- Email: [email protected]
multicystic phenotype
Notch4 overexpression in ameloblastoma correlates with the solid/multicystic phenotype Chong Huat Siar, BDS, MSc, FDSRCPS, FRCPath,a Hitoshi Nagatsuka, DDS, PhD,b Kee Seng Chuah, BDS, MDSc,f Rosario Santos Rivera, DDS, PhD,c Keisuke Nakano, DDS, PhD,d Kok Han Ng, BDS, MSc, FDSRCPS, FRCPath,e and Toshiyuki Kawakami, PhD,g Kuala Lumpur, Malaysia, Okayama and Shiojiri, Japan, and Manila, Philippines UNIVERSITY OF MALAYA, OKAYAMA UNIVERSITY, UNIVERSITY OF THE EAST, MATSUMOTO DENTAL UNIVERSITY, AND INSTITUTE FOR MEDICAL RESEARCH
Objective. Notch signaling has been implicated in cell fate decisions during odontogenesis and tumorigenesis of some odontogenic neoplasms; however, its role in solid/multicystic (SA), unicystic (UA), and recurrent (RA) ameloblastoma remains unclear. The aim of this study was to determine Notch receptor and ligand expressions in these subtypes and to speculate on their significance. Methods. Notch receptors (Notch1, 2, 3, 4) and ligands (Jagged1, 2, and Delta1) were examined immunohistochemically in SA (n ⫽ 23), UA (n ⫽ 22), and RA (n ⫽ 19). Results. Notch4 overexpression in SA (n ⫽ 19/23; 82.6%) compared with UA (n ⫽ 1/22; 4.5%) or RA (n ⫽ 10/19; 52.6%) (P ⬍ .05) suggests positive correlation between Notch4 signaling and ameloblastomas with a solid/multicystic phenotype. Ligand (Jagged1 and Delta1) underexpression compared with their receptors (Notch1, 3, 4) (P ⬍ .05) and nonreactivity for Notch2 and Jagged2 in all 3 subsets suggests that ameloblastoma epithelium belongs to an earlier stage of differentiation (equivalent to inner enamel epithelium of developing tooth germ) before lineage commitment. Conclusion. Present findings suggest that Notch signaling molecules may play differing roles in the acquisition of different ameloblastoma phenotypes. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2010;110:224-233)
Ameloblastoma is the most frequently encountered odontogenic neoplasm of the jaws and it accounts for approximately 11% to 18% of all odontogenic tu-
This study was jointly supported by the University of Malaya Research Grant (No. FS170/2008C) and Grant-in-Aid for Scientific Research (C) (21592326[C]) from the Japan Society for the Promotion of Science. a Professor and Head, Department of Oral Pathology, Oral Medicine, and Periodontology, Faculty of Dentistry, University of Malaya, Kuala Lumpur, Malaysia. b Professor and Chairman, Department of Oral Pathology and Medicine, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University, Okayama, Japan. c Lecturer, College of Dentistry, University of the East, Manila, Philippines. d Associate Professor, Hard Tissue Pathology Unit, Matsumoto Dental University, Graduate School of Oral Medicine, Shiojiri, Japan. e Consultant Oral Pathologist, Formerly Unit of Stomatology, Cancer Research Centre, Institute for Medical Research, Jalan Pahang, Kuala Lumpur, Malaysia. f Graduate student, Department of Oral Pathology, Oral Medicine, and Periodontology, Faculty of Dentistry, University of Malaya, Kuala Lumpur, Malaysia. g Professor and Chairman, Hard Tissue Pathology Unit, Matsumoto Dental University, Graduate School of Oral Medicine, Shiojiri, Japan. Received for publication Nov 23, 2009; returned for revision Feb 12, 2010; accepted for publication Mar 8, 2010. 1079-2104/$ - see front matter © 2010 Mosby, Inc. All rights reserved. doi:10.1016/j.tripleo.2010.03.009
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mors.1-3 Although classified as a benign tumor, the ameloblastoma follows a locally invasive course and has a tendency to recur after many years of apparent cure. According to the 2005 histological classification of tumors by the World Health Organization, ameloblastomas are categorized into 4 distinct clinicopathological variants: solid/multicystic (SA), extraosseous/ peripheral, desmoplastic, and unicystic ameloblastoma (UA).1 The 2 most common subtypes are SA and UA.1-4 SA demonstrates an aggressive biologic behavior by exhibiting a great infiltrative potential and a higher recurrence rate. UA tends to present some characteristics of an odontogenic cyst, occurs at an earlier age, and has a lower recurrence rate.1,5 Notch signaling is an evolutionarily conserved pathway that plays a crucial role in cell fate determination in a variety of tissue types during development as well as postnatally.6-9 In mammals, there are 4 Notch receptors (Notch1, 2, 3, 4) and 5 ligands (Jagged1 and 2 belong to the Serrate family, whereas Delta1, 3, and 4 belong to the Delta family). Notch receptors are structurally homologous transmembrane proteins with distinct differences in their extracellular and intracellular domains (ICD). Serrate and Delta proteins are also structurally related transmembrane proteins with multiple epidermal growth factor (EGF)-like repeats and DSL motif (Delta, Serrate, Lag-2) in their extracellular
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domains.9 The Notch signaling pathway is activated when ligands from neighboring cells bind Notch receptors. This triggers the proteolytic release of the Notch ICD, which translocates to the nucleus to interact with DNA-bound proteins and activates the transcription of selected target genes such as Hes1, Hes5, and Hes7.7-9 Notch signaling operates via local cell-cell interaction and is involved in diverse developmental processes ranging from control of proliferation to apoptosis, differentiation, maintenance of stemness, and cell fate decision as well as odontogenesis.9-13 Dysregulation of Notch signaling has been implicated in some genetic diseases and tumorigenesis.10,11 Recently Notch involvement in the determination of cell fate decisions in some odontogenic neoplasms has received considerable attention.14-17 Notch1 activity has been reported in plexiform and follicular ameloblastoma,14,15,17 ameloblastic carcinoma,15 and ameloblastic fibroma16 but not in the odontogenic myxoma.16 Activity of other Notch members (Notch1, 2, 3) and their ligands (Jagged1 and Delta1) has also been evaluated in acanthomatous and granular cell ameloblastoma.17 These findings suggest that Notch contributes to the control of neoplastic cell differentiation and suppresses neoplastic cell proliferation in odontogenic epithelium.14-17 However to date, data on involvement of Notch receptors and their ligands in UA and recurrent ameloblastoma (RA) remain unknown. The ameloblastoma is a clinically significant odontogenic neoplasm in this region and considerable work has been carried out in the hope of gaining a better understanding on the pathobiology of this tumor.18-22 The aim of this study was to determine Notch receptor and ligand expression patterns in SA, UA, and RA and to speculate on their significance. Four Notch receptors (Notch1, 2, 3, 4) and 3 ligands (Jagged1 and 2 and Delta1) were analyzed. The rationale for examining both Notch receptors and their ligands was to evaluate the potential role of these molecules in the 3 ameloblastoma subtypes. MATERIALS AND METHODS Samples The study sample consisted of 23 cases of primary SA, 22 primary UA, and 19 RA. These were retrieved from the surgical pathology files of the Department of Oral Pathology, Oral Medicine, and Periodontology, Faculty of Dentistry, University of Malaya, Kuala Lumpur, Malaysia. Cases were reviewed and selected according to the criteria of the World Health Organization’s histological classification of tumors.1 Patient characteristics, namely age at presentation, gender, ethnicity, duration, tumor location, and radiologic findings were recorded.
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From the archival formalin-fixed, paraffin-embedded tissue blocks of these cases, new 5-m-thick sections were prepared for staining with hematoxylin-eosin, and for immunohistochemistry with Notch1 to 4, Jagged1, Delta1 (LifeSpan Biosciences Inc, Seattle, WA), and Jagged2 (Abcam Inc, Cambridge, MA). Immunohistochemistry The Envision technique used for the immunohistochemical detection of Notch1 to 4, Jagged1, Jagged2, and Delta1 was as previously described.15 Briefly, deparaffinized sections of 5-m thickness were pretreated for antigen retrieval by microwaving (99°C) in 10 nM of citrate buffer (pH 6, 20 minutes). These sections were then immersed in 0.3% methanol containing 3% hydrogen peroxide for 20 minutes, to block endogenous peroxidase, and rinsed in 0.05 M Trisbuffered saline (TBS) (5 minutes, 2 times) before immersing in blocking solution (Dako Corporation, Carpinteria, CA) for 20 minutes at room temperature. Then the sections were incubated with the primary antibody against each of the Notch family members (mouse anti-human Notch1 monoclonal antibody [LSC16925], rabbit anti-human Notch2 polyclonal antibody [LS-B399], rabbit anti-human Notch3 polyclonal antibody [LS-B1621], rabbit anti-human Notch4 polyclonal antibody [LS-C40785], LifeSpan Biosciences Inc, 1:500 dilution) and ligands (rabbit anti-human Jagged1 polyclonal antibody [LS-C24920] and rabbit anti-mouse Delta1 polyclonal antibody [LS-B72], LifeSpan Biosciences Inc, 1:500 dilution, rabbit antihuman Jagged2 polyclonal antibody [ab60041], Abcam Inc, 1:500 dilution) for 1 hour at room temperature. Immunoreactions were performed using the Envision Kit (Dako Corporation). The antigenic sites were visualized using diaminobenzidine (DAB) substrate chromogen (Dako Corporation) and counterstained with Mayer’s hematoxylin. For negative control, sections were treated as above but without the primary antibody. All the control sections were negative. Positive controls for all markers were from known positive breast cancer tissue sections. Immunohistochemical analysis Semiquantitative analyses as well as microscopic observations were performed on 5 randomly selected areas where ameloblastoma neoplastic cells predominated. For image acquisition, a color charge-coupled device (CCD) camera (MicroPublisher 5.0, Q Imaging, Inc., Surrey, BC, Canada) was mounted on a binocular light microscope (Nikon Eclipse E400, Nikon, Inc., Tokyo, Japan). Images (original magnification ⫻100) were captured and displayed on the computer monitor (Del Trinitron, Windows Professional 2000; Dell,
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Table I. Patient characteristics for solid/multicystic, unicystic and recurrent ameloblastomas Findings Total (n) Gender Age Ethnicity
Duration Locations Radiologic appearances
Histologic subtypes
Solid/multicystic
Unicystic
Recurrent
23 cases Male: 14 (60.9%) Female: 9 (39.1%) Mean: 25.6 y Range: 10-52 y Malays: 16 (69.6%) Chinese: 4 (17.4%) Indians: 2 (8.7%) Others: 1 (4.3%) Mean: 11.1 mo Range: 1 week-6 y Mandible: 22 (95.7%) Maxilla: 1 (4.3%) n ⫽ 15 RLNOS: 5 (33.3%) MLRL: 10 (66.7%) Plexiform (P): 10 (43.5%) Follicular (F): 4 (17.4%) P ⫹ F: 4 (17.4%) Granular cell: 3 (13.0%) Acanthomatous: 2 (8.7%)
22 cases Male: 12 (54.5%) Female: 10 (45.5%) Mean: 25.4 y Range: 3-54 y Malays: 9 (40.9%) Chinese: 10 (45.5%) Indians: 0 (0%) Others: 3 (13.6%) Mean: 9.4 mo Range: 1-36 mo Mandible: 22 (100%) Maxilla: 0 n ⫽ 14 RLNOS: 11 (78.6%) MLRL: 3 (21.4%) Luminal: 15 (68.2%) Intraluminal: 3 (13.6%) Mural: 4 (18.2%)
19 cases Male: 11 (57.9%) Female: 8 (42.1%) Mean age: 36.2 y Range: 13-75 y Malays: 8 (42.1%) Chinese: 6 (31.6%) Indians: 4 (21.1%) Others: 1 (5.2%) Mean: 9 y* Range: 2-20 y* Mandible: 18 (94.8%) Maxilla: 1 (5.2%) n⫽6 RLNOS ⫽ 4 (66.7%) MLRL ⫽ 2 (33.3%) Plexiform (P): 8 (42.1%) Follicular (F): 4 (21.1%) P ⫹ F: 1 (5.2%) Acanthomatous: 1 (5.2%) Luminal: 4 (21.1%) Intraluminal: 1 (5.2%)
RLNOS, radiolucent lesion not otherwise specified; MLRL, multilocular radiolucent lesion. *Refers to the duration between primary treatment and onset of recurrent tumor.
Omaha, NE). Quantification was performed using Image-Pro Express software (Media Cybernetics Inc., Bethesda, MD) for Windows. The level of expression for Notch1, 2, 3, and 4; Jagged1 and 2; and Delta1 was quantified according to the percentage of immunoreactive tumor epithelial cells present: (⫺) negative when none of the tumor epithelial cells were positively stained in the cytoplasm or on the cell membrane; (⫹) mild when staining was present in focal areas (⬍25%); (⫹⫹) moderate when staining was evident in significant areas (25%-50%); and (⫹⫹⫹) strong when staining was present in predominant areas of the tumor (⬎50%). Stromal immunoreactivity was also assessed in a similar manner. Statistical analysis Differences in the percentages of cases with different immunoreactivity levels were analyzed by the Fisher Exact or Mann-Whitney U test for differences between 2 groups or the Kruskal-Wallis test for differences among 3 or more groups. Statistical level of significance was set at P ⬍ .05. RESULTS Patients’ characteristics These details for SA, UA, and RA cases are summarized in Table I.
Immunohistochemical findings Results of immunohistochemical staining and semiquantitative analysis are shown in Table II and illustrated in Figs. 1-4. Notch1 expression. Notch1 was variably expressed in SA, UA, and RA (P ⬎ .05). This positive reaction was more frequently detected in the central than in the peripheral neoplastic epithelial cells. Protein immunolocalization was membranous and/or cytoplasmic. Granular cell and acanthomatous tumor epithelium similarly demonstrated mild Notch1 expression. Stromal components, namely endothelial lining of blood vessels and fibroblasts, also weakly expressed Notch1 in those cases exhibiting Notch1 immunopositivity (Table II, Figs. 1, B, 2, B, and 4, A). Notch2 expression. Notch2 immunoreactivity was absent in all ameloblastoma subtypes analyzed (P ⬎ .05). The stromal vascular structures and fibroblasts were also similarly stained negative (Figs. 1 C, 2C, and 4 B). Notch3 expression. Approximately half of the cases in the SA, UA, and RA groups were nonreactive for Notch3 (P ⬎ .05). In the remaining cases that stained positive for Notch3, the protein was localized more frequently in the cytoplasm and cell membrane of central rather than in peripheral tumor epithelial cells. Granular cell and acanthomatous tumor epithelium heterogeneously expressed Notch3. Stromal endothelium
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Table II. Staining intensity for Notch1, 3, 4; Jagged1; and Delta1 in solid/multicystic, unicystic and recurrent ameloblastomas Notch1 Type of ameloblastoma Solid/multicystic (n ⫽ 23) Plexiform (P) (n ⫽ 10) Follicular (F) (n ⫽ 4) P⫹F (n ⫽ 4) Granular cell (n ⫽ 3) Acanth (n ⫽ 2) PA-like cells (n ⫽ 23) SR-like cells (n ⫽ 23) Unicystic (n ⫽ 22) Luminal (n ⫽ 15) Intraluminal (n ⫽ 3) Mural (n ⫽ 4) PA-like cells (n ⫽ 22) SR-like cells (n ⫽ 22) Recurrent (n ⫽ 19) Plexiform (P) (n ⫽ 8) Follicular (F) (n ⫽ 4) P⫹F (n ⫽ 1) Acanth (n ⫽ 1) Luminal (n ⫽ 4) Intraluminal (n ⫽ 1) PA-like cells (n ⫽ 19) SR-like cells (n ⫽ 19)
Notch3
Notch4
3⫹
2⫹
1⫹
0
3⫹
2⫹
1⫹
0
3⫹ 2⫹
0 0 0 0 0 0 0
2 1 0 1 1 3 5
3 3 3 1 1 5 11
5 0 1 1 0 15 7
1 0 0 0 0 0 1
0 2 2 1 1 2 8
3 1 0 1 1 8 6
6 1 2 1 0 13 8
6 4 4 3 2 18 19
0 0 0 0 0
1 0 0 1 1
4 1 1 1 6
10 2 3 20 15
0 0 0 0 0
0 2 1 0 3
11 1 3 6 13
4 0 0 16 6
0 0 0 0 0 0 0 0
1 0 0 0 1 0 1 2
1 3 1 0 2 0 1 6
6 1 0 1 1 1 17 11
1 1 0 0 0 0 0 2
2 2 1 0 1 0 1 6
2 0 0 0 2 1 4 6
3 1 0 1 1 0 14 5
Jagged1
Delta1
1⫹
0
3⫹
2⫹
1⫹
0
3⫹
2⫹
1⫹
0
4 0 0 0 0 5 4
0 0 0 0 0 0 0
0 0 0 0 0 0 0
0 0 0 0 0 0 0
0 0 0 0 0 0 0
5 4 3 2 2 12 16
5 0 1 1 0 11 7
0 0 0 0 0 0 0
1 1 1 1 1 1 3
1 2 0 1 1 6 7
8 1 3 1 0 16 13
1 0 0 1 1
3 0 0 2 2
9 3 4 5 6
2 0 0 14 13
0 0 0 0 0
0 0 0 0 0
7 2 3 3 12
8 1 1 19 10
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
15 3 4 22 22
6 0 1 0 2 1 8 10
1 3 0 1 1 0 2 5
0 0 0 0 0 0 3 0
1 1 0 0 1 0 6 4
0 0 0 0 0 0 0 0
1 1 0 0 0 0 0 2
1 2 1 0 0 0 2 4
6 1 0 1 4 1 17 13
0 0 0 0 0 0 0 0
1 0 0 0 0 0 1 1
0 1 0 0 0 0 1 1
7 3 1 1 4 1 17 17
P⫹F, mixed plexiform and follicular; Acanth, Acanthomatous; PA, Pre-ameloblast; SR, Stellate reticulum; 3⫹, strong; 2⫹, moderate; 1⫹, mild; 0, none. Note: Notch 2 and Jagged2 were consistently absent in all cases (data not shown in Table).
and fibroblasts also showed positive immunostaining in those cases expressing Notch3 reactivity (Table II, Figs. 1, D, 2D, 3A, and 4, C). Notch4 expression. Notch4 was detected in all 22 cases of SA. A significantly large number of SA (n ⫽ 19/23; 82.6%) demonstrated strong immunoreactivity in more than 90% of the tumor epithelium compared with UA (n ⫽ 1/22; 4.5%) or RA (n ⫽ 10/19; 52.6%) (P ⬍ .05). Within the RA group, strong Notch4 immunostaining was mostly observed in the plexiform subtype (n ⫽ 6/8; 75%) compared with the unicystic variant (n ⫽ 3/5; 60%). In all these Notch4-positive cases, immunoreactivity was membranous and/or cytoplasmic, and was detected more frequently in central than in peripheral neoplastic epithelium. Nuclear immunopositivity was infrequent. Stromal endothelium and fibroblasts also stained strongly positive in those cases that strongly expressed Notch4 (Table II, Figs. 1, E, 2, E, 3, B, and 4, D). Jagged1, Jagged2, and Delta1 expressions. Jagged1 expression level was mild in 60.9% SA, 34.1% UA, and 15.8% RA (P ⬎ .05). Jagged2 was not detected in all 3 subsets (P ⬎ .05). Delta1 was absent in 63.0% SA, 100% UA, and 89.4% RA (P ⬎ .05) (Table II, Figs. 1, F, 2, F-H, 3, C-D, and 4, E-G).
DISCUSSION There are 2 major drawbacks in the present investigation: one was the lack of information on the clinical outcome of SA, UA, and RA cases, and the other was that the original and recurrent tumors from the same patient were not available for comparison of expression levels. Within the limits of this study, a novel observation was high expression of Notch4 protein in primary SA compared with UA and RA. Acanthomatous and granular cell ameloblastoma tumor epithelum alike demonstrated strong Notch4 expression. Within the RA group, Notch4 overexpression was mostly encountered in recurrent tumors with a plexiform histologic pattern. The mechanism(s) that confers this preferential expression pattern in ameloblastomas with a solid/multicystic phenotype is unknown. Moreover, the role of this Notch member in normal odontogenesis remains undetermined. In the present study, Notch4 positivity was predominantly membranous and/or cytoplasmic and this corresponded with the fact that Notch family members including Notch4 are transmembrane proteins with extracellular and intracellular domains. The occasional presence of Notch4 positivity in SA tumor nuclei most likely represented the cleaved Notch4 ICD that has translocated to the nucleus.6,7 However, the signifi-
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Fig. 1. Primary solid/multicystic ameloblastoma showing A, plexiform-type tumor epithelium with a squamous focus (boxed area) (H&E stain; original magnification, ⫻40); B, mild Notch1 in this squamous focus (Anti-Notch1; original magnification ⫻200); C, Notch2 nonreactivity (Anti-Notch2; original magnification ⫻100); and D, moderate Notch3 expression in folliculartype tumor epithelium (Anti-Notch3; original magnification ⫻100). E, Strong Notch4 positivity (Anti-Notch4; original magnification ⫻400) and F, mild Jagged1 in granular cell-type tumor epithelium (Anti-Jagged1; original magnification ⫻400).
cance of this finding remains unknown. In other organ systems, Notch4 overexpression has been reported to correlate with tumor grade in human astrocytomas,23 and overexpressed in pancreatic cancer,24 malignant melanoma,25 and breast cancer.26 It is known that Notch1 to 3 signaling molecules are broadly expressed in diverse cell types, whereas Notch4 expression is restricted to endothelial cells.27 In this study, we observed that stromal endothelium tends to show strong
Notch4 positivity in those cases of solid/multicystic ameloblastoma expressing the same. A plausible explanation is that Notch4 ICD has selective vascular endothelial growth factor-D in both tumor and host stroma, suggesting a differential regulation of cytokines that may impact vascular recruitment and autocrine tumor signaling.28 Only one main study comparing Notch signaling in ameloblastoma and developing tooth germ has been
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Fig. 2. Primary unicystic ameloblastoma showing A, odontogenic lining epithelium exhibiting B, mild Notch1; C, negative Notch2; D, moderate Notch3; E, mild Notch4; F, mild Jagged1; G, negative Jagged 2; and H, negative Delta1 expressions (A, H&E stain; B, Anti-Notch1; C, Anti-Notch2; D, Anti-Notch3; E, Anti-Notch4; F, Anti-Jagged1; G, Anti-Jagged2; H, Anti-Delta1. original magnification, A-H, ⫻200).
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Fig. 3. Recurrent solid/multicystic ameloblastoma showing A, moderate Notch3 and B, strong Notch4 expressions in folliculartype tumor epithelium; C, mild Jagged1 and D, mild Delta1 expressions in plexiform-type tumor epithelium (A, Anti-Notch3; B, Anti-Notch4; C, Anti-Jagged1; D, Anti-Delta1; original magnification, A-D, ⫻40).
reported thus far.17 Transcripts of Notch receptors (Notch1, 2, and 3) and ligands (Jagged1 and Delta1) are reportedly expressed in both normal and neoplastic odontogenic epithelium suggesting that Notch signaling might control cellular differentiation and proliferation in these tissue types.17 The present study demonstrated that Notch receptor and ligand protein expression patterns in the neoplastic odontogenic epithelial cells in SA, UA, and RA seem to recapitulate the expression patterns of these molecules during normal odontogenesis. At the protein level, positive expression for Notch receptors (Notch1 and 3) and ligands (Jagged1 and Delta1) were observed in all 3 subtypes, although at varying intensity levels, whereas Notch2 and Jagged2 proteins were not expressed or barely detectable. Jagged1 expression level was mostly mild and the immunoreactivity was detected more fre-
quently in peripheral than in central tumor epithelium. This distribution pattern correlates with Jagged1 expression profile during odontogenesis, which subdivides dental epithelium in the tooth primordium into 2 distinct juxtaposed epithelial cell subpopulations: ameloblastic (which is inner enamel epithelium destined to give rise to ameloblast) and nonameloblastic (stratum intermedium, stellate reticulum, and outer enamel epithelium) regions.29 On the other hand, Delta1 expression level was predominantly weak to absent in all 3 subtypes of ameloblastoma examined and the immunoreactivity was also more frequently observed in the peripheral than central neoplastic epithelial cells. This expression pattern is comparable with the weak Delta1 expression observed during tooth initiation and morphogenesis.30 However, during cytodifferentiation, its expression is up-regulated in the epithelium-derived
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Fig. 4. A-D, Percentage scores of SA, UA, and RA for Notch receptors (Notch1-4). E-G, Percentage scores of SA, UA, and RA for Notch ligands (Jagged1, Jagged2, and Delta1).
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ameloblasts.30 It is known that during the early stages of normal human tooth development, Notch2 protein was expressed in the dental epithelium, but was absent from proliferating cells of inner enamel epithelium.31 At more advanced stages, Notch2 protein was expressed in enamel-producing ameloblasts. This implies that Notch2 signaling plays a role in the later developmental stages of ameloblast maturation after lineage commitment.31 In the current study, Jagged2 protein was consistently absent in all 3 ameloblastoma subtypes evaluated. In the molar tooth germ, Jagged2 was not expressed by dental epithelium at E13, whereas its expression was up-regulated in fully differentiated ameloblasts at E15.32 In addition, Jagged2 expression in the epithelium of the developing tooth germ correlated with proliferation rather than differentiation.13 Therefore, a likely explanation for the lack of Notch2Jagged2 signaling in ameloblastoma may be that the neoplastic odontogenic epithelial cells of this tumor belong to an earlier stage of development (equivalent to inner enamel epithelium of developing tooth germ) before lineage commitment.13,31,32 Based on the aforementioned findings, we speculated that Jagged1 is the main ligand for Notch receptors during ameloblastoma tumorigenesis; Delta1 probably plays a subsidiary role whereas Jagged2 is not involved. Three cases of RA in the current study were nonreactive for all 4 Notch receptors (Notch1, 2, 3, and 4) and 3 ligands (Jagged1 and 2, and Delta1) evaluated. This finding seems to suggest that cell fate decision in ameloblastoma may occur independent of the Notch signaling pathway. In summary, Notch receptor and ligand expression patterns were evaluated in 3 ameloblastoma subtypes: SA, UA, and RA. Although information on the clinical outcome of these cases is lacking and the original and recurrent tumors from the same patient were not available for comparison of expression levels, present findings suggest that Notch signaling molecules may play differing roles in the acquisition of different ameloblastoma phenotypes. We are grateful to Khoo Guat Sim, Rusnani Kamal, Siti Nurul Huda Ariffin, Richee Rudeen Yasib, Norhasilah Mokhtar, and Noorfaizah Silin for technical and secretarial support.
4. 5.
6. 7. 8. 9.
10. 11. 12.
13. 14.
15.
16.
17. 18. 19. 20.
21.
22.
23.
REFERENCES 1. Gardner DG, Heikinheimo K, Shear M, Philipsen HP, Coleman H. Ameloblastomas. In: Barnes L, Eveson JW, Reichart PA, Sidransky D, editors. World Health Organization classification of tumours: pathology and genetics of head and neck tumours, Lyons: IARC Press; 2005. p. 296-300. 2. Wenig BM. Atlas of head and neck pathology. 2nd ed. New York: Saunders; 2008. p. 131-8. 3. Neville BW, Damm DD, Allen CM. Odontogenic cysts and
24.
25.
tumors. In: Gnepp DR, editor. Diagnostic surgical pathology of the head and neck. 2nd ed. New York: Saunders; 2008. p. 785-838. Robinson L, Martinez MG. Unicytic ameloblastoma: a prognostic distinct entity. Cancer 1977;40:2278-85. Escande C, Chaine A, Menard P, Ernenwein D, Ghouls, Boutattour A, et al. A treatment algorithm for the adult ameloblastomas according to Pitié-Salpêtrière Hospital experience. J Cranio-Maxillofac Surg 2009;37:363-9. Miele L. Notch signaling. Clin Cancer Res 2006;12:1074-9. Fiúza UM, Arias AM. Cell and molecular biology of Notch. J Endocrinol 2007;194:459-74. Fortini ME. Notch signaling: the core pathway and its posttranslational regulation. Dev Cell 2009;16:633-47. Artavanis-Tsakonas S, Rand MD, Lake RJ. Notch signaling: cell fate control and signal integration in development. Science 1999; 284:770-6. Harper JA, Yuan JS, Tan JB, Visan I, Guidos CJ. Notch signaling in development and disease. Clin Genet 2003;64:461-72. Leong KG, Karsan A. Recent insights into the role of Notch signaling in tumorigenesis. Blood 2006;107:2223-33. Mitsiadis TA, Lardelli M, Lendahl U, Thesleff I. Expression of Notch 1, 2 and 3 is regulated by epithelial-mesenchymal interactions and retinoic acid in the developing mouse tooth and associated with determination of ameloblast cell fate. J Cell Biol 1995;130:407-18. Mitsiadis TA, Regaudiat L, Gridlley T. Notch signaling pathway in tooth morphogenesis. Arch Oral Biol 2005;50:137-40. Siar CH, Ng KH, Ariff Z, Muraki E, Shimizu T, Tsujigiwa H, et al. A case report of ameloblastoma of the mandible with examination of Notch signaling. Oral Med Pathol 2006;11:35-9. Nakano K, Siar CH, Tsujigiwa H, Nagatsuka H, Nagai N, Kawakami T. Notch signaling in benign and malignant ameloblastic neoplasms. Eur J Med Res 2008;13:476-80. Nakano K, Chelvanayagam P, Born K, Siar CH, Ng KH, Nagatsuka H, et al. A study of recurrent giant odontogenic myxoma of the mandible with immunohistochemical examination for Notch. Oral Med Pathol 2008;12:53-6. Kumamoto H, Ohki K, Ooya K. Expression of Notch signaling molecules in ameloblastomas. J Oral Pathol Med 2008;37:228-34. Siar CH, Ng KH. Ameloblastoma in Malaysia—a 25-year review. Ann Acad Med Singapore 1993;22:856-60. Ng KH, Siar CH. Peripheral ameloblastoma with clear cell differentiation. Oral Surg Oral Med Oral Pathol 1990;70:210-3. Nakano K, Siar CH, Nagai N, Naito I, Sado Y, Nagatsuka H, et al. Distribution of basement membrane type IV collagen in ameloblastoma: an immunoflourescence study. J Oral Pathol Med 2002;31:494-9. Nagatsuka H, Phuu PH, Tsujigiwa H, Siar CH, Gunduz M, Sugahara T, et al. Heparanase gene and protein expression in ameloblastoma: possible role in local invasion of tumor cells. Oral Oncol 2005;41:542-8. Tsujigiwa H, Nagatsuka H, Phuu PH, Gunduz M, Siar CH, Oida S, et al. Analysis of amelogenin gene (AMGX, AMGY) expression in ameloblastoma. Oral Oncol 2005;41:843-50. Xu P, Yu S, Jiang R, Kang C, Wang C, Jiang H, et al. Differential expression of Notch family members in astrocytomas and medulloblastomas. Path Oncol Res 2009;15:703-10. Miyamoto Y, Maitra A, Ghosh B, Zechner U, Argani P, Iacobuzio-Donahue CA, et al. Notch mediates TGF alpha-induced changes in epithelial differentiation during pancreatic tumorigenesis. Cancer Cell 2003;3:565-76. Nickoloff BJ, Osborne BA, Miele L. Notch signaling as a therapeutic target in cancer: a new approach to the development of cell fate modifying agents. Oncogene 2003;22:6598-608.
OOOOE Volume 110, Number 2 26. Callahan R, Egan SE. Notch signaling in mammary development and oncogenesis. J Mammary Gland Biol Neoplasia 2004;9: 145-63. 27. Wu J, Iwata F, Grass JA, Osborne CS, Elnitski L, Fraser P, et al. Molecular determinants of Notch4 transcription in vascular endothelium. Mol Cell Biol 2005;25:1458-74. 28. O’Neill CF, Urs S, Cinelli C, Lincoln A, Nadeau RJ, León R, et al. Notch2 signaling induces apoptosis and inhibits human MDA-MB231 xenograft growth. Am J Pathol 2007;171:1023-36. 29. Mitsiadis TA, Henrique D, Thesleff I, Lendahl U. Mouse Serrate-1 (Jagged-1) expression in the developing tooth is regulated by epithelial-mesenchymal interactions and fibroblast growth factor-4. Dev 1997;124:1473-83. 30. Mitsiadis TA, Hirsinger E, Lendahl U, Goridis C. Delta-Notch signaling in odontogenesis: correlation with cytodifferentiation and evidence for feedback regulation. Dev Biol 1998:204:420-31.
Siar et al. 233 31. Mitsiadis TA, Romeas A, Lendahl U, Sharpe PT, Farges JC. Notch2 protein distribution in human teeth under normal and pathological conditions. Exp Cell Res 2003;282:101-9. 32. Borkosky SS, Nagatsuka H, Orita Y, Tsujigiwa H, Yoshinobu J, Gunduz M, et al. Sequential expressions of Notch1, Jagged2 and Math1 in molar tooth germ of mouse. Biocell 2008;32: 251-8.
Reprint requests: Chong H. Siar, BDS, MSc, FDSRCPS, FRCPath Department of Oral Pathology, Oral Medicine, and Periodontology Faculty of Dentistry, University of Malaya 50603 Kuala Lumpur, Malaysia [email protected]
Objective. Notch signaling has been implicated in cell fate decisions during odontogenesis and tumorigenesis of some odontogenic neoplasms; however, its role in solid/multicystic (SA), unicystic (UA), and recurrent (RA) ameloblastoma remains unclear. The aim of this study was to determine Notch receptor and ligand expressions in these subtypes and to speculate on their significance. Methods. Notch receptors (Notch1, 2, 3, 4) and ligands (Jagged1, 2, and Delta1) were examined immunohistochemically in SA (n ⫽ 23), UA (n ⫽ 22), and RA (n ⫽ 19). Results. Notch4 overexpression in SA (n ⫽ 19/23; 82.6%) compared with UA (n ⫽ 1/22; 4.5%) or RA (n ⫽ 10/19; 52.6%) (P ⬍ .05) suggests positive correlation between Notch4 signaling and ameloblastomas with a solid/multicystic phenotype. Ligand (Jagged1 and Delta1) underexpression compared with their receptors (Notch1, 3, 4) (P ⬍ .05) and nonreactivity for Notch2 and Jagged2 in all 3 subsets suggests that ameloblastoma epithelium belongs to an earlier stage of differentiation (equivalent to inner enamel epithelium of developing tooth germ) before lineage commitment. Conclusion. Present findings suggest that Notch signaling molecules may play differing roles in the acquisition of different ameloblastoma phenotypes. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2010;110:224-233)
Ameloblastoma is the most frequently encountered odontogenic neoplasm of the jaws and it accounts for approximately 11% to 18% of all odontogenic tu-
This study was jointly supported by the University of Malaya Research Grant (No. FS170/2008C) and Grant-in-Aid for Scientific Research (C) (21592326[C]) from the Japan Society for the Promotion of Science. a Professor and Head, Department of Oral Pathology, Oral Medicine, and Periodontology, Faculty of Dentistry, University of Malaya, Kuala Lumpur, Malaysia. b Professor and Chairman, Department of Oral Pathology and Medicine, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University, Okayama, Japan. c Lecturer, College of Dentistry, University of the East, Manila, Philippines. d Associate Professor, Hard Tissue Pathology Unit, Matsumoto Dental University, Graduate School of Oral Medicine, Shiojiri, Japan. e Consultant Oral Pathologist, Formerly Unit of Stomatology, Cancer Research Centre, Institute for Medical Research, Jalan Pahang, Kuala Lumpur, Malaysia. f Graduate student, Department of Oral Pathology, Oral Medicine, and Periodontology, Faculty of Dentistry, University of Malaya, Kuala Lumpur, Malaysia. g Professor and Chairman, Hard Tissue Pathology Unit, Matsumoto Dental University, Graduate School of Oral Medicine, Shiojiri, Japan. Received for publication Nov 23, 2009; returned for revision Feb 12, 2010; accepted for publication Mar 8, 2010. 1079-2104/$ - see front matter © 2010 Mosby, Inc. All rights reserved. doi:10.1016/j.tripleo.2010.03.009
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mors.1-3 Although classified as a benign tumor, the ameloblastoma follows a locally invasive course and has a tendency to recur after many years of apparent cure. According to the 2005 histological classification of tumors by the World Health Organization, ameloblastomas are categorized into 4 distinct clinicopathological variants: solid/multicystic (SA), extraosseous/ peripheral, desmoplastic, and unicystic ameloblastoma (UA).1 The 2 most common subtypes are SA and UA.1-4 SA demonstrates an aggressive biologic behavior by exhibiting a great infiltrative potential and a higher recurrence rate. UA tends to present some characteristics of an odontogenic cyst, occurs at an earlier age, and has a lower recurrence rate.1,5 Notch signaling is an evolutionarily conserved pathway that plays a crucial role in cell fate determination in a variety of tissue types during development as well as postnatally.6-9 In mammals, there are 4 Notch receptors (Notch1, 2, 3, 4) and 5 ligands (Jagged1 and 2 belong to the Serrate family, whereas Delta1, 3, and 4 belong to the Delta family). Notch receptors are structurally homologous transmembrane proteins with distinct differences in their extracellular and intracellular domains (ICD). Serrate and Delta proteins are also structurally related transmembrane proteins with multiple epidermal growth factor (EGF)-like repeats and DSL motif (Delta, Serrate, Lag-2) in their extracellular
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domains.9 The Notch signaling pathway is activated when ligands from neighboring cells bind Notch receptors. This triggers the proteolytic release of the Notch ICD, which translocates to the nucleus to interact with DNA-bound proteins and activates the transcription of selected target genes such as Hes1, Hes5, and Hes7.7-9 Notch signaling operates via local cell-cell interaction and is involved in diverse developmental processes ranging from control of proliferation to apoptosis, differentiation, maintenance of stemness, and cell fate decision as well as odontogenesis.9-13 Dysregulation of Notch signaling has been implicated in some genetic diseases and tumorigenesis.10,11 Recently Notch involvement in the determination of cell fate decisions in some odontogenic neoplasms has received considerable attention.14-17 Notch1 activity has been reported in plexiform and follicular ameloblastoma,14,15,17 ameloblastic carcinoma,15 and ameloblastic fibroma16 but not in the odontogenic myxoma.16 Activity of other Notch members (Notch1, 2, 3) and their ligands (Jagged1 and Delta1) has also been evaluated in acanthomatous and granular cell ameloblastoma.17 These findings suggest that Notch contributes to the control of neoplastic cell differentiation and suppresses neoplastic cell proliferation in odontogenic epithelium.14-17 However to date, data on involvement of Notch receptors and their ligands in UA and recurrent ameloblastoma (RA) remain unknown. The ameloblastoma is a clinically significant odontogenic neoplasm in this region and considerable work has been carried out in the hope of gaining a better understanding on the pathobiology of this tumor.18-22 The aim of this study was to determine Notch receptor and ligand expression patterns in SA, UA, and RA and to speculate on their significance. Four Notch receptors (Notch1, 2, 3, 4) and 3 ligands (Jagged1 and 2 and Delta1) were analyzed. The rationale for examining both Notch receptors and their ligands was to evaluate the potential role of these molecules in the 3 ameloblastoma subtypes. MATERIALS AND METHODS Samples The study sample consisted of 23 cases of primary SA, 22 primary UA, and 19 RA. These were retrieved from the surgical pathology files of the Department of Oral Pathology, Oral Medicine, and Periodontology, Faculty of Dentistry, University of Malaya, Kuala Lumpur, Malaysia. Cases were reviewed and selected according to the criteria of the World Health Organization’s histological classification of tumors.1 Patient characteristics, namely age at presentation, gender, ethnicity, duration, tumor location, and radiologic findings were recorded.
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From the archival formalin-fixed, paraffin-embedded tissue blocks of these cases, new 5-m-thick sections were prepared for staining with hematoxylin-eosin, and for immunohistochemistry with Notch1 to 4, Jagged1, Delta1 (LifeSpan Biosciences Inc, Seattle, WA), and Jagged2 (Abcam Inc, Cambridge, MA). Immunohistochemistry The Envision technique used for the immunohistochemical detection of Notch1 to 4, Jagged1, Jagged2, and Delta1 was as previously described.15 Briefly, deparaffinized sections of 5-m thickness were pretreated for antigen retrieval by microwaving (99°C) in 10 nM of citrate buffer (pH 6, 20 minutes). These sections were then immersed in 0.3% methanol containing 3% hydrogen peroxide for 20 minutes, to block endogenous peroxidase, and rinsed in 0.05 M Trisbuffered saline (TBS) (5 minutes, 2 times) before immersing in blocking solution (Dako Corporation, Carpinteria, CA) for 20 minutes at room temperature. Then the sections were incubated with the primary antibody against each of the Notch family members (mouse anti-human Notch1 monoclonal antibody [LSC16925], rabbit anti-human Notch2 polyclonal antibody [LS-B399], rabbit anti-human Notch3 polyclonal antibody [LS-B1621], rabbit anti-human Notch4 polyclonal antibody [LS-C40785], LifeSpan Biosciences Inc, 1:500 dilution) and ligands (rabbit anti-human Jagged1 polyclonal antibody [LS-C24920] and rabbit anti-mouse Delta1 polyclonal antibody [LS-B72], LifeSpan Biosciences Inc, 1:500 dilution, rabbit antihuman Jagged2 polyclonal antibody [ab60041], Abcam Inc, 1:500 dilution) for 1 hour at room temperature. Immunoreactions were performed using the Envision Kit (Dako Corporation). The antigenic sites were visualized using diaminobenzidine (DAB) substrate chromogen (Dako Corporation) and counterstained with Mayer’s hematoxylin. For negative control, sections were treated as above but without the primary antibody. All the control sections were negative. Positive controls for all markers were from known positive breast cancer tissue sections. Immunohistochemical analysis Semiquantitative analyses as well as microscopic observations were performed on 5 randomly selected areas where ameloblastoma neoplastic cells predominated. For image acquisition, a color charge-coupled device (CCD) camera (MicroPublisher 5.0, Q Imaging, Inc., Surrey, BC, Canada) was mounted on a binocular light microscope (Nikon Eclipse E400, Nikon, Inc., Tokyo, Japan). Images (original magnification ⫻100) were captured and displayed on the computer monitor (Del Trinitron, Windows Professional 2000; Dell,
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Table I. Patient characteristics for solid/multicystic, unicystic and recurrent ameloblastomas Findings Total (n) Gender Age Ethnicity
Duration Locations Radiologic appearances
Histologic subtypes
Solid/multicystic
Unicystic
Recurrent
23 cases Male: 14 (60.9%) Female: 9 (39.1%) Mean: 25.6 y Range: 10-52 y Malays: 16 (69.6%) Chinese: 4 (17.4%) Indians: 2 (8.7%) Others: 1 (4.3%) Mean: 11.1 mo Range: 1 week-6 y Mandible: 22 (95.7%) Maxilla: 1 (4.3%) n ⫽ 15 RLNOS: 5 (33.3%) MLRL: 10 (66.7%) Plexiform (P): 10 (43.5%) Follicular (F): 4 (17.4%) P ⫹ F: 4 (17.4%) Granular cell: 3 (13.0%) Acanthomatous: 2 (8.7%)
22 cases Male: 12 (54.5%) Female: 10 (45.5%) Mean: 25.4 y Range: 3-54 y Malays: 9 (40.9%) Chinese: 10 (45.5%) Indians: 0 (0%) Others: 3 (13.6%) Mean: 9.4 mo Range: 1-36 mo Mandible: 22 (100%) Maxilla: 0 n ⫽ 14 RLNOS: 11 (78.6%) MLRL: 3 (21.4%) Luminal: 15 (68.2%) Intraluminal: 3 (13.6%) Mural: 4 (18.2%)
19 cases Male: 11 (57.9%) Female: 8 (42.1%) Mean age: 36.2 y Range: 13-75 y Malays: 8 (42.1%) Chinese: 6 (31.6%) Indians: 4 (21.1%) Others: 1 (5.2%) Mean: 9 y* Range: 2-20 y* Mandible: 18 (94.8%) Maxilla: 1 (5.2%) n⫽6 RLNOS ⫽ 4 (66.7%) MLRL ⫽ 2 (33.3%) Plexiform (P): 8 (42.1%) Follicular (F): 4 (21.1%) P ⫹ F: 1 (5.2%) Acanthomatous: 1 (5.2%) Luminal: 4 (21.1%) Intraluminal: 1 (5.2%)
RLNOS, radiolucent lesion not otherwise specified; MLRL, multilocular radiolucent lesion. *Refers to the duration between primary treatment and onset of recurrent tumor.
Omaha, NE). Quantification was performed using Image-Pro Express software (Media Cybernetics Inc., Bethesda, MD) for Windows. The level of expression for Notch1, 2, 3, and 4; Jagged1 and 2; and Delta1 was quantified according to the percentage of immunoreactive tumor epithelial cells present: (⫺) negative when none of the tumor epithelial cells were positively stained in the cytoplasm or on the cell membrane; (⫹) mild when staining was present in focal areas (⬍25%); (⫹⫹) moderate when staining was evident in significant areas (25%-50%); and (⫹⫹⫹) strong when staining was present in predominant areas of the tumor (⬎50%). Stromal immunoreactivity was also assessed in a similar manner. Statistical analysis Differences in the percentages of cases with different immunoreactivity levels were analyzed by the Fisher Exact or Mann-Whitney U test for differences between 2 groups or the Kruskal-Wallis test for differences among 3 or more groups. Statistical level of significance was set at P ⬍ .05. RESULTS Patients’ characteristics These details for SA, UA, and RA cases are summarized in Table I.
Immunohistochemical findings Results of immunohistochemical staining and semiquantitative analysis are shown in Table II and illustrated in Figs. 1-4. Notch1 expression. Notch1 was variably expressed in SA, UA, and RA (P ⬎ .05). This positive reaction was more frequently detected in the central than in the peripheral neoplastic epithelial cells. Protein immunolocalization was membranous and/or cytoplasmic. Granular cell and acanthomatous tumor epithelium similarly demonstrated mild Notch1 expression. Stromal components, namely endothelial lining of blood vessels and fibroblasts, also weakly expressed Notch1 in those cases exhibiting Notch1 immunopositivity (Table II, Figs. 1, B, 2, B, and 4, A). Notch2 expression. Notch2 immunoreactivity was absent in all ameloblastoma subtypes analyzed (P ⬎ .05). The stromal vascular structures and fibroblasts were also similarly stained negative (Figs. 1 C, 2C, and 4 B). Notch3 expression. Approximately half of the cases in the SA, UA, and RA groups were nonreactive for Notch3 (P ⬎ .05). In the remaining cases that stained positive for Notch3, the protein was localized more frequently in the cytoplasm and cell membrane of central rather than in peripheral tumor epithelial cells. Granular cell and acanthomatous tumor epithelium heterogeneously expressed Notch3. Stromal endothelium
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Table II. Staining intensity for Notch1, 3, 4; Jagged1; and Delta1 in solid/multicystic, unicystic and recurrent ameloblastomas Notch1 Type of ameloblastoma Solid/multicystic (n ⫽ 23) Plexiform (P) (n ⫽ 10) Follicular (F) (n ⫽ 4) P⫹F (n ⫽ 4) Granular cell (n ⫽ 3) Acanth (n ⫽ 2) PA-like cells (n ⫽ 23) SR-like cells (n ⫽ 23) Unicystic (n ⫽ 22) Luminal (n ⫽ 15) Intraluminal (n ⫽ 3) Mural (n ⫽ 4) PA-like cells (n ⫽ 22) SR-like cells (n ⫽ 22) Recurrent (n ⫽ 19) Plexiform (P) (n ⫽ 8) Follicular (F) (n ⫽ 4) P⫹F (n ⫽ 1) Acanth (n ⫽ 1) Luminal (n ⫽ 4) Intraluminal (n ⫽ 1) PA-like cells (n ⫽ 19) SR-like cells (n ⫽ 19)
Notch3
Notch4
3⫹
2⫹
1⫹
0
3⫹
2⫹
1⫹
0
3⫹ 2⫹
0 0 0 0 0 0 0
2 1 0 1 1 3 5
3 3 3 1 1 5 11
5 0 1 1 0 15 7
1 0 0 0 0 0 1
0 2 2 1 1 2 8
3 1 0 1 1 8 6
6 1 2 1 0 13 8
6 4 4 3 2 18 19
0 0 0 0 0
1 0 0 1 1
4 1 1 1 6
10 2 3 20 15
0 0 0 0 0
0 2 1 0 3
11 1 3 6 13
4 0 0 16 6
0 0 0 0 0 0 0 0
1 0 0 0 1 0 1 2
1 3 1 0 2 0 1 6
6 1 0 1 1 1 17 11
1 1 0 0 0 0 0 2
2 2 1 0 1 0 1 6
2 0 0 0 2 1 4 6
3 1 0 1 1 0 14 5
Jagged1
Delta1
1⫹
0
3⫹
2⫹
1⫹
0
3⫹
2⫹
1⫹
0
4 0 0 0 0 5 4
0 0 0 0 0 0 0
0 0 0 0 0 0 0
0 0 0 0 0 0 0
0 0 0 0 0 0 0
5 4 3 2 2 12 16
5 0 1 1 0 11 7
0 0 0 0 0 0 0
1 1 1 1 1 1 3
1 2 0 1 1 6 7
8 1 3 1 0 16 13
1 0 0 1 1
3 0 0 2 2
9 3 4 5 6
2 0 0 14 13
0 0 0 0 0
0 0 0 0 0
7 2 3 3 12
8 1 1 19 10
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
15 3 4 22 22
6 0 1 0 2 1 8 10
1 3 0 1 1 0 2 5
0 0 0 0 0 0 3 0
1 1 0 0 1 0 6 4
0 0 0 0 0 0 0 0
1 1 0 0 0 0 0 2
1 2 1 0 0 0 2 4
6 1 0 1 4 1 17 13
0 0 0 0 0 0 0 0
1 0 0 0 0 0 1 1
0 1 0 0 0 0 1 1
7 3 1 1 4 1 17 17
P⫹F, mixed plexiform and follicular; Acanth, Acanthomatous; PA, Pre-ameloblast; SR, Stellate reticulum; 3⫹, strong; 2⫹, moderate; 1⫹, mild; 0, none. Note: Notch 2 and Jagged2 were consistently absent in all cases (data not shown in Table).
and fibroblasts also showed positive immunostaining in those cases expressing Notch3 reactivity (Table II, Figs. 1, D, 2D, 3A, and 4, C). Notch4 expression. Notch4 was detected in all 22 cases of SA. A significantly large number of SA (n ⫽ 19/23; 82.6%) demonstrated strong immunoreactivity in more than 90% of the tumor epithelium compared with UA (n ⫽ 1/22; 4.5%) or RA (n ⫽ 10/19; 52.6%) (P ⬍ .05). Within the RA group, strong Notch4 immunostaining was mostly observed in the plexiform subtype (n ⫽ 6/8; 75%) compared with the unicystic variant (n ⫽ 3/5; 60%). In all these Notch4-positive cases, immunoreactivity was membranous and/or cytoplasmic, and was detected more frequently in central than in peripheral neoplastic epithelium. Nuclear immunopositivity was infrequent. Stromal endothelium and fibroblasts also stained strongly positive in those cases that strongly expressed Notch4 (Table II, Figs. 1, E, 2, E, 3, B, and 4, D). Jagged1, Jagged2, and Delta1 expressions. Jagged1 expression level was mild in 60.9% SA, 34.1% UA, and 15.8% RA (P ⬎ .05). Jagged2 was not detected in all 3 subsets (P ⬎ .05). Delta1 was absent in 63.0% SA, 100% UA, and 89.4% RA (P ⬎ .05) (Table II, Figs. 1, F, 2, F-H, 3, C-D, and 4, E-G).
DISCUSSION There are 2 major drawbacks in the present investigation: one was the lack of information on the clinical outcome of SA, UA, and RA cases, and the other was that the original and recurrent tumors from the same patient were not available for comparison of expression levels. Within the limits of this study, a novel observation was high expression of Notch4 protein in primary SA compared with UA and RA. Acanthomatous and granular cell ameloblastoma tumor epithelum alike demonstrated strong Notch4 expression. Within the RA group, Notch4 overexpression was mostly encountered in recurrent tumors with a plexiform histologic pattern. The mechanism(s) that confers this preferential expression pattern in ameloblastomas with a solid/multicystic phenotype is unknown. Moreover, the role of this Notch member in normal odontogenesis remains undetermined. In the present study, Notch4 positivity was predominantly membranous and/or cytoplasmic and this corresponded with the fact that Notch family members including Notch4 are transmembrane proteins with extracellular and intracellular domains. The occasional presence of Notch4 positivity in SA tumor nuclei most likely represented the cleaved Notch4 ICD that has translocated to the nucleus.6,7 However, the signifi-
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Fig. 1. Primary solid/multicystic ameloblastoma showing A, plexiform-type tumor epithelium with a squamous focus (boxed area) (H&E stain; original magnification, ⫻40); B, mild Notch1 in this squamous focus (Anti-Notch1; original magnification ⫻200); C, Notch2 nonreactivity (Anti-Notch2; original magnification ⫻100); and D, moderate Notch3 expression in folliculartype tumor epithelium (Anti-Notch3; original magnification ⫻100). E, Strong Notch4 positivity (Anti-Notch4; original magnification ⫻400) and F, mild Jagged1 in granular cell-type tumor epithelium (Anti-Jagged1; original magnification ⫻400).
cance of this finding remains unknown. In other organ systems, Notch4 overexpression has been reported to correlate with tumor grade in human astrocytomas,23 and overexpressed in pancreatic cancer,24 malignant melanoma,25 and breast cancer.26 It is known that Notch1 to 3 signaling molecules are broadly expressed in diverse cell types, whereas Notch4 expression is restricted to endothelial cells.27 In this study, we observed that stromal endothelium tends to show strong
Notch4 positivity in those cases of solid/multicystic ameloblastoma expressing the same. A plausible explanation is that Notch4 ICD has selective vascular endothelial growth factor-D in both tumor and host stroma, suggesting a differential regulation of cytokines that may impact vascular recruitment and autocrine tumor signaling.28 Only one main study comparing Notch signaling in ameloblastoma and developing tooth germ has been
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Fig. 2. Primary unicystic ameloblastoma showing A, odontogenic lining epithelium exhibiting B, mild Notch1; C, negative Notch2; D, moderate Notch3; E, mild Notch4; F, mild Jagged1; G, negative Jagged 2; and H, negative Delta1 expressions (A, H&E stain; B, Anti-Notch1; C, Anti-Notch2; D, Anti-Notch3; E, Anti-Notch4; F, Anti-Jagged1; G, Anti-Jagged2; H, Anti-Delta1. original magnification, A-H, ⫻200).
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Fig. 3. Recurrent solid/multicystic ameloblastoma showing A, moderate Notch3 and B, strong Notch4 expressions in folliculartype tumor epithelium; C, mild Jagged1 and D, mild Delta1 expressions in plexiform-type tumor epithelium (A, Anti-Notch3; B, Anti-Notch4; C, Anti-Jagged1; D, Anti-Delta1; original magnification, A-D, ⫻40).
reported thus far.17 Transcripts of Notch receptors (Notch1, 2, and 3) and ligands (Jagged1 and Delta1) are reportedly expressed in both normal and neoplastic odontogenic epithelium suggesting that Notch signaling might control cellular differentiation and proliferation in these tissue types.17 The present study demonstrated that Notch receptor and ligand protein expression patterns in the neoplastic odontogenic epithelial cells in SA, UA, and RA seem to recapitulate the expression patterns of these molecules during normal odontogenesis. At the protein level, positive expression for Notch receptors (Notch1 and 3) and ligands (Jagged1 and Delta1) were observed in all 3 subtypes, although at varying intensity levels, whereas Notch2 and Jagged2 proteins were not expressed or barely detectable. Jagged1 expression level was mostly mild and the immunoreactivity was detected more fre-
quently in peripheral than in central tumor epithelium. This distribution pattern correlates with Jagged1 expression profile during odontogenesis, which subdivides dental epithelium in the tooth primordium into 2 distinct juxtaposed epithelial cell subpopulations: ameloblastic (which is inner enamel epithelium destined to give rise to ameloblast) and nonameloblastic (stratum intermedium, stellate reticulum, and outer enamel epithelium) regions.29 On the other hand, Delta1 expression level was predominantly weak to absent in all 3 subtypes of ameloblastoma examined and the immunoreactivity was also more frequently observed in the peripheral than central neoplastic epithelial cells. This expression pattern is comparable with the weak Delta1 expression observed during tooth initiation and morphogenesis.30 However, during cytodifferentiation, its expression is up-regulated in the epithelium-derived
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Fig. 4. A-D, Percentage scores of SA, UA, and RA for Notch receptors (Notch1-4). E-G, Percentage scores of SA, UA, and RA for Notch ligands (Jagged1, Jagged2, and Delta1).
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ameloblasts.30 It is known that during the early stages of normal human tooth development, Notch2 protein was expressed in the dental epithelium, but was absent from proliferating cells of inner enamel epithelium.31 At more advanced stages, Notch2 protein was expressed in enamel-producing ameloblasts. This implies that Notch2 signaling plays a role in the later developmental stages of ameloblast maturation after lineage commitment.31 In the current study, Jagged2 protein was consistently absent in all 3 ameloblastoma subtypes evaluated. In the molar tooth germ, Jagged2 was not expressed by dental epithelium at E13, whereas its expression was up-regulated in fully differentiated ameloblasts at E15.32 In addition, Jagged2 expression in the epithelium of the developing tooth germ correlated with proliferation rather than differentiation.13 Therefore, a likely explanation for the lack of Notch2Jagged2 signaling in ameloblastoma may be that the neoplastic odontogenic epithelial cells of this tumor belong to an earlier stage of development (equivalent to inner enamel epithelium of developing tooth germ) before lineage commitment.13,31,32 Based on the aforementioned findings, we speculated that Jagged1 is the main ligand for Notch receptors during ameloblastoma tumorigenesis; Delta1 probably plays a subsidiary role whereas Jagged2 is not involved. Three cases of RA in the current study were nonreactive for all 4 Notch receptors (Notch1, 2, 3, and 4) and 3 ligands (Jagged1 and 2, and Delta1) evaluated. This finding seems to suggest that cell fate decision in ameloblastoma may occur independent of the Notch signaling pathway. In summary, Notch receptor and ligand expression patterns were evaluated in 3 ameloblastoma subtypes: SA, UA, and RA. Although information on the clinical outcome of these cases is lacking and the original and recurrent tumors from the same patient were not available for comparison of expression levels, present findings suggest that Notch signaling molecules may play differing roles in the acquisition of different ameloblastoma phenotypes. We are grateful to Khoo Guat Sim, Rusnani Kamal, Siti Nurul Huda Ariffin, Richee Rudeen Yasib, Norhasilah Mokhtar, and Noorfaizah Silin for technical and secretarial support.
4. 5.
6. 7. 8. 9.
10. 11. 12.
13. 14.
15.
16.
17. 18. 19. 20.
21.
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REFERENCES 1. Gardner DG, Heikinheimo K, Shear M, Philipsen HP, Coleman H. Ameloblastomas. In: Barnes L, Eveson JW, Reichart PA, Sidransky D, editors. World Health Organization classification of tumours: pathology and genetics of head and neck tumours, Lyons: IARC Press; 2005. p. 296-300. 2. Wenig BM. Atlas of head and neck pathology. 2nd ed. New York: Saunders; 2008. p. 131-8. 3. Neville BW, Damm DD, Allen CM. Odontogenic cysts and
24.
25.
tumors. In: Gnepp DR, editor. Diagnostic surgical pathology of the head and neck. 2nd ed. New York: Saunders; 2008. p. 785-838. Robinson L, Martinez MG. Unicytic ameloblastoma: a prognostic distinct entity. Cancer 1977;40:2278-85. Escande C, Chaine A, Menard P, Ernenwein D, Ghouls, Boutattour A, et al. A treatment algorithm for the adult ameloblastomas according to Pitié-Salpêtrière Hospital experience. J Cranio-Maxillofac Surg 2009;37:363-9. Miele L. Notch signaling. Clin Cancer Res 2006;12:1074-9. Fiúza UM, Arias AM. Cell and molecular biology of Notch. J Endocrinol 2007;194:459-74. Fortini ME. Notch signaling: the core pathway and its posttranslational regulation. Dev Cell 2009;16:633-47. Artavanis-Tsakonas S, Rand MD, Lake RJ. Notch signaling: cell fate control and signal integration in development. Science 1999; 284:770-6. Harper JA, Yuan JS, Tan JB, Visan I, Guidos CJ. Notch signaling in development and disease. Clin Genet 2003;64:461-72. Leong KG, Karsan A. Recent insights into the role of Notch signaling in tumorigenesis. Blood 2006;107:2223-33. Mitsiadis TA, Lardelli M, Lendahl U, Thesleff I. Expression of Notch 1, 2 and 3 is regulated by epithelial-mesenchymal interactions and retinoic acid in the developing mouse tooth and associated with determination of ameloblast cell fate. J Cell Biol 1995;130:407-18. Mitsiadis TA, Regaudiat L, Gridlley T. Notch signaling pathway in tooth morphogenesis. Arch Oral Biol 2005;50:137-40. Siar CH, Ng KH, Ariff Z, Muraki E, Shimizu T, Tsujigiwa H, et al. A case report of ameloblastoma of the mandible with examination of Notch signaling. Oral Med Pathol 2006;11:35-9. Nakano K, Siar CH, Tsujigiwa H, Nagatsuka H, Nagai N, Kawakami T. Notch signaling in benign and malignant ameloblastic neoplasms. Eur J Med Res 2008;13:476-80. Nakano K, Chelvanayagam P, Born K, Siar CH, Ng KH, Nagatsuka H, et al. A study of recurrent giant odontogenic myxoma of the mandible with immunohistochemical examination for Notch. Oral Med Pathol 2008;12:53-6. Kumamoto H, Ohki K, Ooya K. Expression of Notch signaling molecules in ameloblastomas. J Oral Pathol Med 2008;37:228-34. Siar CH, Ng KH. Ameloblastoma in Malaysia—a 25-year review. Ann Acad Med Singapore 1993;22:856-60. Ng KH, Siar CH. Peripheral ameloblastoma with clear cell differentiation. Oral Surg Oral Med Oral Pathol 1990;70:210-3. Nakano K, Siar CH, Nagai N, Naito I, Sado Y, Nagatsuka H, et al. Distribution of basement membrane type IV collagen in ameloblastoma: an immunoflourescence study. J Oral Pathol Med 2002;31:494-9. Nagatsuka H, Phuu PH, Tsujigiwa H, Siar CH, Gunduz M, Sugahara T, et al. Heparanase gene and protein expression in ameloblastoma: possible role in local invasion of tumor cells. Oral Oncol 2005;41:542-8. Tsujigiwa H, Nagatsuka H, Phuu PH, Gunduz M, Siar CH, Oida S, et al. Analysis of amelogenin gene (AMGX, AMGY) expression in ameloblastoma. Oral Oncol 2005;41:843-50. Xu P, Yu S, Jiang R, Kang C, Wang C, Jiang H, et al. Differential expression of Notch family members in astrocytomas and medulloblastomas. Path Oncol Res 2009;15:703-10. Miyamoto Y, Maitra A, Ghosh B, Zechner U, Argani P, Iacobuzio-Donahue CA, et al. Notch mediates TGF alpha-induced changes in epithelial differentiation during pancreatic tumorigenesis. Cancer Cell 2003;3:565-76. Nickoloff BJ, Osborne BA, Miele L. Notch signaling as a therapeutic target in cancer: a new approach to the development of cell fate modifying agents. Oncogene 2003;22:6598-608.
OOOOE Volume 110, Number 2 26. Callahan R, Egan SE. Notch signaling in mammary development and oncogenesis. J Mammary Gland Biol Neoplasia 2004;9: 145-63. 27. Wu J, Iwata F, Grass JA, Osborne CS, Elnitski L, Fraser P, et al. Molecular determinants of Notch4 transcription in vascular endothelium. Mol Cell Biol 2005;25:1458-74. 28. O’Neill CF, Urs S, Cinelli C, Lincoln A, Nadeau RJ, León R, et al. Notch2 signaling induces apoptosis and inhibits human MDA-MB231 xenograft growth. Am J Pathol 2007;171:1023-36. 29. Mitsiadis TA, Henrique D, Thesleff I, Lendahl U. Mouse Serrate-1 (Jagged-1) expression in the developing tooth is regulated by epithelial-mesenchymal interactions and fibroblast growth factor-4. Dev 1997;124:1473-83. 30. Mitsiadis TA, Hirsinger E, Lendahl U, Goridis C. Delta-Notch signaling in odontogenesis: correlation with cytodifferentiation and evidence for feedback regulation. Dev Biol 1998:204:420-31.
Siar et al. 233 31. Mitsiadis TA, Romeas A, Lendahl U, Sharpe PT, Farges JC. Notch2 protein distribution in human teeth under normal and pathological conditions. Exp Cell Res 2003;282:101-9. 32. Borkosky SS, Nagatsuka H, Orita Y, Tsujigiwa H, Yoshinobu J, Gunduz M, et al. Sequential expressions of Notch1, Jagged2 and Math1 in molar tooth germ of mouse. Biocell 2008;32: 251-8.
Reprint requests: Chong H. Siar, BDS, MSc, FDSRCPS, FRCPath Department of Oral Pathology, Oral Medicine, and Periodontology Faculty of Dentistry, University of Malaya 50603 Kuala Lumpur, Malaysia [email protected]