Journal of the Formosan Medical Association (2013) 112, 756e760

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ORIGINAL ARTICLE Langerhans cells in odontogenic epithelia of odontogenic fibromas

Yang-Che Wu a,b, Yi-Ping Wang a,b, Julia Yu-Fong Chang a,b,c, Hsin-Ming Chen a,b, Andy Sun a,b, Chun-Pin Chiang a,b,d,* a Graduate Institute of Clinical , School of Dentistry, National Taiwan University, Taipei, Taiwan b Department of Dentistry, National Taiwan University Hospital, College of Medicine, National Taiwan University, Taipei, Taiwan c Department of Oral and Maxillofacial Surgery, Division of Oral Pathology, School of Dentistry, University of Washington, Seattle, USA d Graduate Institute of Oral Biology, School of Dentistry, National Taiwan University, Taipei, Taiwan

Received 5 September 2013; received in revised form 29 October 2013; accepted 5 November 2013

KEYWORDS Background/Purpose: Langerhans cell (LC) is an antigen-presenting cell that is very important CD1a; for T-cell-mediated immune reactions. Our previous studies have shown the presence of LCs in immunohisto- some odontogenic tumors and cysts. In this study, we further examined the presence of LCs in chemistry; odontogenic epithelia of 16 odontogenic fibromas (OFs). Langerhans cell; Methods: Anti-CD1a and anti-S-100 immunostains were used to detect the presence of LCs in odontogenic nests or strands of odontogenic epithelia of 16 OFs. epithelium; Results: These 16 OFs included 10 peripheral OFs excised from seven male and three female odontogenic fibroma; patients (mean age, 38 years) and six central OFs (including one recurrent OF) removed from S-100 protein five male patients (mean age, 28 years). Of the 10 peripheral OFs, six were found on the mandibular gingiva and four on the maxillary gingiva. Four central OFs were located in the maxilla and two in the . We found that both anti-CD1a and anti-S-100 immunostains had an equal ability to identify LCs in OFs. Positively stained dendritic LCs could be detected in nests and strands of odontogenic epithelia in nine (six peripheral and three central OFs) of the 16 OFs. In five peripheral OFs, dendritic LCs were found in occasional nests or strands of odon- togenic epithelia. In one peripheral and three central OFs, dendritic LCs could be detected in at least half of the nests or strands of odontogenic epithelium in the tissue section. Conclusion: LCs can be detected in the nests or strands of odontogenic epithelia in approxi- mately 60% of the 10 peripheral OFs and approximately 50% of the six central OFs detected. Copyright ª 2013, Elsevier Taiwan LLC & Formosan Medical Association. All rights reserved.

Conflicts of interest: none. * Corresponding author. Department of Dentistry, National Taiwan University Hospital, Number 1, Chang-Te Street, Taipei 10048, Taiwan. E-mail address: [email protected] (C.-P. Chiang).

0929-6646/$ - see front matter Copyright ª 2013, Elsevier Taiwan LLC & Formosan Medical Association. All rights reserved. http://dx.doi.org/10.1016/j.jfma.2013.11.002 Langerhans cells in odontogenic fibromas 757

Introduction Immunohistochemical staining was performed using a su- persensitive polymer-horseradish peroxidase technique. Briefly, tissues sections were deparaffinized and rehy- Odontogenic fibroma (OF) is a rare drated. Next, they were placed in a plastic slide holder characterized by varying numbers of nests or strands of (Dako, Copenhagen, Denmark) containing 0.01 M citrate odontogenic epithelium in a mature fibrous stroma. OFs can buffer, heated in a microwave oven for 15 minutes to further be divided into central (intraosseous) and periph- retrieve antigenicity, and then treated with 3% H O in eral (extraosseous) OFs according to the anatomic sites 2 2 methanol for 10 minutes to quench endogenous peroxidase involved.1 Our previous study has reported the clinico- activity. After washing in 10 mM Tris-buffered saline (TBS), pathological features of 12 peripheral OFs and three central pH 7.6, the sections were incubated with 10% normal goat OFs in 15 Taiwanese patients. The peripheral OFs seem to serum (BioGenex, San Ramon, CA, USA) to block nonspecific have a predilection for the mandibular gingiva (67%).2 binding. These sections were then incubated overnight at Langerhans cell (LC) is an antigen-presenting cell that is 4 C with mouse anti-S-100 monoclonal antibody (Thermo very important for T-cell-mediated immune reactions. LCs Fisher Scientific, Runcorn, UK) at a dilution of 1:100 or can be demonstrated by anti-S-100 or anti-CD1a immunos- mouse anti-CD1a monoclonal antibody (Abcam plc, Cam- tain in odontogenic epithelia of odontogenic tumors such as bridge, UK) at a dilution of 1:50. BioGenex Super Sensitive the noncalcifying variant of Pindborg tumor,3e5 classical TM detection systems were used for the detection of bound Pindborg tumor,6 central granular cell odontogenic antibodies. After washing in TBS, the sections were treated tumor,7,8 ameloblastoma,9,10 and OF,11,12 as well as in the with a super enhancer reagent for 10 minutes and subse- lining epithelia of radicular cysts,10.13 dentigerous quently with the polymer-horseradish peroxidase reagent cysts,10,13 odontogenic keratocysts,9,13 and calcifying for another 10 minutes. Diaminobenzidine hydrochloride odontogenic cysts.9 Because only a few OF cases have been (0.02%) (Zymed Laboratories, San Francisco, CA, USA) examined for the presence of LCs in odontogenic epithelia containing 0.03% H O was used as a chromogen to visualize of OFs and the results are controversial, this study further 2 2 peroxidase activity. The preparations were counterstained used anti-CD1a and anti-S-100 immunostains to assess the lightly with hematoxylin, mounted with Permount, and presence of LCs in the nests or strands of odontogenic examined by light microscopy. Sections of a human mela- epithelia of 16 OFs. and a human LC histiocytosis that were previously shown to be positive for S-100 and CD1a proteins, respec- Materials and methods tively, were used as positive controls. TBS instead of a primary antibody was used as a negative control. LCs in our samples were considered positive for S-100 or The study group consisted of 16 OF cases retrieved from the CD1a protein expressions when a prominent brown cyto- archives of the Department of Oral Pathology and Oral plasmic staining was observed within nests or strands of Diagnosis, National Taiwan University Hospital, Taipei, odontogenic epithelium. Initially, the sections were scan- Taiwan, from January 2006 to December 2012. Of these 16 ned at low power. For sections that showed heterogeneous OF cases, four (Cases 1, 2, 3, and 11 in Table 1) had been patterns of positive staining, the predominant pattern was reported previously.2 Demographic data, including gender taken into account for scoring. At least five high-power and age of patients, as well as location, size, clinical (200) fields were chosen randomly, and the number of foci diagnosis, treatment, and recurrence of the lesions were of positively stained LCs in nests or strands of odontogenic obtained by reviewing the dental or medical charts. epithelium was counted for each case. The mean number of All surgical specimens were obtained from total excision positively stained LC foci per high-power field was counted or enucleation of the tumor. The specimens were then fixed and graded as follows: , no positively stained LC focus; þ, in 10% neutral formalin for at least 24 hours, dehydrated in 3 positively stained LC foci per high-power field; and þþ, graded alcohol, and then embedded in paraffin. The tissue >3 positively stained LC foci per high-power field. Each of blocks were cut in serial sections of 4 mm thickness, and these assessments was carried out by two investigators stained with hematoxylin and eosin. The histopathological independently. In this study, the interobserver reproduc- diagnosis was confirmed by examination of the hematoxylin ibility was 100%. and eosin-stained tissue sections. Microscopic criteria for the diagnosis of OFs included the presence of nests or strands of odontogenic epithelium and foci of osteoid-, Results cementoid-, or cementicle-like calcifications in a fibrous or myxomatous stroma. Because the clinicopathological find- ings of 12 peripheral OFs and three central OFs have Demographic and clinical data of 16 OF cases in 15 patients already been reported in our previous study,2 this study as well as the mean number of positively stained LC foci focused on the evaluation of the presence of LCs in nests or detected by anti-CD1a and anti-S-100 immunostains in 16 strands of odontogenic epithelia in 16 OFs. OF specimens are presented in Table 1. Case 16 was a recurrent central OF of Case 15, which recurred 8 months after the initial excision of the tumor. Other 14 OF cases Immunohistochemistry for studying LCs showed no recurrence of the lesion after a follow-up period ranging from 10 months to 79 months. The 16 OFs included All the specimens for immunohistochemical staining were 10 peripheral OFs excised from seven male and three fe- fixed in 10% neutral formalin, embedded in paraffin, and male patients (mean age, 38 years) and six central OFs cut in serial sections of 4 mm thickness. removed from five male patients (mean age, 28 years). Of 758 Y.-C. Wu et al.

Table 1 Demographic and clinical data of 16 cases of OF as well as the mean number of positively stained foci of LCs detected by anti-CD1a and anti-S-100 immunostains in 16 OF specimens. Mean number of positively stained LC focia Case No. Age (y)/sex Location (gingiva) Greatest Symptoms Clinical diagnosis Anti-CD1a Anti-S-100 diameter and signs immunostain immunostain (cm) Peripheral odontogenic fibroma 1 40/M No. 13e15 areas 1.5 Swelling þþ 2 34/M No. 32 and No. 33 areas 1.0 Swelling Soft tissue tumor 3 45/M No. 23 and No. 24 areas 1.0 Swelling Pyogenic granuloma þþ 4 45/M No. 22 to No. 24 areas 3.5 Swelling Ossifying fibroma 5 42/M No. 34 and No. 35 areas 1.0 Mass Pyogenic granuloma þþ 6 22/F No. 33 and No. 34 areas 2.0 Swelling Odontogenic fibroma 7 41/F No. 33 and No. 34 areas 1.0 Mass Pyogenic granuloma þþ 8 51/M No. 31 and No. 42 areas 1.6 Mass Pyogenic granuloma 9 35/M No. 33 and No. 34 areas 1.8 Mass Ossifying fibroma þþ 10 23/F No. 11 and No. 12 areas 1.1 Mass Pyogenic granuloma þþ þþ Central odontogenic fibroma 11 10/M No. 47 and No. 48 areas 2.5 Painful swelling 12 51/M No. 14 to No. 16 areas 2.2 Painful Bone tumor þþ þþ swelling 13 42/M No. 14 to No. 17 areas 4.5 Swelling Fibrous dysplasia 14 19/M No. 33 and No. 34 areas 1.5 Swelling Osteoblastoma 15 17/M No. 21 and No. 22 areas 1.5 Swelling Odontogenic tumor þþ þþ 16 18/M No. 21 and No. 22 areas 2.5 Swelling Odontogenic fibroma þþ þþ

LC Z Langerhans cell; OF Z odontogenic fibroma. a The mean number of positively stained LC foci per 200 high-power field was counted and graded as follows: , no positively-stained LC focus; þ, 3 positively stained LC foci per high-power field; and þþ, >3 positively stained LC foci per high-power field. the 10 peripheral OFs, six were found on the mandibular being >3 per high-power field (Table 1, and Fig. 1E and F). gingiva and four on the maxillary gingiva. Four central OFs In addition, the recurrent central OF case (Case 16) had were located in the maxilla and two in the mandible. All the nearly the same number of LCs as those in the original 10 peripheral OFs were excised completely from the gingiva tumor (Case 15; Table 1). and the six central OFs enucleated completely from the maxilla or the mandible. Microscopically, all the peripheral OFs were covered by a Discussion parakeratinized stratified squamous epithelium. Focal sur- face ulceration was found in seven of the 10 peripheral OFs. LCs have been studied in a variety of odontogenic tumors The stromal component of the 16 OFs was predominantly and cysts.3e13 We reported a case of noncalcifying variant fibrocollagenous (13 cases) or predominantly myxomatous of intraosseous calcifying epithelial odontogenic tumor (3 cases). The odontogenic epithelium was usually arranged with the presence of LCs within tumor epithelial nests in in nests or strands (Fig. 1A). Juxtaepithelial hyalinization the right anterior region of the maxilla of a 52-year-old (Fig. 1A) was discernible in six cases. Foci of calcifications Taiwanese woman.3 A review of the English literature including osteoid-, cementoid- (Fig. 1B), and/or revealed two cases of noncalcifying variant of intraosseous cementicle-like materials were noted in all the 16 OFs. calcifying epithelial odontogenic tumor with LCs in the Both anti-CD1a and anti-S-100 immunostains had an anterior and premolar regions of the maxilla.4,5 We thus equal ability to identify LCs in OFs. Positively stained suggested that the noncalcifying, LC-rich variant of calci- dendritic LCs could be detected in nests or strands of fying epithelial odontogenic tumor may have a distinct odontogenic epithelia in nine (6 peripheral and 3 central predilection for occurrence in the anterior and premolar OFs) of the 16 OFs (Table 1). In five LC-positive peripheral regions of the maxilla, in contrast to the classical calcifying OFs, dendritic LCs were found in occasional nests or strands epithelial odontogenic tumors that usually occur in the of odontogenic epithelia, with the mean number of posi- molar and ascending ramus area of the mandible.1 Poom- tively stained LC foci being 3 per high-power field (Table sawat and Punyasingh6 also demonstrated the presence of 1, and Fig. 1C and D). In one peripheral and three central dendritic LCs among sheets of tumor odontogenic epithelial OFs, dendritic LCs could be detected in at least half of the cells of a classical calcifying epithelial odontogenic tumor. nests or strands of odontogenic epithelium in the OF tissue Both Chiang et al7 and Cheng et al8 reported the presence section, with the mean number of positively stained LC foci of LCs in odontogenic epithelia of a case of central granular Langerhans cells in odontogenic fibromas 759

Figure 1 H&E-stained and immunostained histological microphotographs of the OF. (A) An H&E-stained tissue section showing a peripheral OF with nests and strands of odontogenic epithelium in the cellular fibrous stroma. Juxtaepithelial hyalinization around nests or strands of odontogenic epithelium is also noted. (B) An H&E-stained tissue section showing a peripheral OF with foci of cementoid calcification and nests of odontogenic epithelium (arrows) in the cellular fibrous stroma. (C) An anti-CD1a- immunostained section of a peripheral OF showing several brown dendritic LCs in an odontogenic epithelial nest. (D) An anti-S- 100-immunostained serial tissue section of a peripheral OF demonstrating a few brown dendritic LCs in the same odontogenic epithelial nest as shown in Figure 1C. (E) An anti-CD1a-immunostained tissue section of a central OF showing six foci of positively stained LCs in nests or strands of odontogenic epithelium. (F) An anti-S-100-immunostained tissue section of a central OF exhibiting six foci of positively stained LCs in nests or strands of odontogenic epithelium [original magnification: (A and B) 20;(CeF) 40]. H&E Z hematoxylin and eosin; LC Z Langerhans cell; OF Z odontogenic fibroma. cell odontogenic tumor. Mello et al9 studied the presence of inflammatory fibrous connective tissue of one radicular LCs in ameloblastomas, keratocystic odontogenic tumors cyst, one dentigerous cyst, and one odontogenic kerato- (also known as odontogenic keratocysts), and calcifying cyst.13 Eversole11 demonstrated the presence of dendritic cystic odontogenic tumors (also known as calcifying odon- LCs in four of the 65 OFs. Mosqueda-Taylor et al12 found LCs togenic cysts). They found more LCs in calcifying cystic within the epithelial islands of all of the six OFs tested. This odontogenic tumors than in ameloblastomas and kerato- study showed the presence of dendritic LCs in nine of the 16 cystic odontogenic tumors, and suggested that a depletion OFs. The results of aforementioned studies indicate that of LCs might influence the local invasiveness of amelo- LCs can be detected in odontogenic epithelia of some blastomas and keratocystic odontogenic tumors. Moreover, odontogenic tumors and lining epithelia of a few odonto- Murase et al10 showed the presence of LCs in tumor or genic cysts. cystic epithelia and sometimes in stromal connective tissue Interestingly, five of the six peripheral OFs with the in 11 of 61 ameloblastomas, 22 of 40 radicular cysts, and clinical diagnosis of pyogenic granulomas showed the three of 28 dentigerous cysts. Recently, we reported the presence of LCs in nests or strands of odontogenic epithelia presence of LCs in both the lining epithelia and the of their tissue sections. A pyogenic granuloma is a red lesion 760 Y.-C. Wu et al. associated with inflammation. Misdiagnosis of the periph- 3. Wang YP, Lee JJ, Wang JT, Liu BY, Yu CH, Kuo RC, et al. Non- eral OF as a pyogenic granuloma suggested that the clinical calcifying variant of calcifying epithelial odontogenic tumor appearance of the lesion was red and it was probably with Langerhans cells. J Oral Pathol Med 2007;36:436e9. associated with inflammation. We checked the tissue sec- 4. Takata T, Ogawa I, Miyauchi M, Ijuhin N, Nikai H, Fujita M. Non- tions of our six peripheral OFs that were misdiagnosed as calcifying Pindborg tumor with Langerhans cells. J Oral Pathol Med 1993;22:378e83. pyogenic granulomas and the three LC-positive central OFs, 5. Asano M, Takahashi T, Kusama K, Iwase T, Hori M, Yamanoi H, and confirmed that all these nine cases had mild to severe et al. A variant of calcifying epithelial odontogenic tumor with infiltration of inflammatory cells in the connective tissue Langerhans cells. J Oral Pathol Med 1990;19:430e4. 10 stroma. Murase et al found that LCs are usually demon- 6. Poomsawat S, Punyasingh J. Calcifying epithelial odontogenic strated in ameloblastomas, radicular cysts, and dentigerous tumor: an immunohistochemical case study. J Mol Histol 2007; cysts that are accompanied by a high degree of inflamma- 38:103e9. tory infiltration in their lesions. On the contrary, LC- 7. Chiang CT, Hu KY, Tsai CC. Central granular cell odontogenic negative ameloblastomas, radicular cysts, and dentigerous tumor: the first reported case in oriental people and cysts generally lack inflammatory responses.10 Recently, we literature review. J Formos Med Assoc 2012. http: also demonstrated the presence of LCs in the lining //dx.doi.org/10.1016/j.jfma.2012.04.011 [Epub ahead of print]. 8. Cheng SJ, Wang YP, Chen HM, Chiang CP. Central granular cell epithelia of radicular cysts, dentigerous cysts, and odon- odontogenic tumor of the mandible. J Formos Med Assoc 2013; togenic keratocysts that are strongly associated with 112:583e5. 13 inflammation in the underlying fibrous cystic wall. 9. Mello LA, Figueiredo AL, Ramos EA, Gurgel CA, Martins MD, de Therefore, we suggest that the presence of chronic Figueiredo CR, et al. CD1a-positive Langerhans cells and their inflammation in the connective tissue stroma may result in relationship with E-cadherin in ameloblastomas and kerato- a higher detection rate of LCs in nests or strands of odon- cystic odontogenic tumors. J Oral Pathol Med 2013;42:454e61. togenic epithelia in OFs with chronic inflammation than in 10. Murase N, Tatemoto Y, Iwai Y, Okada Y, Mori M. Langerhans cells those without chronic inflammation. in odontogenic tumours and cysts as detected by S-100 protein e Immunohistochemistry is a good method to identify a immunohistochemistry. Basic Appl Histochem 1990;34:135 41. specific cell or marker in oral lesions including oral can- 11. Eversole LR. Odontogenic fibroma, including amyloid and ossifying variants. Head Neck Pathol 2011;5:335e43. cer.14 All previous studies and this study used either anti- 12. Mosqueda-Taylor A, Martı´nez-Mata G, Carlos-Bregni R, CD1a or anti-S-100 immunostain, or both to detect LCs in Vargas PA, Toral-Rizo V, Cano-Valde´z AM, et al. Central odon- 3e13 15 odontogenic tumors and cysts, oral , oral togenic fibroma: new findings and report of a multicentric 15,16 15,16 epithelial dysplasia, oral squamous cell carcinoma, collaborative study. Oral Surg Oral Med Oral Pathol Oral Radiol e oral ,17 20 and LC histiocytosis.1 Although the Endod 2011;112:349e58. anti-CD1a antibody is more specific than the anti-S-100 13. Wu YC, Wang YP, Chang JYF, Chiang CP. Langerhans cells in antibody for LCs, both antibodies have shown an equal lining epithelia of odontogenic cysts. J Formos Med Assoc 2013; ability to identify LCs in this and previous studies. 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