is only mild) and co-localize with p63 nuclear stain (brown). Basal cells of the entire ductal system are positive for p63 (nuclear stain). Blood vessel is mildly positive for calponin (arrow). J, K, L, In J (pseudoglandular stage), p63 (brown) is present in basal cells of initial ductal system; at this stage CD29 is not detected. Blood vessel wall positive for CD29 (red) (arrow). In canalicular phase (K) a few cells positive for CD29 (red) are observed around the more developed glandular structure; p63+ cells (brown) are seen in the basal layer of the branched ductal system. In terminal bud phase (L) CD29+ cells (red) (myoepithelial cells) are seen around the acinar lobules and intercalated ducts and co-localize with p63 nuclear stain (brown). Blood vessel positive for CD29 (arrow). 2010 Blackwell Publishing Ltd, Histopathology, 57, 410–417.
416
R F Ianez et al.
detected in myoepithelial cells around the terminal sacs of the salivary glands up to the intercalated duct, This confirms the interface role of myoepithelial cells that probably mediate intercommunication via CD29 between epithelial cells and mesenchymal tissue during the development of salivary glands and in adult life. This is crucial for maintenance of tissue homeostasis. Furthermore, in a previous study of salivary gland development, our group suggested that b1-integrin might be related to progenitor cells in the intercalated ducts in the early stages of salivary gland morphogenesis.25 However, this possibility is still subject to confirmation, as in the later stages of salivary gland differentiation a great number of myoepithelial and basal cells express this protein. Therefore we might consider CD29 as a sensitive but non-specific marker of myoepithelial cells, since it is also present in other cells of the ductal system. The protein p63 has also been increasingly employed as a myoepithelial cell marker, especially in mammary glands. Members of the p63 family are reported to be predominantly expressed by progenitors (basally located stem ⁄ reserve cells) in the epithelium of many human organs.21 Our results have demonstrated the presence of p63 in proliferative layers of the covering oral epithelium and in glandular structures of developing salivary glands (myoepithelial and basal cells of the glandular system). Other workers have verified similar results in adult salivary glands.26–28 The presence of p63 is also reported in basal cells of skin and in myoepithelial cells of mammary glands. The protein is also a marker of basal cells of the prostate and is regarded as a basic requirement for the development of the organ according to Signoretti et al.29 In view of these results, we speculate that p63 could be useful as a marker of the most primitive and undifferentiated cells within benign and malignant tumours and, if this is confirmed, may be useful as a tool to aid differential diagnosis of difficult lesions. Some authors have also hypothesized that the population of cells positive for p63 in many tissues may include stem cells and that it can also occur in the salivary glands.30–32 Indeed, in some specimens included in our study, p63+ cells were detected in the acinar ⁄ intercalated duct junction, and this fact may be related to the presence of progenitor ⁄ reserve cells at this site. However, its expression in the population of myoepithelial cells affects the analysis of this possibility, and the controversy of the relationship between luminal and myoepithelial cells with respect to primitive cells of the salivary glands remains. The proteins GFAP, caldesmon and CD10 were not detected in myoepithelial cells in any phase of
development of salivary glands samples analysed in this study, contrary to some studies that have considered these proteins to be markers of myoepithelial cells.19,33 Santos et al.34 have shown that GFAP is expressed in certain cases of myoepithelial tumours by immunohistochemistry, including soft tissue myoepitheliomas, which are related to cartilaginous differentiation, and suggest that this protein contributes significantly to the diagnosis of soft tissue myoepitheliomas with cartilaginous differentiation. Other studies have reported that GFAP does not constitute a good marker for myoepithelial cells of various glands—breast, sweat glands and salivary glands.15,20,35 The absence of caldesmon in myoepithelial cells of developing salivary glands also concurs with published data. In 2005 and 2006, Furuse et al.18 and Leibl and Moinfar,19 respectively, demonstrated that myoepithelial cells of normal salivary glands and tumour originating in the salivary glands do not express this protein. CD10 protein was not detected in developing glandular structures in our study and was present only in blood vessel walls, concordant with results published by Kalof et al.33 Lack of CD10 was reported in most developing breast specimens examined in the study of Jolicoeur et al.21 Neves et al.36 also report that myoepithelial cells in normal human salivary glands are negative for this marker. However, in the review on salivary gland pathology by Cheuk and Chan,37 CD10 is considered to be a myoepithelial cell marker with low specificity. Considering all this conflicting information on CD10, other studies are needed to relate this protein to myoepithelial cells or to rule out the possibility of CD10 as a useful marker for these cells. In summary, our immunohistochemical study on salivary gland myoepithelial differentiation has shown that this process is complex and tightly time and space regulated in which several markers of myoepithelial cells could be valuable for research and diagnostic purposes. Amongst these markers, the proteins SMA, calponin, CD29, S100 and p63, which are present from the earliest stage of maturation of this cell type, were the most valuable; however, they are not exclusive proteins of myoepithelial cells, and can be present in other cell types.
Competing interests None to declare.
References 1. Dale AC. Salivary gland. In Ten Cate AR ed. Oral histology, 4th edn. St Louis: Mosby, 1994; 312–333. 2010 Blackwell Publishing Ltd, Histopathology, 57, 410–417.
Salivary gland myoepithelial maturation
2. Klein RM. Development, structure and function of salivary glands. In Avery JK ed. Oral development and histology, 2nd edn. New York: Thieme, 1994; 352–379. 3. Redman RS. Myoepithelium of salivary glands. Microsc. Res. Tech. 1994; 27; 25–45. 4. Ogawa Y. Immunohistochemistry of myoepithelial cells in the salivary glands. Prog. Histochem. Cytochem. 2003; 38; 343–426. 5. Tucker AS. Salivary gland development. Semin. Cell Dev. Biol. 2007; 18; 237–244. 6. Sassa´ Benedete AP, Sobral AP, Lima DM et al. Expression of transforming growth factor-beta 1, -beta 2, and -beta 3 in human developing teeth: immunolocalization according to the odontogenesis phases. Pediatr. Dev. Pathol. 2008; 11; 206–212. 7. Dardick I, Rippstein P, Skimming L et al. Immunohistochemistry and ultrastructure of myoepithelium and modified myoepithelium of the ducts of human major salivary glands: histogenetic implications for salivary gland tumours. Oral Surg. Oral Med. Oral Pathol. 1987; 64; 703–715. 8. Dardick I, Parks WR, Little J et al. Characterization of cytoskeletal proteins in basal cells of human parotid salivary gland ducts. Virchows Arch. A Pathol. Anat. Histopathol. 1988; 412; 525–532. 9. Dardick I, Burford-Mason AP. Pathology of the salivary glands: the contribution of electron microscopy. Microsc. Res. Tech. 1994; 27; 46–60. 10. Takai Y, Dardick I, Mackay A et al. Diagnostic criteria for neoplastic myoepithelial cells in pleomorphic adenomas and myoepitheliomas. Immunocytochemical detection of musclespecific actin, cytokeratin 14, vimentin and glial fibrillary acidic protein. Oral Surg. Oral Med. Oral Pathol. 1995; 79; 330–341. 11. Erlandson RA, Cardon-Cardo C, Higgins PJ. Histogenesis of benign pleomorphic adenoma (mixed tumor) of the major salivary glands. An ultrastructural and immunohistochemical study. Am. J. Surg. Pathol. 1984; 8; 803–820. 12. Kahn HJ, Baumal R, Marks A et al. Myoepithelial cells in salivary gland tumors. An immunohistochemical study. Arch. Pathol. Lab. Med. 1985; 109; 190–195. 13. Draeger A, Nathrath WBJ, Lane EB et al. Cytokeratins, smooth muscle actin and vimentin in human normal salivary gland and pleomorphic adenomas. Immunohistochemical studies with particular reference to myoepithelial and basal cells. APMIS 1991; 99; 405–415. 14. Araujo VC, Carvalho YR, Araujo NS. Actin versus vimentin in myoepithelial cells of salivary gland tumors. A comparative study. Oral Surg. Oral Med. Oral Pathol. 1994; 77; 387–391. 15. Ogawa Y, Yamauchi S, Ohnishi A et al. Immunohistochemistry of myoepithelial cells during development of the rat salivary glands. Anat. Embryol. (Berl.) 1999; 200; 215–228. 16. Okura M, Hiranuma T, Tominaga G et al. Expression of S-100 protein and glial fibrillary acid protein in cultured submandibular gland epithelial cells and salivary gland tissues. Am. J. Pathol. 1996; 148; 1709–1716. 17. Maruyama S, Cheng J, Shingaki S et al. Establishment and characterization of pleomorphic adenoma cell systems: an in-vitro demonstration of carcinomas arising secondarily from adenomas in the salivary gland. BMC Cancer 2009; 9; 247. 18. Furuse C, Sousa SO, Nunes FD et al. Myoepithelial cell markers in salivary gland neoplasms. Int. J. Surg. Pathol. 2005; 13; 57–65. 19. Leibl S, Moinfar F. Mammary NOS-type sarcoma with CD10 expression: a rare entity with features of myoepithelial differentiation. Am. J. Surg. Pathol. 2006; 30; 450–456.
2010 Blackwell Publishing Ltd, Histopathology, 57, 410–417.
417
20. Grandi D, Campanini N, Becchi G et al. On the myoepithelium of human salivary glands. An immunocytochemical study. Eur. J. Morphol. 2000; 38; 249–255. 21. Jolicoeur F, Gaboury LA, Oligny LL. Basal cells of second trimester fetal breasts: immunohistochemical study of myoepithelial precursors. Pediatr. Dev. Pathol. 2003; 6; 398–413. 22. Dardick I, Stratis M, Parks WR et al. S-100 protein antibodies do not label normal salivary gland myoepithelium. Am. J. Pathol. 1991; 138; 619–628. 23. Zarbo RJ, Hatfield JS, Trojanowski JO et al. Immunoreactive glial fibrillary acidic protein in normal and neoplastic salivary glands: a combined immunohistochemical and immunoblot study. Surg. Pathol. 1998; 1; 55–63. 24. Foschini MP, Scarpellini F, Gown AM et al. Differential expression of myoepithelial markers in salivary sweat and mammary glands. Int. J. Surg. Pathol. 2000; 8; 29–37. 25. Lourenc¸o SV, Lima DM, Uyekita SH et al. Expression of beta-1 integrin in human developing salivary glands and its parallel relation with maturation markers: in situ hybridisation and immunofluorescence study. Arch. Oral Biol. 2007; 52; 1064– 1071. 26. Bilal H, Handra-Luca A, Bertrand JC et al. P63 is expressed in basal and myoepithelial cells of human normal and tumour salivary gland tissues. J. Histochem. Cytochem. 2003; 51; 133– 139. 27. Yang A, Kaghad M, Wang Y et al. p63, a p53 homolog at 3q2729, encodes multiple products with transactivating, deathinducing, and dominant-negative activities. Mol. Cell 1998; 2; 305–316. 28. Barbareschi M, Pecciarini L, Cangi MG et al. p63, a p53 homologue, is a selective nuclear marker of myoepithelial cells of the human breast. Am. J. Surg. Pathol. 2001; 25; 1054– 1060. 29. Signoretti S, Waltregny D, Dilks J et al. p63 is a prostate basal cell marker and is required for prostate development. Am. J. Pathol. 2000; 157; 1769–1775. 30. Fuchs E, Raghavan S. Getting under the skin of epidermal morphogenesis. Nat. Rev. Genet. 2002; 3; 199–209. 31. Mills AA, Zheng B, Wang XJ et al. p63 is a p53 homologue required for limb and epidermal development. Nature 1999; 398; 708–713. 32. Yang A, Schweitzer R, Sun D. p63 essential for regenerative proliferation in limb, craniofacial and epithelial development. Nature 1999; 398; 714–718. 33. Kalof AN, Tam D, Beatty B et al. Immunostaining patterns of myoepithelial cells in breast lesions: a comparison of CD10 and smooth muscle myosin heavy chain. J. Clin. Pathol. 2004; 57; 625–629. 34. Santos GC, Carvalho KC, Falzoni R et al. Glial fibrillary acidic protein in tumor types with cartilaginous differentiation. Mod. Pathol. 2009; 22; 1321–1327. 35. Hainfellner JA, Voigtla¨nder T, Stro¨bel T et al. Fibroblasts can express glial fibrillary acidic protein (GFAP) in vivo. J. Neuropathol. Exp. Neurol. 2001; 60; 449–461. 36. Neves C de O, Soares AB, Costa AF et al. CD10 (neutral endopeptidase) expression in myoepithelial cells of salivary neoplasms. Appl. Immunohistochem. Mol. Morphol. 2010; 18; 172–178. 37. Cheuk W, Chan JK. Advances in salivary gland pathology. Histopathology 2007; 51; 1–20.
