RAPID COMMUNICATION |
Correspondence to: Pierre J. Fouret, Service d'Anatomie et de Cytologie Pathologiques, Groupe Hospitalier Pitié-Salpêtrière, 83 bou-levard de l'Hôpital, 75634 Paris Cedex 13, France. E-mail: pierre.fouret@psl.ap-hop-paris.fr
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Summary |
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p63 is essential for epithelial cell survival and may function as an oncogene. We examined by immunohistochemistry p63 expression in human normal and tumor salivary gland tissues. In normal salivary glands, p63 was expressed in the nuclei of myoepithelial and basal duct cells. Among 68 representative salivary gland tumors, 63 displayed p63 reactivity. In all tumor types differentiated towards luminal and myoepithelial lineages (pleomorphic adenomas, basal cell adenomas, adenoid cystic carcinomas, and epithelialmyoepithelial carcinomas), p63 was expressed in myoepithelial cells, whereas luminal cells were always negative. Similarly, in mucoepidermoid carcinomas, basal, intermediate, and squamous cells expressed p63, in contrast to luminal mucous cells. p63 reactivity was also restricted to basal cells in Warthin tumors and oncocytomas. Myoepitheliomas and myoepithelial carcinomas all expressed p63. The only five negative tumors were three of four acinar cell carcinomas and two of three adenocarcinomas. In conclusion, p63 is expressed in the nuclei of normal human salivary gland myoepithelial and basal duct cells. p63 expression is retained in the modified myoepithelial and basal cells of human salivary gland tumors, which suggests a role for p63 in oncogenesis of these complex tumors.
(J Histochem Cytochem 51:133139, 2003)
Key Words: p63, salivary gland, myoepithelial cell, tumor, immunohistochemistry
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Introduction |
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The p63 gene, mapping to 3q27-29, is the third member of the p53 family (, TA-ß, and TA-
) contain the N-terminal transactivation (TA) domain, activate transcription from p53-responsive promoters, and can induce apoptosis (
Np63 isoforms lack the TA domain and may function as a dominant-negative factor that inhibits the transactivation functions of p53 and TAp63 proteins (
The p63 -/- mice are characterized by agenesis of stratified squamous epithelia and their derivatives, including mammary, sebaceous, and salivary glands, and defects in limb and craniofacial structures (
In mouse embryos, p63 is expressed primarily within the ectoderm (
Somatic p63 mutations are exceedingly rare in human tumors (
Salivary glands comprise two lineages: one epithelial luminal lineage and one myoepithelial lineage. Myo-epithelial cells express -smooth muscle actin (
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Materials and Methods |
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Tissue Samples
We investigated by immunohistochemistry six normal and 68 tumor paraffin-embedded samples from human salivary gland: 15 pleomorphic adenomas, four basal cell adenomas, four oncocytomas, four myoepitheliomas, one canalicular adenoma, four Warthin tumors, 11 adenoid cystic carcinomas, nine mucoepidermoid carcinomas, four acinar cell carcinomas, one basal adenocarcinoma, two epithelialmyoepithelial carcinomas, three myoepithelial carcinomas, three adenocarcinomas, NOS, two polymorphous low-grade adenocarcinomas, and one carcinoma ex pleomorphic adenoma. All tumor samples contained salivary gland parenchyma or squamous epithelium adjacent to the tumor. Paraffin-embedded normal human breast tissue from reduction mammoplasty was used as a positive control for p63 immunostaining. Frozen tissue samples from normal human skin and submandibular gland were also immunostained.
Study cases were retrieved from the files of the Service d'Anatomie et de Cytologie Pathologiques, Groupe Hospitalier Pitié-Salpêtrière, Paris, France. All cases were reviewed by two pathologists (PF and AHL) and were classified using the diagnostic criteria of the World Health Organization International Histological Classification of Tumors (
Primary Antibodies
Two different anti-p63 antibodies were used: the mouse anti-human monoclonal antibody (MAb) 4A4 (NeoMarkers; Freemont, CA) was raised against amino acids 1205 mapping at the NH2 terminus of Np63; the mouse anti-human MAb 7JUL (Novocastra; Newcastle upon Tyne, UK) was raised against amino acids 319410. Both MAbs recognize all the different p63 isoforms. MAb 4A4 gave the strongest staining on paraffin sections but failed to localize p63 on frozen sections. Immunoreactivity with MAb 7JUL was similar to 4A4 staining on paraffin sections and could be detected on frozen sections. The mouse anti-human monoclonal antibody CKB1 (Sigma; St. Louis, MO), which recognizes cytokeratin 14, was used in frozen sections to stain myoepithelial cells.
Immunohistochemical Analysis
Immunohistochemical staining was performed according to standard procedures. Antigen retrieval was performed by bain-marie heating for 30 min at 98C in 10 mmol/liter citrate buffer, pH 6.0 (BioGenex; San Ramon, CA). Endogenous peroxidase activity was blocked by incubation in 3% H2O2 for 5 min. MAb 4A4 was applied at room temperature (RT) for 1 hr at a 1:200 dilution. The detection step was performed using an LSAB+ kit and DAB (DAKO; Carpinteria, CA) as chromogen. Slides were counterstained with hematoxylin. A positive control (normal human breast tissue) was included in each analysis. As negative staining control, the antibody anti-p63 was substituted for a mouse monoclonal anti-IgG of the same subclass (anti-IgG1; Sigma).
Double Immunofluorescence Staining
The 7JUL (anti-p63) and CKB1 (anti-cytokeratin 14) antibodies were used to realize double immunofluorescence staining in acetone-fixed frozen tissue sections of human normal salivary gland. A sequential method was performed. We used the Vectastain Universal ABC-AP kit (Vector Laboratories; Burlingame, CA) with a red substrate (Fast Red; DAKO) to detect p63 first. Nonspecific binding was blocked by horse serum. Slides were incubated overnight at 4C with MAb 7JUL at a 1:50 dilution. Fast Red was applied for 30 min. As a negative control for this staining, the 7JUL antibody was substituted for a mouse MAb of the same subclass (anti-IgG1). After rinsing, slides were incubated with goat serum and directly incubated with the CKB1 antibody at a 1:50 dilution for 60 min at RT. After washing, cytokeratin 14 signals were detected by a second FITC-conjugated antibody (FITC-conjugated AffiniPure F(ab')2 fragment goat anti-mouse IgM µ-chain-specific; Jackson ImmunoResearch Laboratories, West Grove, PA) at a 1:100 dilution for 30 min. Slides were then rinsed and coverslipped with Immu-Mount (Chandon; CML, Nemours, France). As a negative staining control MAb CKB1 was substituted for a mouse monoclonal anti-IgM (Sigma). The preparations were analyzed with a Zeiss Axioplan 2 Imaging microscope. p63 detected with Fast Red substrate was visualized using a conventional rhodamine filter set, resulting in a red emission. To observe double staining, an FITC filter set was used, resulting in an orange emission for p63 and a green emission for cytokeratin 14. Digital images were acquired using Axiovision 3.0 software.
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Results |
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p63 Expression in Normal Salivary Gland
p63-reactive nuclei were seen in basal cells around luminal cells in intercalated, striated, and some interlobular ducts (Fig 1). Small, often elongated nuclei at the periphery of the acini were also p63-reactive. The location of these p63-reactive nuclei suggested that the p63-reactive cells in the acini were myoepithelial cells. Luminal duct cells and acinar cells were unreactive.
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To confirm that p63-reactive nuclei belonged to myoepithelial cells, we realized a double immunofluorescence staining for p63 and cytokeratin 14. The p63-reactive nuclei were seen closely associated with cytokeratin 14-expressing cell processes. Although this association was seen in most cells, a few isolated p63-reactive cells were negative for cytokeratin 14.
p63 Expression in Benign Epithelial Tumors
Fifteen of 15 pleomorphic adenomas demonstrated p63 nuclear staining in the modified myoepithelial cells (Fig 2a2i). p63 reactivity was present in chondromyxoid areas as well as cellular areas, and did not correlate with tumor type (whether or not the tumor was predominantly myxoid or cellular). Tumor data are summarized in Table 1. Immunoreactivity was diffuse in 13 cases, although there was great variability in the approximate proportions of reactive tumor cells (between 10% and 90% of tumor cells). Focal positivity was observed in two cases. One had a large negative myxoid area; the other comprised a uniformly reactive myxoid zone and a large cellular zone in which only very few reactive cells were seen. All cell types were reactive, including stellate, plasmacytoid, spindle, clear cells, and cells appearing to be in transition from one type to another. The modified myoepithelial cells surrounding duct-like structures were often the most obviously reactive. p63 reactivity was also strong and consistent in basaloid cells at the periphery of squamous islands. In contrast, p63 reactivity was always absent in cuboidal or columnar epithelial cells lining duct lumens.
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The neoplastic cells of the four myoepitheliomas demonstrated p63 immunoreactivity. p63 protein was more consistently seen in spindle cells, whereas plasmacytoid and clear cells, which were predominant in one case, were occasionally reactive.
p63 reactivity was prominent in palisading nuclei of the dark cells along the peripheral stromal interface of the epithelial nests in four of four basal cell adenomas. Staining of the larger light cells was more variable. All cuboidal duct cells surrounding small lumens were negative.
Immunoreactivity was found in several foci of basaloid-appearing cells in one case of canalicular adenoma of the upper lip. A few columnar cells were also p63-reactive.
Four of four Warthin tumors demonstrated strong and constant p63 reactivity in basal cells. The palisading nuclei of luminal columnar cells were unreactive, although a strong nonspecific cytoplasmic staining could be seen. Scattered stromal lymphoid cells exhibited nuclear reactivity. Oncocytes in four oncocytomas also displayed moderate to strong nonspecific cytoplasmic staining, but specific immunoreactivity could not be observed in their centrally placed nuclei. However, variable numbers of smaller reactive nuclei were seen at the stromal interface of the oncocytic cell clusters.
p63 Expression in Primary Malignant Epithelial Tumors
The 11 adenoid cystic carcinomas showed a mixture of cribriform, tubular, and solid growth patterns (Fig 2j2t). Whatever the predominant growth pattern, the cell immunoreactivity was very consistent from one tumor to another. Abluminal cells of myoepithelial differentiation were diffusely and strongly reactive for p63. In contrast, the luminal cells were unreactive.
The nine mucoepidermoid carcinomas were predominantly cystic in four cases and solid in five cases. Using the AFIP criteria (
The two epithelialmyoepithelial carcinomas had a similar immunohistochemical staining profile, with clear myoepithelial cells being strongly reactive, whereas the darker luminal cells were unreactive.
Three of four acinar cell carcinomas, in which various numbers of acinar, intercalated duct-like, vacuolated, and nonspecific glandular cells were observed, were entirely negative for p63. However, in one papillarycystic case, intercalated duct-type epithelial cells were immunoreactive.
The three myoepithelial carcinomas exhibited focal reactivity in 1030% of tumor cells.
Most basaloid tumor cells were strongly reactive in one case of basal cell adenocarcinoma.
Two polymorphous low-grade adenocarcinoma cases exhibited diffuse nuclear reactivity. Some of these p63-reactive cells lined central lumens in small tubular structures.
Of three adenocarcinoma cases, NOS, one poorly differentiated case demonstrated p63 reactivity in few scattered tumor nuclei. p63 protein was not detected in two other cases, although in one case residual p63-immunoreactive myoepithelial cells were seen around an intraductal component.
One case of carcinoma ex pleomorphic adenoma showed scattered p63-reactive tumor nuclei.
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Discussion |
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Normal human salivary gland myoepithelium is reactive with antibodies to cytokeratins and muscle-specific actin. Among the several subtypes of cytokeratin, cytokeratin 14 is expressed most consistently and is specific for myoepithelial and ductal basal cells (
In contrast to p53 mutant mice, mice lacking the p63 gene have severe developmental defects involving all squamous epithelia and their derivatives, including skin, hair, and teeth, as well as mammary, lachrymal, and salivary glands (
This report is the first to determine the frequent expression of p63 in a large series of human salivary gland tumors encompassing the most commonly encountered tumor types as well as some less common neoplasms. Among other human tumors, p63 expression is frequently found in squamous-cell carcinomas, urothelial carcinomas, thymomas, and in a subset of non-Hodgkin lymphomas (
One of the interesting findings in our study is the correlation between cell type and p63 expression in tumors with differentiation along myoepithelial (p63-reactive) and luminal (p63-unreactive) lineages such as pleomorphic adenoma, basal cell adenoma, adenoid cystic carcinoma, mucoepidermoid carcinoma, and epithelialmyoepithelial carcinoma. Nevertheless, it should be noted that "aberrant" p63 expression can be seen in some tumor cells having little or no myoepithelial differentiation, such as columnar cells in canalicular adenoma, intercalated ductal cells in acinar cell carcinoma, and a few nonspecific glandular cells in adenocarcinoma, NOS. Notwithstanding these exceptions, p63 expression seems related to the fact that salivary gland tumor tissues retain the compartmentalization and differentiation seen in their normal counterpart.
Despite striking sequence similarities and conservation of functional domains among p53 family members, p63 is not a classical tumor suppressor gene (N variants may function as a dominant-negative factor that blocks the transcription-activating ability of TAp63, p53, or the related gene p73 (
Np63 may function as an oncogene (
Mesenchymal elements, including fibroblasts, myofibroblasts, endothelial cells, and smooth muscle cells, have undetectable p63 (
In conclusion, p63 is expressed in the nuclei of normal human salivary gland myoepithelial and basal duct cells as well as the modified myoepithelial and basal cells of human salivary gland tumors. A better characterization of salivary gland myoepithelial cells may provide valuable information regarding maintenance of this tissue, histogenesis and oncogenesis of salivary gland tumors, and may have clinical utility for the diagnosis.
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Acknowledgments |
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We thank Edith Joly, Annette Lesot, and Nicole Vignot for excellent technical assistance.
Received for publication September 6, 2002; accepted September 23, 2002.
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