Odontogenic tumors: a retrospective clinicopathological study from two Italian centers

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1 PATHOLOGICA 2017;109:35-46 Review - Study Group GIPaTeC Odontogenic tumors: a retrospective clinicopathological study from two Italian centers C. RUBINI 1, M. MASCITTI 2, A. SANTARELLI 2, A. TEMPESTA 3, L. LIMONGELLI 3, G. FAVIA 3, E. MAIORANO 4 1 Department of Biomedical Sciences and Public Health, Marche Polytechnic University, Ancona, Italy; 2 Department of Clinical Specialistic and Dental Sciences, Marche Polytechnic University, Ancona, Italy; 3 Interdisciplinary Department of Medicine, Section of Odontostomatology, University of Bari Aldo Moro, Italy; 4 Department of Emergency and Organ Transplantation, Section of Pathological Anatomy, University of Bari Aldo Moro, Italy Key words Odontogenic tumors Keratocystic odontogenic tumor Ameloblastoma Peripheral odontogenic tumor Summary Objective. The aim of this retrospective study was to perform an epidemiological analysis of all odontogenic tumors treated in the University Hospitals Ospedali Riuniti in Ancona and Policlinico in Bari, from 1990 to Materials and methods. A retrospective survey of 277 patients treated for odontogenic tumors from 1990 to 2015 was performed. Data were retrieved from the archives of the above quoted Sections of Pathology. The lesions were classified according to 2005 WHO histological classification, and the following variables were analyzed: age, sex, histopathological diagnosis, site distribution, tumor size, and relapses. Peripheral odontogenic tumors were analyzed considering these lesions separately from their central counterparts. Results. In a total of 344 surgical specimens, there were 277 primary tumors and 67 recurrences. As regards primary lesions, there were 185 odontogenic keratocysts (keratocystic odontogenic tumors) (66.8%), 49 ameloblastomas (17.7%), and 40 other benign odontogenic tumors (14.4%). As to malignant tumors, only 3 ameloblastic carcinomas were found (1.1%). The mean age was 46.7 years, with a M:F ratio of 1.8:1. The mandible was the most common site of localization, with 211 cases (76.2%). Also, 21 cases of peripheral odontogenic tumors were found, ameloblastomas being the most common (8 cases, 38.1%). Conclusions. There is a wide variety of cysts, some of which are subject to variations according to sex, localization, and age. Odontogenic tumors are rare neoplasms and appear to show variations according to sex, localization, and age, and may be useful to the clinicians who need to make clinical judgments before biopsy about the most probable diagnosis. Introduction The term odontogenic tumors comprises a group of neoplasms and hamartomatous lesions derived from epithelial, ectomesenchymal and/or mesenchymal cells of tissues involved in the odontogenesis or remnants of tissues that take part in the formation of tooth 1. Due to their histological origin, these lesions are found exclusively within the maxillofacial region, more precisely in two locations: intraosseous (or centrally located) and extraosseous (or peripherally located, in the gingiva and alveolar mucosa) 2. The first ascertained description of an odontogenic tumor has been made by the father of modern dentistry, Pierre Fauchard, in 1746, while the first attempt to classify these lesions was published by Pierre Broca in About a century later, a consensus-based classification sponsored by World Health Organization (WHO) was published 4, while the last edition of this classification came out 11 years ago (Tab. I) 1. Odontogenic tumors are relatively rare lesions, accounting for about 2% of all maxillofacial specimens sent for diagnosis to pathology services. Just to give an idea of the rarity of these lesions, it was estimated that odontogenic tumors represent approximately % of all tumors in the human body 5. Almost all of these tumors are benign since, according to the literature, malignant neoplasms represent a small proportion, ranging from 0% to 6.1%, of all odontogenic lesions 6. In view of its biological behaviour, odontogenic keratocyst was reclassified in 2005 by WHO as a neoplastic lesions named keratocystic odontogenic tumor, though such terminology has neither been universally accepted Correspondence Marco Mascitti, via Tronto 10, Ancon, Italy - Tel Fax marcomascitti86@ hotmail.it

2 36 C. RUBINI ET AL. Tab. I. Classification of odontogenic tumors (WHO 2005). Odontogenic tumor Benign tumours Odontogenic epithelium with mature, fibrous stroma without odontogenic ectomesenchyme 1. Ameloblastoma: - Solid/multicystic type - Extraosseous/peripheral type - Desmoplastic type - Unicystic type 2. Squamous odontogenic tumour 3. Calcifying epithelial odontogenic tumour 4. Adenomatoid odontogenic tumour 5. Keratocystic odontogenic tumour Odontogenic epithelium with odontogenic ectomesenchyme, with or without hard tissue formation 1. Ameloblastic fibroma 2. Ameloblastic fibrodentinoma 3. Ameloblastic fibro-odontoma 4. Odontoma (complex and compound types) 5. Odontoameloblastoma 6. Calcifying cystic odontogenic tumour 7. Dentinogenic ghost cell tumour Mensechyme and/or odontogenic ectomesenchyme with or without odontogenic epithelium 1. Odontogenic fibroma 2. Odontogenic myxoma/myxofibroma 3. Cementoblastoma Malignant tumours Odontogenic carcinomas 1. Metastasizing ameloblastoma 2. Ameloblastic carcinoma (primary and secondary types) 3. Primary intraosseous squamous cell carcinoma: - solid type - derived from keratocystic odontogenic tumour - derived from odontogenic cysts 4. Clear cell odontogenic carcinoma 5. Ghost cell odontogenic carcinoma Odontogenic sarcomas 1. Ameloblastic fibrosarcoma 2. Ameloblastic fibrodentino and fibro-odontosarcoma nor commonly used worldwide and, most probably, the forthcoming WHO classification will re-consider such change. Nevertheless, one of the main effects of this new classification is the change in the epidemiology of these lesions, as well as of odontogenic cysts. In fact, since odontomas, ameloblastomas and keratocystic odontogenic tumors represents more than 80% of all odontogenic neoplasms, it is deduced that most of these tumors are exceedingly rare 3. The etiology and the oncogenic processes in odontogenic tumors are still unknown, apart from indications that several genetic factors and proteins seem to be involved in differentiation and proliferation of odontogenic epithelial cells 7 8. As to studies of odontogenic tumor development, a heuristic role was played by the view that the interactions between epithelial and ectomesenchymal tissue elements occurring during normal tooth development also operate to a certain extent in these lesions 9. Clinically, these neoplasms are typically asymptomatic, although they may cause mandibular /maxillary expansion, teeth displacement, root resorption, and bone loss. The fact that most of these tumors remain painless is the main reason for patients not complaining until the lesions have reached large sizes 10. Therefore, the knowledge of features such as age, location, and radiographic appearance of the odontogenic tumors can be valuable to develop more precise clinical differential diagnoses. Several studies have described the presentation of odontogenic tumors, with special attention to their clinical manifestations and histopathologic features However, due to the rarity of these lesions, most of these studies are case reports, case series, or small single-institution reviews, resulting in lack of meaningful information about the epidemiology of these lesions, particularly with regards to rare histological variants. On the contrary, extensive reviews performed by reference centres, with specialized pathologist for these lesions, can provide a comprehensive region-specific epidemiology. The aim of this retrospective study was to perform an epidemiological analysis comprehensive of all odontogenic tumors diagnosed and surgically treated in the University Hospitals Ospedali Riuniti in Ancona and Policlinico in Bari, Italy, from 1990 to In this investigation, we focused on the analysis of the most frequent neoplastic lesions: ameloblastoma and odontogenic keratocyst (i.e., keratocystic odontogenic tumor, KOT). Furthermore, our results were compared with those reported in the literature. Material and methods This study was conducted in accordance with the Ethical Principles for Medical Research Involving Human Subjects statement of the Helsinki Declaration. The present retrospective study considered 277 patients operated for odontogenic tumors from 1990 to Data were retrieved and catalogued from clinical records and from the archives of the above Sections of Pathology by a single operator, in order to ensure uniformity of the collected data. Because the 25-year period considered in this study, during which the classification of these lesions has changed, the histopathological slides of odontogenic tumors were re-evaluated by two pathologists to confirm the diagnosis, according to the current

3 ODONTOGENIC TUMORS: A RETROSPECTIVE CLINICOPATHOLOGICAL STUDY FROM TWO ITALIAN CENTERS 37 WHO histopathological criteria 1. In case of controversies, a third pathologist was consulted in order to reach a unanimous consensus. In the present study, the following data were considered for each case: age, sex, histopathological diagnosis, site distribution, dimensions and relapses. The regions of onset (mandible and maxilla) were further subdivided into two regions (anterior and posterior). In case of multiple tumors or tumors occupying both the anterior and posterior regions, only the most widely affected part of jawbone was taken into account. Furthermore, peripheral odontogenic tumors were analyzed considering these lesions separately from their central counterparts. The data analysis was performed using GraphPad Prism software version 6.00 for Windows (GraphPad Software, San Diego California USA, The Chi-square test and Fisher s exact test were applied to analyze the statistical significance of the data when applicable. The level of statistical significance was set at p < Results Overall 277 patients were treated for odontogenic neoplasms and recorded at the University Hospitals Ospedali Riuniti in Ancona and Policlinico in Bari, Italy, from 1990 to surgical specimens were retrieved from the archives of the Sectionsof Pathology, corresponding to 277 primary tumors and 67 recurrences. The mean age at diagnosis of the primary tumor was 46.7 years, ranging from 5 to 98 years, with a standard deviation (SD) of Among all cases, 177 (63.9%) were males (mean age of 47.4 years), and 100 (36.1%) were females (mean age of 45.6 years), with a M:F ratio of 1.8:1 (p < 0.05). Also, 26 cases of pediatric patients (< 18 years) were identified, with a mean age of 13.8 years (SD of 3.1) and a slight female predominance (M:F ratio of 0.9:1), while cases of odontogenic tumors in children less than 10 years of age are exceedingly rare, with only 3 cases in our series. Regarding the localization, the mandible (211 cases, 76.2%) was more commonly involved than the maxilla (66 cases, 23.8%) (p < 0.05). The posterior region of the jaws (156 cases in the mandible and 36 in the maxilla) was more frequently affected than the anterior region (55 cases in the mandible and 30 in the maxilla) (p < 0.05), while no difference was found as to the affected side (141 cases on the right side of the jaws, 136 cases on the left side). Data on the size of the primary tumor were available for 228 patients, while 49 cases did not present this data. A mean tumor size of 2.3 cm was found, with a SD of 1.3. Fiftyone patients developed tumor recurrence, and 9 of them showed multiple recurrences over the years. The mean age at the first recurrence was 49.6 years, with a SD of Men were mostly affected (M:F ratio of 1.7:1), with a mean age of onset greater than women (53 years vs 43.9 years), reflecting the sex and age distributions of primary tumors. Regarding the localization, the majority of cases occurred in the mandible (41 cases, 80.4%), especially in the posterior region (26 cases). Only 10 cases involved the maxilla (19.6%), showing no specific preferred site. As to the size of the first tumor recurrences, 6 cases did not present this data, while the remaining 45 patients showed a mean tumor size of 2.4 cm, with a SD of 1.6. The size of the subsequent tumor recurrences was also analyzed, showing a mean tumor size of 2.8 cm, with a SD of 1.4, indicating a progressive increase in tumor size. Noteworthy, 2 pediatric patients were diagnosed with the Nevoid Basal Cell Carcinoma Syndrome (NBCCS or Gorlin syndrome), and showed the greatest number of recurrences (4 and 5, respectively). Table II shows the distribution of primary odontogenic tumors, both benign and malignant, in relation Tab. II. Lesion Frequency Male Female M:F Age Site distribution Num. (%) Num. (%) Num. (%) Ratio Mean (SD) Maxilla Mandible KOT 185 (66.8) 118 (66.7) 67 (67) 1.8: (19.1) Am 49 (17.7) 35 (19.8) 14 (14) 2.5: (19.1) CCOT 6 (2.2) 4 (2.3) 2 (2) 2:1 68 (30.9) 3 3 CEOT 4 (1.4) 2 (1.1) 2 (2) 1: (15.7) 2 2 AOT 2 (0.7) 1 (0.6) 1 (1) 1:1 38 (18.4) 0 2 SOT 1 (0.4) 1 (0.6) 0 (0) DGCT 2 (0.7) 1 (0.6) 1 (1) 1: (40.3) 0 2 Cb 2 (0.7) 1 (0.6) 1 (1) 1: (24.7) 0 2 Od 7 (2.5) 4 (2.3) 3 (3) 1.3: (20.3) 1 6 AF 4 (1.4) 2 (1.1) 2 (2) 1: (10.6) 1 3 OF 7 (2.5) 2 (1.1) 5 (5) 0.4: (21.2) 3 4 OM 5 (1.8) 3 (1.7) 2 (2) 1.5: (16.4) 2 3 Am Ca 3 (1.1) 2 (1.1) 1 (1) 2: (17.8) 2 1 Total 277 (100) 177 (100) 100 (100) 1.8: (20.4) Primary odontogenic tumors distribution. KOT = Odontogenic keratocyst (keratocystic odontogenic tumor); Am = Ameloblastoma; CCOT = Calcifying Cistic Odontogenic Tumor; CEOT = Calcifying Epithelial Odontogenic Tumor; AOT = Adenomatoid Odontogenic Tumor; SOT = Squamous Odontogenic Tumor; DGCT = Dentinogenic Ghost Cell Tumor; Cb = Cementoblastoma; Od = Odontoma; AF = Ameloblastic Fibroma; OF = Odontogenic Fibroma; OM = Odontogenic Myxoma; Am Ca = Ameloblastic Carcinoma.

4 38 C. RUBINI ET AL. Tab. III. Lesion Frequency Male Female M:F Age Site distribution Recurrence Num. (%) Num. (%) Num. (%) ratio Mean (SD) Maxilla Mandible Mean (SD) KOT 36 (70.6) 32 (78.1) 4 (40) 8: (18.6) (32) Am 11 (21.6) 7 (17.1) 4 (40) 1.7: (19.5) (39) CEOT 2 (3.9) 1 (2.4) 1 (10) 1: (23.3) (8) OF 1 (1.9) 0 (0) 1 (10) Am Ca 1 (1.9) 1 (2.4) 0 (0) Total 51 (100) 41 (100) 10 (100) 4.1: (19.1) (34) Recurrent odontogenic tumors distribution (first recurrence). KOT = Odontogenic keratocyst (keratocystic odontogenic tumor); Am = Ameloblastoma; CEOT = Calcifying Epithelial Odontogenic Tumor; OF = Odontogenic Fibroma; Am Ca = Ameloblastic Carcinoma. to sex, age, and localization. Odontogenic keratocysts (KOT) accounted for most of the lesions, with 185 cases (66.8%), while ameloblastoma was the second most common odontogenic tumor of this series, with 49 cases (19.4%). Taken together, all the other tumor types represented 15.5% of all odontogenic tumors, with 43 cases. Only 3 cases of malignant odontogenic tumors were identified, accounting for 1.1% of all lesions. Table III shows the distribution of recurrent odontogenic tumors in correlation with sex, age, localization, and time to recurrence. As for the primary tumors, the odontogenic keratocyst (KOT) was the most common, with 36 cases (70.6%), followed by ameloblastoma (11 cases, 21.6%), calcifying epithelial odontogenic tumor (2 cases, 3.9%), odontogenic fibroma (1 case, 1.9%), and 1 case of ameloblastic carcinoma (1.9%). The current data show that recurrences appeared at a mean time of 48 months, with a SD of 34, without significant differences between the single lesions (p > 0.05). Table IV focuses on the results concerning peripheral odontogenic lesions. A total of 21 cases were retrieved from the archives. The mean age at diagnosis was 55.4 years, ranging from 14 to 87 years, with a SD of Males were more frequently affected (15 cases, 76.2%) than females (6 cases, 23.8%) and the mandible more frequently than the maxilla (13 vs. 8 cases, respectively). Regarding single lesions, ameloblastomas were the most common lesions, with 9 cases (42.9%). Among pediatric patients (Tab. V), the mandible was more frequently affected than the maxilla, with 14 and 12 cases, respectively. As for odontogenic tumors in adulthood, odontogenic keratocysts (KOT) were the Tab. IV. Lesion Frequency Male Female M:F Age Site distribution Num. (%) Num. (%) Num. (%) ratio Mean (SD) Maxilla Mandible Am 8 (38.1) 6 (40) 2 (13.3) 3:1 63 (9) 2 6 CCOT 3 (14.3) 3 (20) 0 (0) 84 (3.6) 1 2 CEOT 2 (9.5) 1 (6.7) 1 (16.7) 1:1 31 (25.5) 1 1 AOT 1 (4.8) 1 (6.7) 0 (0) 38 (18.4) 0 1 DGCT 1 (4.8) 1 (6.7) 0 (0) AF 1 (4.8) 0 (0) 1 (16.7) OF 3 (14.3) 1 (6.7) 2 (13.3) 0.5:1 47 (7.8) 2 1 OM 2 (9.5) 2 (13.3) 0 (0) 43 (5.6) 1 1 Total 21 (100) 15 (100) 6 (100) 3.2: (21.8) 8 13 Peripheral odontogenic tumors distribution. In this table, maxilla and mandible stand for mucosa of upper and lower regions, respectively. Am = Ameloblastoma; CCOT = Calcifying Cistic Odontogenic Tumor; CEOT = Calcifying Epithelial Odontogenic Tumor; AOT = Adenomatoid Odontogenic Tumor; DGCT = Dentinogenic Ghost Cell Tumor; AF = Ameloblastic Fibroma; OF = Odontogenic Fibroma; OM = Odontogenic Myxoma. Tab. V. Lesion Frequency Male Female M:F Age Site distribution Num. (%) Num. (%) Num. (%) ratio Mean (SD) Maxilla Mandible KOT 18 (69.2) 7 (58.3) 11 (78.6) 0.6: (18.6) 8 10 Am 3 (11.5) 1 (8.3) 2 (14.3) 0.5: (6.6) 1 2 Od 2 (7.7) 2 (16.7) 0 (0) 11.5 (3.5) 0 2 CEOT 2 (7.7) 1 (8.3) 1 (7.1) 1: (0.7) 1 1 CCOT 1 (3.9) 1 (8.3) 0 (0) Total 26 (100) 12 (100) 14 (100) 0.9: (3.1) Odontogenic tumors distribution in paediatric patients (< 18 years). KOT = Odontogenic keratocyst (keratocystic odontogenic tumor); Am = Ameloblastoma; Od = Odontoma; CCOT = Calcifying Cistic Odontogenic Tumor; CEOT = Calcifying Epithelial Odontogenic Tumor.

5 ODONTOGENIC TUMORS: A RETROSPECTIVE CLINICOPATHOLOGICAL STUDY FROM TWO ITALIAN CENTERS 39 most common, with 18 cases (69.2%), followed by 3 cases of ameloblastoma, 2 odontomas, 2 calcifying epithelial odontogenic tumor, and 1 calcifying cystic odontogenic tumor. Odontogenic keratocysts (KOT) were the most frequently diagnosed, with 185 cases (66.8% of all tumors) and a M:F ratio of 1.8:1 (Tab. II) (p < 0.05). This tumor represent the most common lesion both in adults and in paediatric patients (69.2%), reaching statistical significance (Tabs. II, V). Regarding the site of onset, odontogenic keratocysts (KOT) were significantly more frequent in the mandible (p < 0.05), with 144 cases localized in this region (77.8% of total), especially in the posterior region (114 cases); 41 cases were found in the maxilla (22.2% of total), equally distributed among the anterior and posterior regions. The mean age was 44.8 years, ranging from 10 and 85 years (SD of 19.1). Regarding sex distribution, males showed a higher frequency between third and sixth decades, whereas the female population presented a more homogeneous distribution (Fig. 1). Regarding histological features, the inflammatory infiltrate was present in 100 cases, while 15 cases showed the presence of satellite cysts (the socalled daughter cysts ). The size of primary tumors was identifiable in 166 cases (Tab. VI): 15 cases were less than 1 cm size, 61 cases ranged between 1 and 2 cm, while 90 cases showed a mass larger than 2 cm. Odontogenic keratocysts (KOT) also showed the highest recurrence rates, with 36 cases (Tab. III); men were significantly more affected by tumor recurrences, with a M:F ratio of 8:1. The mean age was 47.7 years, slightly higher than the mean age at first diagnosis. This is in agreement with the mean time of recurrence, that was 45 months (SD of 32). Regarding the site of onset, seems that the mandible was more affected than the primary tumor. The presence of inflammatory infiltrate in recurrent odontogenic keratocysts (KOT) was significantly lower compared to the primary tumors, since only 10 cases showed this histological feature (p < 0.05), and only 2 cases showed the presence of satellite cysts in the wall of the original tumor. Regarding the tumor size, data from 31 patients with recurrent tumors were available (Tab. VI). The second most frequent lesion was ameloblastoma, accounting 49 cases (17.7% of all primary tumors) and a M:F ratio of 2.5:1 (Table II) (p < 0.05). Furthermore, this lesion was the most represented peripheral odontogenic tumor, with a total of 8 cases (38.1%) (Tab. IV). Regarding the site of onset, ameloblastomas occurred mainly in the mandible, with 38 cases (77.5%) (p < 0.05), mainly affecting the posterior region. The mean age was 55.3 years, ranging from 17 and 91 years (SD of 19.1), significantly higher than that of keratocystic odontogenic tumor (p < 0.05). Figure 2 displays the age distribu- Fig. 1. Distribution of Odontogenic keratocyst (KOT) according to sex and age. Tab. VI. Lesion Dimensions Inflammatory infiltrate Satellite cysts < 1 cm 1-2 cm > 2 cm Yes No Yes No Primary KOT Recurrent KOT Comparison of tumor size and histological features between primary and recurrent keratocystic odontogenic tumor. KOT = Odontogenic keratocyst (keratocystic odontogenic tumor).

6 40 C. RUBINI ET AL. Fig. 2. Distribution of ameloblastomas according to sex and ages. Tab. VII. Lesion Dimensions Histological subtypes < 1 cm 1-2 cm > 2 cm Solid/multicystic Unicystic Peripheral Desmoplastic Primary Am Recurrent Am Comparison of tumor size and histological subtypes between primary and recurrent ameloblastoma. Am = Ameloblastoma. tion of ameloblastomas; female patients showed a peak frequency in fifth decade, whereas males presented two distinct frequency peaks (fifth and seventh decades). All the histological subtypes of ameloblastoma were found (Tab. VII): the solid / multicystic type was the most frequent, with 21 cases (42.9%), followed by the unicystic (19 cases, 38.8%), peripheral (8 cases, 16.3%), and desmoplastic (1 case, 2%) types. The data regarding the size of primary ameloblastomas were found in 37 cases: 3 cases were less than 1 cm size, 7 cases ranged between 1 and 2 cm, while 27 cases showed a mass larger than 2 cm. As to the inflammatory infiltrate, this feature was present in two cases only, one primary and one recurrent tumor. All other benign odontogenic tumors (namely calcifying cystic odontogenic tumor, calcifying epithelial odontogenic tumor, adenomatoid odontogenic tumor, squamous odontogenic tumor, dentinogenic ghost cell tumor, cementoblastoma, odontoma, ameloblastic fibroma, odontogenic fibroma, and odontogenic myxoma), showed a very low frequency, reaching a total of 40 cases (14.4% of all primary tumors). More detailed informations are shown in Tables II, III, IV, and V. Among the malignant lesions, only three cases of ameloblastic carcinoma were retrieved (1.1% of all tumors), while no other histological type of odontogenic carcinoma or sarcoma were found. Discussion The current classification of odontogenic neoplasms was published in the 2005 edition of the WHO classification of head and neck tumors (Tab. I) 1. Compared to the previous edition, several important changes, including new nomenclature and classification of benign and malignant neoplasms, were introduced. Furthermore, it is worth mentioning that odontogenic cysts were not considered in the 2005 classification, therefore the 1992 classification is still relevant for these common lesions of the jaws and maxillofacial region 13. Odontogenic tumors are rare lesions, and their frequency is less than 3% of oro-maxillofacial specimens submitted to an oral pathology service, as reported by epidemiological data in the literature However, ethno-geographic variation in the frequency of these tumors is well known, with several studies conducted in Africa and Asia that show a frequency ranging from 3.9% to 9.6% of all oro-maxillofacial lesions Malignant tumors represent a small portion of all odontogenic lesions, although there is a quite wide range in the literature, ranging from 0% to 6.1% of total 18. Our results agree with those from Europe and America, showing a very low frequency (1.1%). Furthermore, it is noteworthy the fact that many odontogenic carcinomas and sarcomas are not present in our archive, stressing that some odontogenic malignant tumors are exceedingly rare.

7 ODONTOGENIC TUMORS: A RETROSPECTIVE CLINICOPATHOLOGICAL STUDY FROM TWO ITALIAN CENTERS 41 According to the 2005 WHO classification, the most common tumors are ameloblastoma, keratocystic odontogenic tumors (also known as odontogenic keratocyst), and odontomas. The present study found odontogenic keratocysts (KOT) to be the most common neoplasm, accounting for 66.8%, followed by ameloblastoma (17.7%), odontoma (2.5%), and odontogenic fibroma (2.5%). Our results differ significantly from those reported in the literature. The reason could be given to ethno-geographic variation in the frequency of this lesion. Another explanation is that, for a number of reasons (i.e. accessibility to public health, the high frequency of accidentally diagnosis, a greater attention to public health) these results might reflect that patients are more likely to undergo surgery to remove such lesions. Differences were found with data reported in some Asian and African countries, were ameloblastoma seems to be the most frequent tumor On the contrary, several other studies in North and South America reported that odontoma is the most common odontogenic tumor These results confirm that ameloblastomas are less frequent in Caucasians, but a series of bias must be taken into account. First of all, in many developing countries with social and health problems, odontomas, because of their asymptomatic and self-limiting nature, could not be removed due to the patient s choice. In fact, many patients believe such kind of lesions would not deserve medical consultation. Secondly, in many cases surgical removal of these lesions is performed in dental offices, and odontomas may not be sent for histopathological analysis Considering the age of onset, odontogenic tumors showed a mean age of 46.7 years, considerably higher than that found by Reichart et al. 25. This difference is partly explained by the fact that the mean age at initial diagnosis in industrialized countries is significantly higher than in developing countries. The analysis of sex distribution showed male predominance, with a M:F ratio of 1.8:1. These results are in agreement with those reported by several authors 28. On the other hand, many other studies found a more balanced M:F ratio or a slight female predominance. Instead, when considering only pediatric patients, there was a slight majority of females (M:F ratio of 0.9:1), without significant differences. Regarding the anatomic localization of the odontogenic tumors, mandible was the site most often affected (76.2%). These results were consistent with those reported by all other authors, emphasizing the greater susceptibility of the mandible to develop odontogenic tumors. Odontogenic keratocyst (KOT) The keratocystic odontogenic tumor, i.e. odontogenic keratocyst, is a benign odontogenic tumor that arises from cells of the dental lamina. This lesion is characterized by a parakeratinized stratified squamous epithelium and exhibits an infiltrative growth pattern 29. Due to its aggressive nature and several genetic alterations of tumor suppressor genes (such as p16, p53, and PTCH1) 30-32, the WHO reclassified this lesion as a tumor, while its orthokeratinized variant, called orthokeratinized odontogenic cyst, has become a cyst in the strict sense. One of the main effects of the 2005 classification is the change in the epidemiology of these lesions, making the keratocystic odontogenic tumor one of the most common neoplasm of odontogenic origin. Our results show that this tumor was the most frequently diagnosed lesion, significantly higher than that reported by other Authors These lesions may be found at any age, but most cases are diagnosed in people between the second and the fifth decades of life. As expected, in our series the patients with this tumor are in a very broad range of ages (10-85 years), with a mean age was 44.8 years. The results of this study are in agreement with the data from the literature, that showed a slight male predilection and a frequent posterior mandibular involvement. In fact, the molar area, the mandibular angle and the ramus were the most common locations of odontogenic keratocysts (KOT), accounting for 61.6% of all cases. Due to the fact that smaller lesions are typically asymptomatic, their discovery is often accidental, during the course of routine radiographic examinations. Larger tumors usually are associated with pain, swelling, and secondary infection, but sometimes they can also be asymptomatic, without causing bone deformities 33. This explains why most of the odontogenic keratocysts (KOT) (90 of these 166 cases with known size, 54.2%) showed a diameter larger than 2 cm. Radiographically, the odontogenic keratocysts (KOT) usually appears as a unilocular radiolucent area with well-defined borders, often with sclerotic margins, while larger lesions may be multilocular. The presence of an un-erupted tooth can be found in the lesion up to 40% of cases, mimicking a dentigerous cyst 34. Histologically, the odontogenic keratocysts (KOT) consists of a cyst lined by parakeratinized stratified squamous epithelium which can have a thickness up to seven cell layers. The diagnostic feature is a hyperchromatic basal cell layer, which exhibits prominent palisading of basal cells (Fig. 3A). The wall of the lesion is composed of fibrous connective tissue, often containing an inflammatory infiltrate (Fig. 3B). Several other histological features can be observed, such as the presence of satellite cysts (the so-called daughter cysts ), formed by the budding of the basal layer into the underlying connective, and foci of proliferative odontogenic epithelium (Fig. 3C, 3D) 29. Inflammatory infiltration was common in primary odontogenic keratocysts (KOT), while satellite cysts were present in 15 cases only, including the two cases affected by the Gorlin syndrome (Tab. VI). Patients with multiple odontogenic keratocysts (KOT), especially if they show satellite cysts, should be suspected to have the NBCCS. This is an autosomal dominant condition caused by mutation of PTCH gene on chromosome 9q 35. Patients with this syndrome are prone to developing multiple basal cell carcinomas. In our series, 2 pediatric patients were diagnosed with the NBCCS, also showing a high recurrence rate and, in fact, both patients relapsed 4 times over several years.

8 42 C. RUBINI ET AL. Fig. 3. (A) Solid / multicystic ameloblastoma with a follicular growth pattern. Columnar cells are seen in the peripheral part, while the central part shows stellate reticulum-like cells. (H&E stain, 40X). (B) Unicystic ameloblastoma with a plexiform growth pattern and endoluminal growth (H&E stain, 10X). (C) Peripheral ameloblastoma. Odontogenic epithelium in close proximity to the surface epithelial cells of the mucosa. (immunohistochemical stain for cytokeratin, 20X). (D) Desmoplastic ameloblastoma. Irregularly shaped tumor islands are seen in an abundant collagenous stroma. (H&E stain. 20X). The treatment of choice for this tumor is enucleation and curettage, but an aggressive surgical treatment, consisting in removal of surrounding bone tissue and use of Carnoy s solution, is recommended for odontogenic keratocysts (KOT) in the context of NBBCS, for larger and/ or recurrent lesions. In this study, the recurrence rate was 19.5%, slightly less than that reported by some Authors 36. A 5-year follow-up is recommended, although this tumor may recur 10 to 15 years after removal. Ameloblastoma Ameloblastoma is a benign and slow-growing tumor of odontogenic epithelial origin, without odontogenic ectomesenchyme or its principal product, the dentine. In our series, ameloblastoma was the second most common odontogenic tumor (17.7%). This frequency is significantly lower than that reported by other Authors, confirming that this tumor is less frequent in Caucasians compared to other ethnicities There are four variants of this neoplasm: solid / multicystic type, extraosseous / peripheral type, desmoplastic type and unicystic type (Fig. 4A-D). The solid / multicistic variant is the most common type of ameloblastoma, representing about 1% of head and neck tumors 39. In our series, we found 21 cases of this subtype, without significant differences with unicystic type. These results were consistent with those reported by Buchner et al. 15, while other Authors showed a significantly higher frequency 40. It is a rare tumor in childhood, as confirmed by our results (3 cases), and occurs typically in the posterior region of mandible. Regarding sex predilection, some Authors reported no sex predilection 25, contrary to our results (M:F 2.5:1). Clinically, it is often asymptomatic, even if it becomes large, and pain may be caused by inflammatory changes following superinfection. Due to the fact that this tumor is clinically silent, smaller lesions are often accidentally detected by routine dental examination 41. As for the keratocystic odontogenic tumors, the majority of ameloblastomas (27 of these 33 cases with known size,

9 ODONTOGENIC TUMORS: A RETROSPECTIVE CLINICOPATHOLOGICAL STUDY FROM TWO ITALIAN CENTERS 43 Fig. 4. (A) Odontogenic keratocyst (KOT). Epithelial cells show a typical inversion of apical-to-basal polarity. (B) Odontogenic keratocyst (KOT). On the left of the picture, fibrous connective tissue is devoid of inflammation. On the right side, focal inflammatory infiltrate is present. (C, D) Odontogenic keratocyst (KOT). Satellite cysts ( daughter cysts ) are seen in the wall of the lesion, containing keratin-like material. 73%) were diagnosed when they were 2 cm or larger (Tab. VII). Radiographically, the most typical aspect is a multilocular radiolucent, well-demarcated and sometimes corticated, but it is not diagnostic. Therefore, any radioopacity seen within the lesion, suggesting the presence of mineralized tissue, is inconsistent with a diagnosis of ameloblastoma 42. Macroscopically, the tumor area is grayish and does not contain hard tissue, with sparsely haemorrhagic and necrotic areas (Fig. 5A, 5B). Histologically, the solid/multicystic ameloblastoma consists of odontogenic epithelium with two growth patterns: follicular and plexiform. The first one shows islands or follicles of various size, surrounded by connective tissue, resembling the stellate reticulum of the enamel organ (Fig. 4A). The plexiform pattern is characterized by a network of anastomosing strands of ameloblastic epithelium (Fig. 4B). Although different, these two patterns can coexist in the same lesion in various proportion, and there is no evidence that they differ in their natural history or response to treatments, although some reports suggested a higher tendency for recurrence in follicular rather than in plexiform ameloblastomas 43. Four main cell types are recognized within the histopathological range of the solid/multicystic ameloblastoma: stellate reticulum-like cell type, acanthomatous cell type, granular cell type, and basal cell type 42. In this tumor, the presence of microcysts and squamous metaplasia of the central areas of the epithelium (when this feature is extensive, the term acanthomatous ameloblastoma is applied) can be seen 25. Other histological features, such as horn pearls or mucous cell metaplasia, are seldom detected

10 44 C. RUBINI ET AL. Fig. 5. (A, B) Solid/multicystic ameloblastoma, sagittal and transversal sections, respectively. The tumor shows white-grey areas alternating with sparsely haemorrhagic areas. The unicystic ameloblastoma is a histological variant characterized by an odontogenic cystic neoplasm with a single large lumen with an ameloblastomatous ingrowth. In our series, we found 19 cases of unicystic ameloblastoma, without significant differences with solid / multicystic variant. On the contrary, other Authors showed a significantly lower frequency of this histological type, representing less than 20% of all ameloblastomas Unicystic ameloblastoma is characterized by an earlier age of onset than the other variants. In fact, in accordance with data in the literature, this variant presented in younger patients, although without statistically significant differences (p > 0.05). The reason for the earlier age of onset is probably due to the fact that usually this variant is in association with an unerupted tooth. This feature raises the possibility that unicistic ameloblastoma Fig. 6. (A) Unicystic ameloblastoma showing typical luminal lining of ameloblast-like cells (H&E stain, 40X).(B) Unicystic ameloblastoma with endoluminal growth (H&E stain, 20X).(C) Unicystic ameloblastoma with intramural growth (H&E stain, 10X).

11 ODONTOGENIC TUMORS: A RETROSPECTIVE CLINICOPATHOLOGICAL STUDY FROM TWO ITALIAN CENTERS 45 develops from the dental follicle or from a dentigerous cyst. There is a marked predominance for the posterior part of the mandible. As for the solid/multicystic variant, the jaw swelling is the most common symptom, even though the majority of cases are found during radiographic examinations undertaken for other purposes 47. Radiographically, although all lesions show a unicystic aspect at the histological level, they do not necessarily present as a unilocular lesion on radiograms, but the typical aspect is a well-corticated unilocular radiolucency 48. Histologically, this is an encapsulated fluid-filled cyst lined by ameloblastomatous epithelium. More precisely, the development of ameloblastoma is only detectable in focal areas, while the remaining cystic surface resembles a dentigerous cyst. Three variants have been reported in literature, based upon the relationship of the epithelium to the wall: luminal (or intraepithelial) variant, intraluminal (or plexiform) variant, and mural (or intramural) variant (Fig. 6A-C) In our report, the intramural variant was the most common type, with 10 cases (52.6%), in accordance with Philipsen et al. 47. Desmoplastic ameloblastoma is a rare histological variant, described for the first time in More than 100 cases are reported in the literature, and several studies hypothesized that the relative frequency of this lesion is between 5% and 12% of all ameloblastomas 50. The radiological aspects of this variant are not homogeneous, usually appearing as mixed radiolucent-radiopaque lesions with ill-defined margins. The histopathology resembles that of conventional ameloblastoma in some aspects, with small islands and cords of odontogenic epithelium in a dense stroma, rich in collagen (Fig. 4D). Some studies found increased production of transforming growth factor-β in these lesions, suggesting a role of this cytokine in the development of desmoplasia 51. In our series, only one case of desmoplastic amelobastoma was found. Peripheral odontogenic tumors Mention should be made of peripheral odontogenic tumors, also known as extraosseous odontogenic tumors. They are a rare variant of odontogenic neoplasms that occur in the soft tissues covering the tooth-bearing portion of the jaws 52. They may present as painless, slowgrowing gingival swellings, usually in adult patients. There is little information in the literature regarding the relative frequency of these lesions, due to the rarity of these tumor variants. Buchner et al. reported that these lesions are very rare, accounting for about 0.05% of all biopsy specimens submitted to an oral pathology biopsy service 15. Peripheral odontogenic fibroma is the most common lesion, with more than 180 cases reported in literature, followed by peripheral ameloblastoma 53. On the contrary, in our series ameloblastoma was the most common peripheral lesion, accounting for 8 cases. Noteworthy, 2 cases of peripheral odontogenic mixoma were found in our series. This is an extremely rare tumor, since only a few cases are reported in the literature 54. A consequence of their peripheral location, conservative treatment, such as curettage, is the first choice of these tumor variants. Nevertheless, although very rarely, these tumors may recur. In our series, three cases of recurrent peripheral ameloblastomas have been identified. Conclusions In conclusion, the present study is a population-based survey from central/southern Italy that reflects the distribution of odontogenic tumors. 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