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CEOT Variants or Entities: Time for a Rethink? A Case Series with Review of the Literature

CEOT Variants or Entities: Time for a Rethink? A Case Series with Review of the Literature The first detailed description of calcifying epithelial odontogenic tumor (CEOT) are ascribed to Jens Pindborg, but this tumor was described some years previously. Subsequently, CEOT was included in the 1971 WHO classification of odontogenic tumors and a since then number of variants have been described, which have added confusion to the diagnostic criteria. We aimed to survey the literature on the variants of CEOT, in parallel with a review of our single institution experience of CEOTs. Cases identified were collated, including available clinical, radiological and histological information and then reviewed, taking into account changes in the understanding and classifications of odontogenic tumors since initial diagnosis. We identified 26 cases from 1975 to 2017 for which histological material was available. Of these, only 13 (50%) showed the “classic” histological appearance, whilst two cases were identified as recognized variants. In 11 cases, other diagnoses or a differential diagnosis were preferred, with no agreed diagnosis in four of these. The proliferation fraction (Ki67) in the 10 cases tested was 2.1% ± 0.18. These findings illustrate the diagnostic challenges in this group of tumors and highlight the gaps in knowledge. Techniques, such as EWSR1 gene cytogenetic analysis, may be helpful in cases with clear cells. However, in other areas of controversy, including the non-calcifying and Langerhans cell rich variants, further investigation, perhaps utilizing sequencing technologies may be needed to refine the classification. Owing to the relative rarity of these lesions it would be beneficial if future work could be pursued as an international collaboration. Keywords Odontogenic · Tumor · CEOT · Calcifying epithelial odontogenic tumor · Clear cell · Amyloid · EWSR1 Introduction and Review of the Literature as adamantoblastoma [4], ameloblastoma of unusual type with calcification [5 ], malignant odontoma [6], and cystic Jens Pindborg described the calcifying epithelial odonto- complex odontoma [7]. In 1963, the term ‘Pindborg tumor’ genic tumor (CEOT), a rare epithelial odontogenic tumor, was first used by Shafer and this is a well-recognized epo- in detail in 1958 [1]. Many authorities suggest, however, nym for this neoplasm [8]. Twenty years after the original that the first description was by Thoma and Goldman ten CEOT description, Pindborg and Franklin reviewed 113 years previously, who termed it adenoid-type adamantoblas- cases reported in the literature [9]. toma [2], although earlier descriptions do exist [3]. Vari- Since the original descriptions, the number of cases has ous synonyms have been used to describe this lesion, such continued to increase and, to date, more than 362 cases have been reported [10]. According to this recent review of pub- lished cases, there was an almost equal distribution among * Keith D. Hunter males and females and the peak age of occurrence of central k.hunter@sheffield.ac.uk lesions was in the 3rd and 4th decades, similar to that pre- Unit of Oral and Maxillofacial Medicine, Pathology sented in our recent series of odontogenic tumors [11]. The and Surgery, School of Clinical Dentistry, University majority occurred in the body of the mandible, but some of Sheffield, 19 Claremont Crescent, Sheffield S10 2TA, UK were large lesions, extending widely antero-posteriorly and Department of Oral Pathology, Faculty of Dental Sciences, involving the ramus [10, 11]. Most presentations are intra- University of Peradeniya, Peradeniya, Sri Lanka osseous but in 1966, Pindborg described an extra-osseous/ Oral Pathology and Biology, University of Pretoria, Pretoria, peripheral CEOT [12]. South Africa Vol:.(1234567890) 1 3 Head and Neck Pathology (2021) 15:186–201 187 Radiologically, CEOTs vary from small, unilocular radio- of clinically aggressive behavior, malignant transformation lucent lesions to extensive multilocular, mixed radio-dense with multiple recurrences and cases with metastasis have lesions often associated with an impacted tooth (in 61% of been reported [10, 16]. central cases [10]). Some authors have considered the pres- The histological hallmarks of the “classic” CEOT are ence of radio-opaque flecks in the pericoronal tissues of an sheets of polyhedral epithelial cells with distinct cell bor- impacted tooth (as originally described by Pindborg) as char- ders, prominent intercellular bridges, nuclear pleomorphism, acteristic for CEOT [13]. Half of the central lesions show and few mitoses (Fig. 1) [1, 9, 12]. Also common are con- evidence of cortical bone perforation whilst 40% of periph- centric calcifications (Liesegang rings) and the presence of eral CEOTs have subjacent bone erosion [10]. On Computed deposits of amorphous ‘amyloid-like’ eosinophilic material tomography (CT) scans, there is diffuse high attenuation, which stains with Congo Red (Fig.  2) and demonstrates suggesting calcification and/or ossification. On magnetic apple-green birefringence on polarization. This material is resonance imaging (MRI), CEOT is a hypointense tumor largely PAS negative prior to calcification [9 ]. on T1-weighted images and a mixed hyper intense tumor on It has been suggested that CEOTs originate from remnants T2-weighted images [14]. CT scans and 3D reconstructions of the dental lamina [17] or stratum intermedium [18]. Two may be useful in delineating the extent of the lesion, which cell types have been demonstrated by electron microscopy: is essential for surgical treatment planning [15]. Whilst polyhedral epithelial cells and myoepithelial-like cells con- CEOT is considered a benign epithelial neoplasm, evidence taining electron-dense tonofilament bundles, electron-dense Fig. 1 Photomicrograph illus- trating the histological features described the original publica- tion by Pindborg [1] Fig. 2 Photomicrograph of the characteristic appearance of CEOT amyloid, as stained by Congo Red (a) 1 3 188 Head and Neck Pathology (2021) 15:186–201 bodies, and fine lamina dense filaments [19]. Immunohisto- Immunohistochemical analysis of the expression of Ki67 chemically, the polyhedral cells of CEOT express laminins (Rabbit polyclonal Abcam ab16667 at 1:50; to assess the 1 and 5, cytokeratins, fibronectin and vimentin [20]. High proliferation fraction) and Amelogenin/AMELX (Rabbit levels of alkaline phosphatase and ATPase localization to monoclonal, Abcam ab129418 at 1:150; to assess ameloblas- the cell membrane are significant findings [21]. The amyloid tic differentiation) was conducted on 10 and 8 cases respec- material has been shown to contain a number of ameloblast tively, where sufficient formalin fixed paraffin embedded associated proteins, most consistently Odontogenic Amelo- (FFPE) material remained. Slides were dewaxed and rehy- blast-Associated Protein (ODAM) [22]. drated before quenching of endogenous peroxidase using Apart from the classic features, a number of CEOT vari- H O . Heat-induced epitope retrieval in 0.01 M sodium cit- 2 2 ants have been reported, with various proportions of clear rate was undertaken before blocking with normal serum. cells, Langerhans cells and some cases without calcification. After primary antibody incubation, biotinylated secondary Furthermore, hybrid tumors with adenomatoid odontogenic antibodies were used and specific staining demonstrated tumor or ameloblastoma [10, 23, 24], and cystic/microcystic using the Vector Nova Red kit (Vector Laboratories Inc, variants have been reported [25, 26]. Ai-Ru et al. proposed a Burlingame, CA, USA). Ki67 was assessed as % of cells sub-classification comprising four histological patterns, indi- positive and AMELX expression was assessed using a modi- cating that some tumors might show a cribriform appearance fied quickscore method [29], with a maximum possible score without clear cell borders; others may contain multinucle- of 24. ated giant cells or cells with abundant eosinophilic cyto- plasm or clear/vacuolated cells with centrally placed nuclei [27]. However, this sub-classification was based on only nine Results cases and has not been widely adopted or otherwise assessed in a larger study population. Thirty two cases had been coded as CEOT in the diagnostic In this case series, we aimed to review all of our diagno- database from 1975 to 2017. Histological slides (H&E and ses of CEOT in the diagnostic archive (either definitive or in Congo Red) were available for 26 cases (Table 1). In one differential diagnosis) and review them in light of the three additional case, whilst a differential diagnosis of CEOT was WHO classifications published during this time (1991, 2005 suggested in the incisional biopsy, the resection showed an and 2017) and the current literature on this entity. unequivocally malignant odontogenic tumor. This case was excluded. A variety of other histochemical (largely PAS) and immunohistochemical stains were available in some cases, conducted as part of the original diagnostic work-up. Of Materials and Methods the 26 cases, 18 were referral/consult cases, so the FFPE blocks were not available for further analysis. In 15 cases, a The diagnostic database of the department of Oral and Max- definitive diagnosis of CEOT had been made, whilst in the illofacial Pathology, Charles Clifford Dental Hospital/School remaining 11, it was part of a differential diagnosis. of Clinical Dentistry, University of Sheffield, was searched The age range was 23–74  years with a mean age of for cases either with the diagnostic code of CEOT (as a 42 ± 2.6 (Table  1). There was an equal gender distribu- definitive diagnosis) or by keyword search where CEOT tion. 62% occurred in the mandible and, of the mandibular was raised as a differential diagnosis in more challenging tumors, the majority were in the posterior mandible (54%). cases from 1975 to 2017. Clinical information including age, Of those in the maxilla, 3/10 (30%) involved the maxillary gender and location of the tumor were recorded, and plain sinus. The majority of CEOTs were intraosseous (18/26; film radiology was reviewed where available. Very limited 69%), whilst 8 were peripheral lesions (31%). Association clinical follow-up data was available, and none of the cases with unerupted teeth was not consistently recorded. for which this was available recurred. Histologically, a variety of appearances were seen Given the passage of time since the original diagnoses in and many cases met the criteria for diagnosis originally the series (a span of 42 years: and three intervening WHO described by Pindborg (13/26; 50%), but a number of other classifications), the original slides were re-evaluated using histological appearances were also observed. Clear cell clus- contemporary diagnostic criteria, with attention to the 2017 ters (of varying extent) were observed in 46% (12/26), more WHO classification of odontogenic lesions [28]. Hematoxy - commonly in peripheral tumors (6/8; 75%). Out of the total lin and Eosin and Congo Red stained sections of the selected sample, 10 cases had no identifiable calcifications (Table  2). cases from the database were re-evaluated by 3 experienced Three of the cases (7, 24 and 26) contained dentin-like mate- OMF Pathologists (PMS, KDH and SAK), and consensus rial (dentinoid). diagnoses recorded. Cases with multiple biopsies (incisional The relationship of the review diagnoses to the origi- and resection) were considered as single cases. nal diagnoses is presented in Table 2. Of the 26 cases, 14 1 3 Head and Neck Pathology (2021) 15:186–201 189 Table 1 Demographic and Case no Year of Dx Age Sex Site Central/peripheral histological data of the cohort of 26 CEOTs 1 1975 32 Male Not known Central 2 1978 38 Female Not known Central 3 1980 50 Female Not known Peripheral 4 1982 38 Male Mid Mandible Central 5 1988 25 Male Mid to post mandible Peripheral 6 1992 23 Male Ant to mid maxilla Peripheral 7 1993 39 Female Mid to post mandible Central 8 1993 31 Female Ant to mid mandible Central 9 1997 44 Male Mid to post mandible Central 10 1998 52 Male Mid mandible Central 11 1999 49 Female Mid maxilla Central 12 2003 32 Female Ant mandible Peripheral 13 2004 69 Female Mid maxilla Central 14 2004 25 Male Maxillary antrum Central 15 2007 48 Female Post mandible Central 16 2008 53 Male Maxillary antrum Central 17 2009 30 Male Post maxilla Central 18 2010 47 Male Mid to post mandible Peripheral 19 2010 27 Female Ant to mid mandible Peripheral 20 2011 46 Male Mid to post maxilla Central 21 2011 49 Male Mid to post mandible Central 22 2012 74 Female Ramus of mandible Central 23 2013 52 Male Mid Mandible Central 24 2015 32 Female Ant maxilla Peripheral 25 2015 55 Female Maxillary antrum Central 26 2016 34 Female Maxillary antrum Peripheral Dx diagnosis were confirmed as CEOT (12 “classic” CEOT, and 2 of 18 (Fig. 3b), indicating that this may be of use, similar to the clear cell variant of CEOT). In 6 cases, CEOT was ODAM, in demonstrating ameloblastic differentiation in part of a differential diagnosis, which variably included the epithelial cells. central odontogenic fibroma, clear cell odontogenic car - cinoma (CCOC), sclerosing odontogenic carcinoma and odontogenic carcinoma with dentinoid. In two cases, other diagnoses were favored (one clear cell odontogenic carci- Discussion noma, and one ameloblastoma with clear cells), and four were odontogenic tumors which were difficult to classify A summary of the main histological variants of CEOT, with no consensus achieved. which have been described in the literature, is presented Immunohistochemistry for Ki67 expression was avail- in Table 3 and a summary of the histochemical and immu- able for 10 of the cases with a mean of 2.1% of positive nohistochemical staining characteristics of these different cells (SEM = 0.18; range 1–6%; Fig. 3a). This reinforces cell types is presented in Table 4. In addition to these main the concept that despite frequent nuclear and pleomor- variants, others, such as melanin-containing lesions have phism, the proliferation rate is low. There was no discern- also been described [24, 30]. The reported variation in ible pattern of ki67 expression with regard to histological clinical outcomes may represent a spectrum of biological subtype, nor in those cases where a malignant diagnosis behavior in CEOT, but conversely may merely represent was considered. The lowest (1%) and highest (6%) Ki67 a group of heterogeneous entities which have, for various expression were both found in “classic” subtypes. AMELX reasons discussed below, been classified together as “vari- (amelogenin) was expressed in the epithelium in all 8 ants” of CEOT, which are briefly reviewed below. cases tested, with the histoscore varying between 5 and 1 3 190 Head and Neck Pathology (2021) 15:186–201 1 3 Table 2 Histological features of the cohort of 26 CEOTs Case no Epithelium Distinct Prominent Eosino- Nuclear/cel- Mitotic Calcifications/ Amyloid Clear cells Original diagnosis Review consensus IHC description cellular intercellular philic lular pleomor- figures Liesegang diagnosis outline bridges cytoplasm phism rings 1 Nests Y Y Y Y N Y Y N Typical CEOT CEOT 2 Nests Y N Y Y N N Y N Typical CEOT CEOT ki67 6% 3 Small nests and Y N Y Y N N Y Y (focal) Unusual, maybe CEOT vs OdF thin strands CEOT or OD hamartoma 4 Nests Y Y Y Y N Y Y N Typical CEOT CEOT 5 Small nests and N N Y Y N Y (focal) N Y (focal) Unusual, CEOT CEOT vs CCOC ki67 5% thin strands (preferred) vs OdF 6 Small nests and Y N Y Y N N Y Y (focal) CEOT CEOT vs OdF thin strands 7 Sheets and thin Y N Y Y N Y* N Y CEOT, clear cell No consensus ki67 < 1% strands variant 8 Sheets and small Y Y Y Y N Y Y N Typical CEOT CEOT ki67 2% nests 9 Sheets and thin N N Y Y N N Y N Unusual OT, No consensus strands CEOT vs OF 10 Sheets and thin Y N Y Y N Y Y Y (focal) CEOT CEOT, clear cell ki67 < 1% strands variant 11 Small nests and Y N Y Y N N Y N CEOT CEOT vs OdF/ ki67 2% thin strands SOC 12 Small nests and Y Y N Y N Y Y Y CEOT CEOT thin strands 13 Small nests Y N Y Y N Y Y Y CEOT CEOT ki67 < 1% (min) 14 Small nests Y Y Y Y N Y Y Y (focal) CEOT, maybe CEOT ki67 1% arising from dentigerous cyst 15 Small nests Y Y Y Y N Y Y N Typical CEOT CEOT 16 Sheets and thin Y N Y Y N N N Y Unusual, maybe Ameloblastoma strands CEOT variant with clear cells 17 Small nests and Y Few ? Y N N N N Unusual, perhaps No consensus thin strands non-calcifying CEOT 18 Small nests and Y In some areas Y Y N Y Equiv Y (most) Clear cell CEOT CEOT, clear cell thin strands variant 19 Small nests and Y Y Y Y N N Y N CEOT CEOT thin strands 20 Small nests Y N Y Y N Y Y Y (focal) CEOT CEOT vs OdF (few) (few) Head and Neck Pathology (2021) 15:186–201 191 Fig. 3 Photomicrograph of Ki67 (a) and AMELX expression (b) in a selected CEOT case from the cohort Clear Cell Variant In 1967, Abrams and Howell described the first case of a CEOT with a clear cell component [31]. Many case reports and series have followed, some of which are summarized in Table 3. Most of the clear cell CEOTs are intraosseous lesions and are most commonly found in the mandible [10]. The mean age is 44 years, which is 8 years older than for conventional CEOT. Unlike conventional CEOT, there is a female predilection and an association with unerupted teeth was found in only six out of the 24 patients, compared with nearly 50% of the conventional CEOTs. It has been sug- gested that clear cell CEOTs are clinically more aggressive as they tend to perforate the cortex and recur more frequently than other CEOT variants [32–34]. In almost all the reported cases, there were areas with histological features of conventional CEOT including poly- hedral sheets of epithelial cells with prominent intercellular bridges, amyloid-like material and calcifications. The clear cells contain PAS positive material which is diastase labile, 1 3 Table 2 (continued) Case no Epithelium Distinct Prominent Eosino- Nuclear/cel- Mitotic Calcifications/ Amyloid Clear cells Original diagnosis Review consensus IHC description cellular intercellular philic lular pleomor- figures Liesegang diagnosis outline bridges cytoplasm phism rings 21 Sheets Y N (few) Y Y N Y Y Y Unusual, perhaps No consensus ki67 < 1% CEOT 22 Sheets Y N (few) Y Y N Y Y N CEOT CEOT 23 Small nests and Y N Y Y N N Y N CEOT CEOT thin strands 24 Sheets and thin N N Y Y N Y* Equiv n CEOT CEOT vs OC strands (few) with dentinoid 25 Small nests and Y N Y Y N Y Y N CEOT CEOT thin strands 26 Small nests and Y N Y Y N Y* Y Y (most) OT, perhaps CEOT, Clear Cell Ki67 < 1% thin strands CEOT variant vs OC with dentinoid OT odontogenic tumor, OdF odontogenic fibroma, OC odontogenic carcinoma, CCOC clear cell odontogenic carcinoma, SOC sclerosing odontogenic carcinoma, equiv equivocal *Calcifications assessed as “dentinoid” 192 Head and Neck Pathology (2021) 15:186–201 1 3 Table 3 A summary of reported cases and case series of variants of CEOT Authors Age/sex Location Radiographic features Histopathological findings C/P CEOT cystic variant Gopalakrishnan et al. [26] 15M Left posterior maxilla Unilocular radiolucency with radiopacities Cyst lining varying from NKSSE to thick- C ened epithelium with characteristics of CEOT Channappa et al. [48] 30M Left posterior maxilla Unilocular radiolucency with calcifications in Cyst lined by odontogenic epithelium, major- C association with impacted tooth #13 ity with uniform thickness, with classic features of CEOT Urias Barreras et al. [49] 31M Left Posterior mandible Unilocular radiopaque/lucent area Lining of odontogenic epithelium with C necrosis, featuring clear cells (PASD posi- tive and osteodentin Dantas et al. [50] 22M Right posterior mandible Unilocular, mixed radiodensity lesion, root Microcystic lined by typical CEOT with C resorption abundant clear cells Sánchez-Romero et al. [25] 42F Right posterior mandible Well-defined mixed radiodense lesion in rela- Microcystic compartments of varying size C tion to an un-erupted third molar and occasional clear cells with classic features of CEOT CEOT clear cell variant Abrams and Howell [31] 50M Posterior mandible Unilocular mixed radiodense/radiolucent Prominent clear cells with classic features of C CEOT Anderson et al. [37] 68F Left mandible molar area Unilocular radiolucent/radiopacity Prominent clear cells with classic features of C CEOT Oikarinen et al. [59] 36F Mandible Left molar to right premolar Multiloculated radiolucent with radiopaque Prominent clear cells with classic features of C central region CEOT. Amyloid diagnosed under electron microscopy Yamaguchi et al. [60] 36M Right mandible from anterior to premolar Unilocular radiolucency Prominent clear cells with classic features of C region CEOT. PAS positive granules in clear cells Ai-Ru et al. [27] 64F Anterior mandible Not recorded Prominent clear cells with classic features of C CEOT Asano et al. [43] 44F Right maxilla Unilocular, radiolucent area with root resorp- Islands that frequently contained clear cells C tion with typical features of CEOT Schmidt-Westhausen et al. [36] 38M Right premolar to left incisor region in Radiolucency with diffuse radiopacities in Central necrosis of large epithelial islands C mandible part of the lesion and clusters of clear cells Hicks et al. [61] 59F Right posterior mandible Unilocular mixed radiolucency and radiopac- Prominent clear cells with classic features of C ity CEOT Kumamoto et al. [62] 14F Right maxillary 3rd molar region Unilocular radiolucency impacted upper right Prominent clear cells, few mitotic figures and C 3rd molar typical features of CEOT Anavi et al. [33] 27M Left mandibular canine and first premolar Unilocular well-circumscribed radiolucency Sheets of clear cells, amyloid and few small C oval calcifications Germanier et al. [63] 44F Right angle of the mandible enclosing the Multiloculated radiolucency with calcifica- Clear cells in some places and with typical C 3rd molar tions CEOT Mohtasham et al. [64] 18M Right anterior maxilla Radiolucency with calcification Scattered clear cells with typical features of C CEOT Head and Neck Pathology (2021) 15:186–201 193 1 3 Table 3 (continued) Authors Age/sex Location Radiographic features Histopathological findings C/P Rangel et al. [32] 65M Right mandible between lateral incisor and Unilocular radiolucency with radio-opacities Significant portion of cells are clear and C canine other areas with typical features of CEOT Sahni et al. [65] 52M Right maxilla Mixed radiodensity lesion Areas of clear cells within epithelial islands C and with typical features of CEOT Chen et al. [66] 59F Posterior mandible/ ramus Unilocular radiolucency Nests of clear cells in a pseudoglandular C pattern. Other areas with typical features of CEOT Turatti et al [67] 25F Left mandible Unilocular radiolucency with root displace- Sheets and nests of clear cells with areas of C ment calcifications and amyloid Rydin et al. [68] 40F Left mandible Unilocular radiolucency with scattered Central portion of the tumor composed C calcifications of clear cells and periphery with typical CEOT Chatterjee et al. [69] 73F Left maxillary molar region A large mixed radiodense/RL area spearing Typical CEOT with Clear cells. PAS positive C maxillary antrum Sabir et al. [70] 63F Angle of the mandible Radiolucent lesion in ramus distal to 3rd Almost all islands are clear cells amyloid in C molar between Júnior et al. [71] 42M Mandibular symphysial region Unilocular radiolucency with patchy radio Most clusters with clear cells and abundant C density small calcifications and amyloid Wertheimer et al. [35] 20M Right maxillary gingiva Premolar region cup-shaped area Typical areas of CEOT with some areas with P clear cells Ai-Ru et al. [27] 32F mandibular gingiva No signs of bone involvement Typical areas of CEOT with some areas with P 47F clusters of clear cells Houston and Fowler [72] 27M Gingiva of right posterior mandible Underlying bone was normal Prominent clear cells with classic features of P CEOT Orsini et al. [73] 32M Maxillary gingiva NA Typical areas of CEOT with some areas with P clusters of clear cells Mesquita et al. [74] 48F Right maxilla, canine region NA Polyhedral and clear epithelial cells associ- P ated with amyloid-like deposition Anavi et al. [33] 27M Left mandible Alveolar crest resorption Sheets of clear cells, focal mild atypia with P amyloid in between cells and clusters de Oliveira et al. [75] 43F Lesion 1: Left mandible Superficial cupping in canine area some clusters are composed with clear cells P Lesion 2: Left maxilla with typical features of CEOT Habibi et al. [76] 70F Left maxilla Normal underlying alveolar bone Typical areas of CEOT with some areas with P clusters of clear cells Gadodia et al. [77] 18M Left mandible Alveolar crest resorption Scattered clear cells with classic features of P CEOT CEOT Non-calcified with Langerhans cells Asano et al. [43] 44F Right maxilla Unilocular radiolucency Less cellular, clear cells within polyhedral C cell clusters. Birbeck granules seen. No calcification 194 Head and Neck Pathology (2021) 15:186–201 1 3 Table 3 (continued) Authors Age/sex Location Radiographic features Histopathological findings C/P Takata et al. [44] 58M Left maxillary canine premolar region Unilocular radiolucency Scattered small islands of epithelial cells. C Within islands many spherical bodies seen. Amyloid present. S-100 positive. Birbeck granules identified Wang et al. [78] 52F Right maxilla, central incisor canine region Unilocular radiolucency Small nests of polyhedral cells and amyloid C deposition. Clear cells present. CD1a posi- tive cells are frequent. No calcification Wang et al. [79] 38M Right mandible Unilocular radiolucency with patchy radi- Small nests and cords of epithelial cells. C 39F Left maxilla opacities Few clear cells. Amyloid present. CD1a + , P Birbeck granules identified. No calcifica- tion Afroz et al. [40] 20F Right maxilla, lateral incisor area Normal underlying alveolar bone Scattered small islands of polygonal cells and P occasional clear cells. Amyloid present. No calcifications. Clear cells confirmed as Langerhans cells (S100) Chen et al. [45] 40F Maxilla Unilocular radiolucency with root resorption Small nests and cords of epithelial islands Both C 58M Multilocular radiolucency with root resorp- with some clear cells. Amyloid present, tion CD1a + , langerin + , No calcification Tseng et al. [80] 24M Left maxilla, canine premolar area Unilocular radiolucency with root resorption Strands and island of epithelial cells and C in canine and premolar some clear cells. Scant amyloid, CD1a + , No calcification Santosh et al. [81] 44M Left anterior maxilla Large unilocular radiolucency Bland epithelial islands with admixed C amyloid. CD1a + cells. No calcification was present Combined epithelial odontogenic tumor. CEOT /AOT Damm et al. [18] 18M Mandible Unilocular predominantly radiolucent, one A cystic tumor lined with areas of typical C 15F case with radiopacities AOT. And some CEOT-like areas Bingham et al. [82] 14F Right mandible Unilocular radiolucent lesion related to Cystic tumor with multiple intraluminal C impacted first premolar tooth nodules. Some typical AOT and others are CEOT. Amyloid positive. Calcifications noted Takeda and Kudo [83] 17F Right maxilla between incisors Unilocular radiolucent lesion with flakes of Encapsulated solid tumor with areas of typi- C radio densities cal AOT and CEOT. Amyloid positive Siar and Ng [51] 13–28 3 in maxilla, 2 in mandible Radiolucent lesion Thick walled cystic tumor lined with areas All C 2M, 3F of typical AOT and variable amounts of CEOT-like areas Ledesma et al. [84] 10–21 9 in maxilla (most canine region), 2 mandible Radiolucent lesion most related to impacted Typical AOT areas with CEOT-like areas of 11 C 10F, 2M canine tooth. Some have radiopacities variable sizes 1 P Miyake et al. [85] 16F Left maxilla, canine region Radiolucent lesion related to impacted canine Encapsulated solid tumor composed with C tooth areas of typical AOT and CEOT. Amyloid positive Head and Neck Pathology (2021) 15:186–201 195 consistent with glycogen, and does not stain with Alcian Blue [35]. This finding is consistent with suggestions that the clear cells form by epithelial cell degradation [36, 37]. Although the presence of typical areas of conventional CEOT, with minor cellular atypia and absence of mitoses helps in diagnosis, special stains and cytogenetics may be helpful in arriving at a final diagnosis. CEOTs with promi- nent clear cells must be diagnosed with caution, as many clear cell neoplasms are malignant and further investigations are needed to exclude clear cell malignancies such as CCOC and other carcinomas with a clear cell component (for exam- ple, of renal or salivary origin) [38]. It is unclear to what extent difficulties in distinguishing clear cell CEOTs from CCOC has contributed to the reported apparent increased aggressiveness of clear cell CEOT. Non‑Calcified and Langerhans Cell‑Rich Variants of CEOT The non-calcified variant of CEOT is the least reported variant (Table  3). To date, eight intraosseous cases and two extraosseous cases of non-calcified CEOT have been reported [39, 40]. The absence of calcification in CEOT may be due to the relative immaturity of the lesion, as long- standing tumors tend to have more calcifications than young, underdeveloped ones [41]. In a study of 19 patients with CEOT by Azevedo et al., the age of patients at the time of diagnosis was linked to the amount of calcification; older patients showing more calcifications [42]. This variant of CEOT usually appears as a radiolucent area on radiographs that may be misdiagnosed as an odontogenic cyst. Many of these cases contain Langerhans cells (LC), which are antigen-presenting immune cells that are normally found in oral epithelium but have also been described in conventional CEOT in small numbers. If abundant, LC-rich lesions are considered a variant of CEOT [43, 44]. They appear histologically as clear cells, which contain Birbeck granules, within the tumor’s conventional pattern of poly- hedral sheets of epithelial cells and amyloid-like material. Five of the cases reported so far were without associated calcification, all of whom presented in patients of Asian origin [45]. However, a Langerhans cell–rich case with cal- cification has been reported in one black individual [46], challenging the concept that ‘all CEOTs with a Langerhans cell component are non-calcified variants’. Diagnosis of this variant is based on either electron microscopic examination of the LC structure or positive staining of LCs for S100 and CD1a [46]. The natural history of this variant is not well described. Histological examination was important in all of the reported cases of non-calcified CEOT, in order to evalu- ate the presence of the classic features of epithelial sheets and amyloid-like material. In one reported case there was 1 3 Table 3 (continued) Authors Age/sex Location Radiographic features Histopathological findings C/P Rosa et al. [86] 17 Anterior mandible Unilocular radiolucent lesion with radio- A cystic tumor with solid mural nodules with C opacity centrally typical AOT and CEOT areas. Amyloid positive C Central, P Peripheral 196 Head and Neck Pathology (2021) 15:186–201 Table 4 Histochemical and immunohistochemical stains in CEOT Epithelial cells Amyloid- like Calcification Clear cells Langerhans cell Stromal cells material Histochemical stains  Congo red ✔  Thioflavin T ✔  PAS ✔  Tryptophan ✔ IHC stains  Pan-cytokeratin ✔ ✔  Cytokeratin cocktail ✔  EGFR ✔  p63 ✔ ✔  CK7 ✔ ✔  CK14 ✔ ✔  CK8 ✔ ✔  CK13 ✔ ✔  CK19 ✔ ✔  Vimentin ✔  Ameloblast-associated protein ✔ ✔ ✔  Amelotin ✔  Ameloblastin ✔  Amelogenin ✔ ✔  S100 protein ✔  CD1a ✔  Langerin ✔  Enamelin ✔  Syndecan-1 ✔ ✔ ✔ (CD138)  E-Cadherin ✔  Amyloid A ✔ ✔ ✔ The information has been gathered from references [9, 20–22, 35, 67, 87, 42] If not calcified a “poorly differentiated non-calcified CEOT” [41]. Others Cystic/Microcystic Variant contained Langerhans cells. Takata et al. reported a case with a histologic appearance consistent with “pattern four” Recently, a number of reports of cystic and microcystic vari- in the Ai-Ru subtypes of conventional CEOT [44]. It was ants of CEOT have been published. The initial report was suggested by Kaushal et al. that the non-calcified variant of a large cystic lesion in a 15 year-old male, in which the of CEOT behaves more aggressively than calcified CEOTs lining demonstrated CEOT features [26]. The lesion was [39]. However, this contrasted with suggestions made in enucleated. A number of similar cases have been reported previous studies that most non-calcified CEOTs contain [48–50], and subsequently, a microcystic variant has also Langerhans cells, which may indicate a less aggressive been described [25]. In this lesion, a pseudo-glandular lesion. More research in non-calcified CEOT cases with appearance was reported in association with otherwise rather and without LCs is required to address this issue. There has conventional CEOT histology. The natural history of these been recent discussion regarding the nature of these non- lesions is not known, but there have been no reports of recur- calcifying, Langerhans cell-rich lesions [47]. This issue will rences so far. be explored further later. 1 3 Head and Neck Pathology (2021) 15:186–201 197 beyond the scope of this review, however, as with ameloblas- Combined CEOTA ‑ denomatoid Odontogenic Tumor tic carcinoma, this is fraught with difficulty. A combination of the use of a proliferation marker, such as Ki67, with his- Although it is not a variant of CEOT, Adenomatoid odonto- genic tumor (AOT) is worth mentioning in this context, as tological features of malignancy may be useful, but this has not been assessed in a cohort of these lesions. some contain CEOT-like areas. AOT is a separate odonto- genic tumor with its own distinctive histological features. In In our cohort, the “classic” appearance, as described in the initial Pindborg paper [1], was found in only 13/26 cases 1983 Damm et al. reported an AOT that contained CEOT- like features and named it ‘combined epithelial odontogenic (50%). In our series, we defined this as a tumor demonstrat- ing the described epithelial features (polyhedral cells with tumor’ [18]. Philipsen and Reichart reported 24 AOTs with some areas of CEOT-like components [23]. None of clear boundaries), and containing amyloid, in keeping with the WHO 2017 classification [28]. Other features, such as these combined AOTs /CEOT were dominated by CEOT- like areas. According to Ng and Siar, the behavior of these calcification and nuclear pleomorphism were variably pre- sent. Tumors with these histological features present little forms of AOT was no different from that of the conventional AOT and suggested they were benign hamartomas without difficulty in diagnosis. Two other tumors were diagnosed as clear cell CEOT as, although they were dominated by a any evidence of CEOT-like aggressive behavior, and none recurred [51]. Thus, combined CEOT-AOTs should be man- clear cell population, they also contained areas of “classic” CEOT, with amyloid. aged as conventional AOTs. The designation of these cases as variants of CEOT has The main differential diagnosis to be considered in the tumors with a significant clear cell component is Clear Cell resulted in a dramatic widening of the histological spectrum of appearances that fall under the diagnostic umbrella of Odontogenic Carcinoma (CCOC). CCOC is an intraosseous malignant neoplasm consisting of sheets, nests and cords of CEOT, far beyond the original histological description [1]. Furthermore, there are some odontogenic tumors that do polygonal to round clear cells, usually separated by fibrous septa and often showing peripheral palisading [53]. The not fit very well into the diagnostic criteria of the existing classification. This includes a number of lesions containing lesional clear cells are usually PAS positive, diastase sen- sitive and negative for mucicarmine (mucin). Congo Red dentinoid and dispersed nests of tumor cells within a hyalin- ized stroma, which can share some histological features of (amyloid) is also negative. Histologically, CC-CEOTs that contain few epithelial islands with clear cells in an eosino- CEOT. This raises an important issue as to the usefulness of tumor sub-classifications that develop incrementally, without philic homogenous stroma need careful investigations in order to confirm them as CEOT. It is mandatory to identify periodic review of the variations in histological appearances in other tumors and integration of new insights from other the presence of amyloid for conr fi mation. Metastatic tumors that contain clear cells are most likely renal cell carcinoma, molecular features including genomic analyses. It also raises questions regarding the usefulness of historical surveys of clear cell breast carcinoma or thyroid carcinoma and, there- fore, immunomarkers such as RCC, CD10, PAX8, ER/PR, variants of this tumor, as, given progress in knowledge of the biology of odontogenic tumors, some variants which have TTF-1 are useful [54]. In difficult cases or small biopsies, fluorescence in situ been labelled as part of the CEOT family, may not be so. In the present report, 26 sequentially accessioned cases hybridization (FISH) for EWSR1 gene rearrangement can be used to resolve this dilemma. EWSR1 gene rearrange- from a single Oral and Maxillofacial Pathology Diagnostic Service from 1975 to 2017 have been analyzed. In these ment is absent in CEOT, clearly separating CC-CEOT from CCOC. Bilodeau et  al. analyzed 12 CCCa and 8 cases, diverse histomorphology was seen, but the index diag- nosis was of a CEOT, or CEOT was included in the differen- CCOCs for EWSR-ATF1 FISH with 92% and 63% posi- tive respectively. Subsequent Congo Red staining revealed tial diagnosis. The whole cohort has been reviewed taking into account a number of other entities which have been that two of the CCOC that were negative for EWSR1 rear- rangement contained amyloid; therefore these were more described since the original diagnoses were made, particu- larly those in the early years of the cohort. In one case the likely to be hypocellular CEOTs rather than CCOC with hyalinized stroma [55]. A key element in this analysis is resection specimen showed an odontogenic malignancy, with necrosis, a high mitotic rate and areas of de-differentiation. the availability of tissue which has not been decalcified. Unfortunately, a combination of unavailability of FFPE We excluded this as there was limited evidence of CEOT in the biopsy or resection. However, this does raise the issue of blocks, very old tissue and a high frequency of decalcifi- cation in our cohort meant that EWRSR1 rearrangement malignant CEOT, which we did not identify in the review of our diagnostic archive. A small number of individual case studies were either not possible, or failed, in our cohort. In cases where a differential diagnosis was agreed after reports have been published, most of which show areas of conventional CEOT with associated malignant transforma- review, four included odontogenic fibroma (OdF) and scle - rosing odontogenic carcinoma as differential diagnoses. tion [16, 52]. A detailed discussion of diagnostic features is 1 3 198 Head and Neck Pathology (2021) 15:186–201 On H&E, these cases resemble “pattern 4” in the subtypes likely require a collaborative international approach to col- described by Ai-Ru et al. [27], with dominance of a fibrous lect sufficiently large cohorts of these cases allow a more stroma component. The difficulties in distinguishing these comprehensive molecular characterization of this group of entities have been recently discussed in the literature and lesions. In this way, more variants may be defined as other are very relevant to addressing the issues of the uncer- entities, whilst the true spectrum of CEOT is established. tain nature of the non-calcifying CEOT variants. As high- Such analysis may also aid in defining the histogenesis of lighted recently by Ide et al. [47], differential diagnosis these lesions. of odontogenic fibroma (OdF) has been raised in these This will not be without its challenges: many of the cases lesions and, indeed, there is much to suggest (including a of CEOT are decalcified, which may significantly com- lack of recurrence) that they may represent odontogenic promise the quality of genomic information which can be fibromas, rather than non-calcifying CEOTs. This is rein- obtained from these specimens. To this end, careful con- forced in the case series reported by Eversole [56], where sideration will have to be given to a concerted international a small number of the 65 OdFs described contained both effort to collect samples which have been optimally col- ODAM positive amyloid and Langerhans cells. It is worth lected, stored and processed. The development of an inter- noting that this issue was raised in the 1971 WHO clas- national prospective database, with associated availability of sification, in relation to the differential diagnosis of non- both fixed and fresh material, which has not undergone harsh calcifying CEOT and cellular OdF [57]. decalcification will be needed, and this could be coordinated We considered sclerosing odontogenic carcinoma as a via various international specialist societies. This will then differential diagnosis in some cases (Table  1). This tumor allow for a program of translational research, which can has now been added to the WHO classification [28], but include multi-omics analyses of these tumors. is somewhat controversial, and clear diagnostic criteria have not been established. Perineural invasion was not Author Contributions KH, PS and CF contributed to the study concep- seen in any of these cases where this was considered as a tion and design. The literature review was undertaken by BSMSS and diagnosis. RA. Material preparation, data collection and analysis were performed Three of these cases contained dentinoid. The signifi- by BSMSS, PS, RA, SAH and KH. The first draft of the manuscript was written by BSMSS and KH and all authors commented on previ- cance of this is unclear, but in two cases, we included ous versions of the manuscript. All authors read and approved the final odontogenic carcinoma with dentinoid in the differential manuscript. diagnosis, as these tumors presented some features similar to the case reports of this entity [58]. In particular, this Funding No external funding was received for this project. was considered in cases where the original diagnosis was rather uncertain, where CEOT was a suggested diagnosis Compliance with Ethical Standards whilst acknowledging the tumor was difficult to classify. This indicates that the classification, and what may be con - Conflict of interest None of the authors have any conflict of interest to declare. sidered to fall within the diagnostic remit of CEOT, may further evolve as other odontogenic entities are described Ethics Approval Approval for the project was granted by West Glasgow and their diagnostic criteria established. Research Ethics Committee (Reference: 08/S0709/70). All cases were pseudoanonymised before analysis. Open Access This article is licensed under a Creative Commons Attri- Conclusion bution 4.0 International License, which permits use, sharing, adapta- tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, The development of diagnostic criteria for a tumor is an provide a link to the Creative Commons licence, and indicate if changes iterative process and the description and acceptance of were made. The images or other third party material in this article are tumor variants is limited to some degree by the lack of included in the article’s Creative Commons licence, unless indicated appropriate molecular tools to confirm or refute the plac- otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not ing of a particular tumor into its place on the classifica- permitted by statutory regulation or exceeds the permitted use, you will tion. The description of a number of the variants of CEOT need to obtain permission directly from the copyright holder. To view a very much falls into this trap. Whilst some of the vari- copy of this licence, visit http://creativ ecommons .or g/licenses/b y/4.0/. ants are most likely true variants of CEOT, it is becoming increasingly apparent that others are most likely a part of the spectrum of other odontogenic entities. 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Combined epithelial odontogenic 2007;38:103–9. tumor-adenomatoid odontogenic tumor and calcifying epithe- lial odontogenic tumor: report of a case. J oral Maxillofac Surg Publisher’s Note Springer Nature remains neutral with regard to 1986;44:574–7. jurisdictional claims in published maps and institutional affiliations. 83. Takeda Y, Kudo K. Adenomatoid odontogenic tumor associated with calcifying epithelial odontogenic tumor. Int J Oral Maxillofac Surg. 1986;15:469–73. 1 3 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Head and Neck Pathology Springer Journals

CEOT Variants or Entities: Time for a Rethink? A Case Series with Review of the Literature

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1936-0568
DOI
10.1007/s12105-020-01200-9
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Abstract

The first detailed description of calcifying epithelial odontogenic tumor (CEOT) are ascribed to Jens Pindborg, but this tumor was described some years previously. Subsequently, CEOT was included in the 1971 WHO classification of odontogenic tumors and a since then number of variants have been described, which have added confusion to the diagnostic criteria. We aimed to survey the literature on the variants of CEOT, in parallel with a review of our single institution experience of CEOTs. Cases identified were collated, including available clinical, radiological and histological information and then reviewed, taking into account changes in the understanding and classifications of odontogenic tumors since initial diagnosis. We identified 26 cases from 1975 to 2017 for which histological material was available. Of these, only 13 (50%) showed the “classic” histological appearance, whilst two cases were identified as recognized variants. In 11 cases, other diagnoses or a differential diagnosis were preferred, with no agreed diagnosis in four of these. The proliferation fraction (Ki67) in the 10 cases tested was 2.1% ± 0.18. These findings illustrate the diagnostic challenges in this group of tumors and highlight the gaps in knowledge. Techniques, such as EWSR1 gene cytogenetic analysis, may be helpful in cases with clear cells. However, in other areas of controversy, including the non-calcifying and Langerhans cell rich variants, further investigation, perhaps utilizing sequencing technologies may be needed to refine the classification. Owing to the relative rarity of these lesions it would be beneficial if future work could be pursued as an international collaboration. Keywords Odontogenic · Tumor · CEOT · Calcifying epithelial odontogenic tumor · Clear cell · Amyloid · EWSR1 Introduction and Review of the Literature as adamantoblastoma [4], ameloblastoma of unusual type with calcification [5 ], malignant odontoma [6], and cystic Jens Pindborg described the calcifying epithelial odonto- complex odontoma [7]. In 1963, the term ‘Pindborg tumor’ genic tumor (CEOT), a rare epithelial odontogenic tumor, was first used by Shafer and this is a well-recognized epo- in detail in 1958 [1]. Many authorities suggest, however, nym for this neoplasm [8]. Twenty years after the original that the first description was by Thoma and Goldman ten CEOT description, Pindborg and Franklin reviewed 113 years previously, who termed it adenoid-type adamantoblas- cases reported in the literature [9]. toma [2], although earlier descriptions do exist [3]. Vari- Since the original descriptions, the number of cases has ous synonyms have been used to describe this lesion, such continued to increase and, to date, more than 362 cases have been reported [10]. According to this recent review of pub- lished cases, there was an almost equal distribution among * Keith D. Hunter males and females and the peak age of occurrence of central k.hunter@sheffield.ac.uk lesions was in the 3rd and 4th decades, similar to that pre- Unit of Oral and Maxillofacial Medicine, Pathology sented in our recent series of odontogenic tumors [11]. The and Surgery, School of Clinical Dentistry, University majority occurred in the body of the mandible, but some of Sheffield, 19 Claremont Crescent, Sheffield S10 2TA, UK were large lesions, extending widely antero-posteriorly and Department of Oral Pathology, Faculty of Dental Sciences, involving the ramus [10, 11]. Most presentations are intra- University of Peradeniya, Peradeniya, Sri Lanka osseous but in 1966, Pindborg described an extra-osseous/ Oral Pathology and Biology, University of Pretoria, Pretoria, peripheral CEOT [12]. South Africa Vol:.(1234567890) 1 3 Head and Neck Pathology (2021) 15:186–201 187 Radiologically, CEOTs vary from small, unilocular radio- of clinically aggressive behavior, malignant transformation lucent lesions to extensive multilocular, mixed radio-dense with multiple recurrences and cases with metastasis have lesions often associated with an impacted tooth (in 61% of been reported [10, 16]. central cases [10]). Some authors have considered the pres- The histological hallmarks of the “classic” CEOT are ence of radio-opaque flecks in the pericoronal tissues of an sheets of polyhedral epithelial cells with distinct cell bor- impacted tooth (as originally described by Pindborg) as char- ders, prominent intercellular bridges, nuclear pleomorphism, acteristic for CEOT [13]. Half of the central lesions show and few mitoses (Fig. 1) [1, 9, 12]. Also common are con- evidence of cortical bone perforation whilst 40% of periph- centric calcifications (Liesegang rings) and the presence of eral CEOTs have subjacent bone erosion [10]. On Computed deposits of amorphous ‘amyloid-like’ eosinophilic material tomography (CT) scans, there is diffuse high attenuation, which stains with Congo Red (Fig.  2) and demonstrates suggesting calcification and/or ossification. On magnetic apple-green birefringence on polarization. This material is resonance imaging (MRI), CEOT is a hypointense tumor largely PAS negative prior to calcification [9 ]. on T1-weighted images and a mixed hyper intense tumor on It has been suggested that CEOTs originate from remnants T2-weighted images [14]. CT scans and 3D reconstructions of the dental lamina [17] or stratum intermedium [18]. Two may be useful in delineating the extent of the lesion, which cell types have been demonstrated by electron microscopy: is essential for surgical treatment planning [15]. Whilst polyhedral epithelial cells and myoepithelial-like cells con- CEOT is considered a benign epithelial neoplasm, evidence taining electron-dense tonofilament bundles, electron-dense Fig. 1 Photomicrograph illus- trating the histological features described the original publica- tion by Pindborg [1] Fig. 2 Photomicrograph of the characteristic appearance of CEOT amyloid, as stained by Congo Red (a) 1 3 188 Head and Neck Pathology (2021) 15:186–201 bodies, and fine lamina dense filaments [19]. Immunohisto- Immunohistochemical analysis of the expression of Ki67 chemically, the polyhedral cells of CEOT express laminins (Rabbit polyclonal Abcam ab16667 at 1:50; to assess the 1 and 5, cytokeratins, fibronectin and vimentin [20]. High proliferation fraction) and Amelogenin/AMELX (Rabbit levels of alkaline phosphatase and ATPase localization to monoclonal, Abcam ab129418 at 1:150; to assess ameloblas- the cell membrane are significant findings [21]. The amyloid tic differentiation) was conducted on 10 and 8 cases respec- material has been shown to contain a number of ameloblast tively, where sufficient formalin fixed paraffin embedded associated proteins, most consistently Odontogenic Amelo- (FFPE) material remained. Slides were dewaxed and rehy- blast-Associated Protein (ODAM) [22]. drated before quenching of endogenous peroxidase using Apart from the classic features, a number of CEOT vari- H O . Heat-induced epitope retrieval in 0.01 M sodium cit- 2 2 ants have been reported, with various proportions of clear rate was undertaken before blocking with normal serum. cells, Langerhans cells and some cases without calcification. After primary antibody incubation, biotinylated secondary Furthermore, hybrid tumors with adenomatoid odontogenic antibodies were used and specific staining demonstrated tumor or ameloblastoma [10, 23, 24], and cystic/microcystic using the Vector Nova Red kit (Vector Laboratories Inc, variants have been reported [25, 26]. Ai-Ru et al. proposed a Burlingame, CA, USA). Ki67 was assessed as % of cells sub-classification comprising four histological patterns, indi- positive and AMELX expression was assessed using a modi- cating that some tumors might show a cribriform appearance fied quickscore method [29], with a maximum possible score without clear cell borders; others may contain multinucle- of 24. ated giant cells or cells with abundant eosinophilic cyto- plasm or clear/vacuolated cells with centrally placed nuclei [27]. However, this sub-classification was based on only nine Results cases and has not been widely adopted or otherwise assessed in a larger study population. Thirty two cases had been coded as CEOT in the diagnostic In this case series, we aimed to review all of our diagno- database from 1975 to 2017. Histological slides (H&E and ses of CEOT in the diagnostic archive (either definitive or in Congo Red) were available for 26 cases (Table 1). In one differential diagnosis) and review them in light of the three additional case, whilst a differential diagnosis of CEOT was WHO classifications published during this time (1991, 2005 suggested in the incisional biopsy, the resection showed an and 2017) and the current literature on this entity. unequivocally malignant odontogenic tumor. This case was excluded. A variety of other histochemical (largely PAS) and immunohistochemical stains were available in some cases, conducted as part of the original diagnostic work-up. Of Materials and Methods the 26 cases, 18 were referral/consult cases, so the FFPE blocks were not available for further analysis. In 15 cases, a The diagnostic database of the department of Oral and Max- definitive diagnosis of CEOT had been made, whilst in the illofacial Pathology, Charles Clifford Dental Hospital/School remaining 11, it was part of a differential diagnosis. of Clinical Dentistry, University of Sheffield, was searched The age range was 23–74  years with a mean age of for cases either with the diagnostic code of CEOT (as a 42 ± 2.6 (Table  1). There was an equal gender distribu- definitive diagnosis) or by keyword search where CEOT tion. 62% occurred in the mandible and, of the mandibular was raised as a differential diagnosis in more challenging tumors, the majority were in the posterior mandible (54%). cases from 1975 to 2017. Clinical information including age, Of those in the maxilla, 3/10 (30%) involved the maxillary gender and location of the tumor were recorded, and plain sinus. The majority of CEOTs were intraosseous (18/26; film radiology was reviewed where available. Very limited 69%), whilst 8 were peripheral lesions (31%). Association clinical follow-up data was available, and none of the cases with unerupted teeth was not consistently recorded. for which this was available recurred. Histologically, a variety of appearances were seen Given the passage of time since the original diagnoses in and many cases met the criteria for diagnosis originally the series (a span of 42 years: and three intervening WHO described by Pindborg (13/26; 50%), but a number of other classifications), the original slides were re-evaluated using histological appearances were also observed. Clear cell clus- contemporary diagnostic criteria, with attention to the 2017 ters (of varying extent) were observed in 46% (12/26), more WHO classification of odontogenic lesions [28]. Hematoxy - commonly in peripheral tumors (6/8; 75%). Out of the total lin and Eosin and Congo Red stained sections of the selected sample, 10 cases had no identifiable calcifications (Table  2). cases from the database were re-evaluated by 3 experienced Three of the cases (7, 24 and 26) contained dentin-like mate- OMF Pathologists (PMS, KDH and SAK), and consensus rial (dentinoid). diagnoses recorded. Cases with multiple biopsies (incisional The relationship of the review diagnoses to the origi- and resection) were considered as single cases. nal diagnoses is presented in Table 2. Of the 26 cases, 14 1 3 Head and Neck Pathology (2021) 15:186–201 189 Table 1 Demographic and Case no Year of Dx Age Sex Site Central/peripheral histological data of the cohort of 26 CEOTs 1 1975 32 Male Not known Central 2 1978 38 Female Not known Central 3 1980 50 Female Not known Peripheral 4 1982 38 Male Mid Mandible Central 5 1988 25 Male Mid to post mandible Peripheral 6 1992 23 Male Ant to mid maxilla Peripheral 7 1993 39 Female Mid to post mandible Central 8 1993 31 Female Ant to mid mandible Central 9 1997 44 Male Mid to post mandible Central 10 1998 52 Male Mid mandible Central 11 1999 49 Female Mid maxilla Central 12 2003 32 Female Ant mandible Peripheral 13 2004 69 Female Mid maxilla Central 14 2004 25 Male Maxillary antrum Central 15 2007 48 Female Post mandible Central 16 2008 53 Male Maxillary antrum Central 17 2009 30 Male Post maxilla Central 18 2010 47 Male Mid to post mandible Peripheral 19 2010 27 Female Ant to mid mandible Peripheral 20 2011 46 Male Mid to post maxilla Central 21 2011 49 Male Mid to post mandible Central 22 2012 74 Female Ramus of mandible Central 23 2013 52 Male Mid Mandible Central 24 2015 32 Female Ant maxilla Peripheral 25 2015 55 Female Maxillary antrum Central 26 2016 34 Female Maxillary antrum Peripheral Dx diagnosis were confirmed as CEOT (12 “classic” CEOT, and 2 of 18 (Fig. 3b), indicating that this may be of use, similar to the clear cell variant of CEOT). In 6 cases, CEOT was ODAM, in demonstrating ameloblastic differentiation in part of a differential diagnosis, which variably included the epithelial cells. central odontogenic fibroma, clear cell odontogenic car - cinoma (CCOC), sclerosing odontogenic carcinoma and odontogenic carcinoma with dentinoid. In two cases, other diagnoses were favored (one clear cell odontogenic carci- Discussion noma, and one ameloblastoma with clear cells), and four were odontogenic tumors which were difficult to classify A summary of the main histological variants of CEOT, with no consensus achieved. which have been described in the literature, is presented Immunohistochemistry for Ki67 expression was avail- in Table 3 and a summary of the histochemical and immu- able for 10 of the cases with a mean of 2.1% of positive nohistochemical staining characteristics of these different cells (SEM = 0.18; range 1–6%; Fig. 3a). This reinforces cell types is presented in Table 4. In addition to these main the concept that despite frequent nuclear and pleomor- variants, others, such as melanin-containing lesions have phism, the proliferation rate is low. There was no discern- also been described [24, 30]. The reported variation in ible pattern of ki67 expression with regard to histological clinical outcomes may represent a spectrum of biological subtype, nor in those cases where a malignant diagnosis behavior in CEOT, but conversely may merely represent was considered. The lowest (1%) and highest (6%) Ki67 a group of heterogeneous entities which have, for various expression were both found in “classic” subtypes. AMELX reasons discussed below, been classified together as “vari- (amelogenin) was expressed in the epithelium in all 8 ants” of CEOT, which are briefly reviewed below. cases tested, with the histoscore varying between 5 and 1 3 190 Head and Neck Pathology (2021) 15:186–201 1 3 Table 2 Histological features of the cohort of 26 CEOTs Case no Epithelium Distinct Prominent Eosino- Nuclear/cel- Mitotic Calcifications/ Amyloid Clear cells Original diagnosis Review consensus IHC description cellular intercellular philic lular pleomor- figures Liesegang diagnosis outline bridges cytoplasm phism rings 1 Nests Y Y Y Y N Y Y N Typical CEOT CEOT 2 Nests Y N Y Y N N Y N Typical CEOT CEOT ki67 6% 3 Small nests and Y N Y Y N N Y Y (focal) Unusual, maybe CEOT vs OdF thin strands CEOT or OD hamartoma 4 Nests Y Y Y Y N Y Y N Typical CEOT CEOT 5 Small nests and N N Y Y N Y (focal) N Y (focal) Unusual, CEOT CEOT vs CCOC ki67 5% thin strands (preferred) vs OdF 6 Small nests and Y N Y Y N N Y Y (focal) CEOT CEOT vs OdF thin strands 7 Sheets and thin Y N Y Y N Y* N Y CEOT, clear cell No consensus ki67 < 1% strands variant 8 Sheets and small Y Y Y Y N Y Y N Typical CEOT CEOT ki67 2% nests 9 Sheets and thin N N Y Y N N Y N Unusual OT, No consensus strands CEOT vs OF 10 Sheets and thin Y N Y Y N Y Y Y (focal) CEOT CEOT, clear cell ki67 < 1% strands variant 11 Small nests and Y N Y Y N N Y N CEOT CEOT vs OdF/ ki67 2% thin strands SOC 12 Small nests and Y Y N Y N Y Y Y CEOT CEOT thin strands 13 Small nests Y N Y Y N Y Y Y CEOT CEOT ki67 < 1% (min) 14 Small nests Y Y Y Y N Y Y Y (focal) CEOT, maybe CEOT ki67 1% arising from dentigerous cyst 15 Small nests Y Y Y Y N Y Y N Typical CEOT CEOT 16 Sheets and thin Y N Y Y N N N Y Unusual, maybe Ameloblastoma strands CEOT variant with clear cells 17 Small nests and Y Few ? Y N N N N Unusual, perhaps No consensus thin strands non-calcifying CEOT 18 Small nests and Y In some areas Y Y N Y Equiv Y (most) Clear cell CEOT CEOT, clear cell thin strands variant 19 Small nests and Y Y Y Y N N Y N CEOT CEOT thin strands 20 Small nests Y N Y Y N Y Y Y (focal) CEOT CEOT vs OdF (few) (few) Head and Neck Pathology (2021) 15:186–201 191 Fig. 3 Photomicrograph of Ki67 (a) and AMELX expression (b) in a selected CEOT case from the cohort Clear Cell Variant In 1967, Abrams and Howell described the first case of a CEOT with a clear cell component [31]. Many case reports and series have followed, some of which are summarized in Table 3. Most of the clear cell CEOTs are intraosseous lesions and are most commonly found in the mandible [10]. The mean age is 44 years, which is 8 years older than for conventional CEOT. Unlike conventional CEOT, there is a female predilection and an association with unerupted teeth was found in only six out of the 24 patients, compared with nearly 50% of the conventional CEOTs. It has been sug- gested that clear cell CEOTs are clinically more aggressive as they tend to perforate the cortex and recur more frequently than other CEOT variants [32–34]. In almost all the reported cases, there were areas with histological features of conventional CEOT including poly- hedral sheets of epithelial cells with prominent intercellular bridges, amyloid-like material and calcifications. The clear cells contain PAS positive material which is diastase labile, 1 3 Table 2 (continued) Case no Epithelium Distinct Prominent Eosino- Nuclear/cel- Mitotic Calcifications/ Amyloid Clear cells Original diagnosis Review consensus IHC description cellular intercellular philic lular pleomor- figures Liesegang diagnosis outline bridges cytoplasm phism rings 21 Sheets Y N (few) Y Y N Y Y Y Unusual, perhaps No consensus ki67 < 1% CEOT 22 Sheets Y N (few) Y Y N Y Y N CEOT CEOT 23 Small nests and Y N Y Y N N Y N CEOT CEOT thin strands 24 Sheets and thin N N Y Y N Y* Equiv n CEOT CEOT vs OC strands (few) with dentinoid 25 Small nests and Y N Y Y N Y Y N CEOT CEOT thin strands 26 Small nests and Y N Y Y N Y* Y Y (most) OT, perhaps CEOT, Clear Cell Ki67 < 1% thin strands CEOT variant vs OC with dentinoid OT odontogenic tumor, OdF odontogenic fibroma, OC odontogenic carcinoma, CCOC clear cell odontogenic carcinoma, SOC sclerosing odontogenic carcinoma, equiv equivocal *Calcifications assessed as “dentinoid” 192 Head and Neck Pathology (2021) 15:186–201 1 3 Table 3 A summary of reported cases and case series of variants of CEOT Authors Age/sex Location Radiographic features Histopathological findings C/P CEOT cystic variant Gopalakrishnan et al. [26] 15M Left posterior maxilla Unilocular radiolucency with radiopacities Cyst lining varying from NKSSE to thick- C ened epithelium with characteristics of CEOT Channappa et al. [48] 30M Left posterior maxilla Unilocular radiolucency with calcifications in Cyst lined by odontogenic epithelium, major- C association with impacted tooth #13 ity with uniform thickness, with classic features of CEOT Urias Barreras et al. [49] 31M Left Posterior mandible Unilocular radiopaque/lucent area Lining of odontogenic epithelium with C necrosis, featuring clear cells (PASD posi- tive and osteodentin Dantas et al. [50] 22M Right posterior mandible Unilocular, mixed radiodensity lesion, root Microcystic lined by typical CEOT with C resorption abundant clear cells Sánchez-Romero et al. [25] 42F Right posterior mandible Well-defined mixed radiodense lesion in rela- Microcystic compartments of varying size C tion to an un-erupted third molar and occasional clear cells with classic features of CEOT CEOT clear cell variant Abrams and Howell [31] 50M Posterior mandible Unilocular mixed radiodense/radiolucent Prominent clear cells with classic features of C CEOT Anderson et al. [37] 68F Left mandible molar area Unilocular radiolucent/radiopacity Prominent clear cells with classic features of C CEOT Oikarinen et al. [59] 36F Mandible Left molar to right premolar Multiloculated radiolucent with radiopaque Prominent clear cells with classic features of C central region CEOT. Amyloid diagnosed under electron microscopy Yamaguchi et al. [60] 36M Right mandible from anterior to premolar Unilocular radiolucency Prominent clear cells with classic features of C region CEOT. PAS positive granules in clear cells Ai-Ru et al. [27] 64F Anterior mandible Not recorded Prominent clear cells with classic features of C CEOT Asano et al. [43] 44F Right maxilla Unilocular, radiolucent area with root resorp- Islands that frequently contained clear cells C tion with typical features of CEOT Schmidt-Westhausen et al. [36] 38M Right premolar to left incisor region in Radiolucency with diffuse radiopacities in Central necrosis of large epithelial islands C mandible part of the lesion and clusters of clear cells Hicks et al. [61] 59F Right posterior mandible Unilocular mixed radiolucency and radiopac- Prominent clear cells with classic features of C ity CEOT Kumamoto et al. [62] 14F Right maxillary 3rd molar region Unilocular radiolucency impacted upper right Prominent clear cells, few mitotic figures and C 3rd molar typical features of CEOT Anavi et al. [33] 27M Left mandibular canine and first premolar Unilocular well-circumscribed radiolucency Sheets of clear cells, amyloid and few small C oval calcifications Germanier et al. [63] 44F Right angle of the mandible enclosing the Multiloculated radiolucency with calcifica- Clear cells in some places and with typical C 3rd molar tions CEOT Mohtasham et al. [64] 18M Right anterior maxilla Radiolucency with calcification Scattered clear cells with typical features of C CEOT Head and Neck Pathology (2021) 15:186–201 193 1 3 Table 3 (continued) Authors Age/sex Location Radiographic features Histopathological findings C/P Rangel et al. [32] 65M Right mandible between lateral incisor and Unilocular radiolucency with radio-opacities Significant portion of cells are clear and C canine other areas with typical features of CEOT Sahni et al. [65] 52M Right maxilla Mixed radiodensity lesion Areas of clear cells within epithelial islands C and with typical features of CEOT Chen et al. [66] 59F Posterior mandible/ ramus Unilocular radiolucency Nests of clear cells in a pseudoglandular C pattern. Other areas with typical features of CEOT Turatti et al [67] 25F Left mandible Unilocular radiolucency with root displace- Sheets and nests of clear cells with areas of C ment calcifications and amyloid Rydin et al. [68] 40F Left mandible Unilocular radiolucency with scattered Central portion of the tumor composed C calcifications of clear cells and periphery with typical CEOT Chatterjee et al. [69] 73F Left maxillary molar region A large mixed radiodense/RL area spearing Typical CEOT with Clear cells. PAS positive C maxillary antrum Sabir et al. [70] 63F Angle of the mandible Radiolucent lesion in ramus distal to 3rd Almost all islands are clear cells amyloid in C molar between Júnior et al. [71] 42M Mandibular symphysial region Unilocular radiolucency with patchy radio Most clusters with clear cells and abundant C density small calcifications and amyloid Wertheimer et al. [35] 20M Right maxillary gingiva Premolar region cup-shaped area Typical areas of CEOT with some areas with P clear cells Ai-Ru et al. [27] 32F mandibular gingiva No signs of bone involvement Typical areas of CEOT with some areas with P 47F clusters of clear cells Houston and Fowler [72] 27M Gingiva of right posterior mandible Underlying bone was normal Prominent clear cells with classic features of P CEOT Orsini et al. [73] 32M Maxillary gingiva NA Typical areas of CEOT with some areas with P clusters of clear cells Mesquita et al. [74] 48F Right maxilla, canine region NA Polyhedral and clear epithelial cells associ- P ated with amyloid-like deposition Anavi et al. [33] 27M Left mandible Alveolar crest resorption Sheets of clear cells, focal mild atypia with P amyloid in between cells and clusters de Oliveira et al. [75] 43F Lesion 1: Left mandible Superficial cupping in canine area some clusters are composed with clear cells P Lesion 2: Left maxilla with typical features of CEOT Habibi et al. [76] 70F Left maxilla Normal underlying alveolar bone Typical areas of CEOT with some areas with P clusters of clear cells Gadodia et al. [77] 18M Left mandible Alveolar crest resorption Scattered clear cells with classic features of P CEOT CEOT Non-calcified with Langerhans cells Asano et al. [43] 44F Right maxilla Unilocular radiolucency Less cellular, clear cells within polyhedral C cell clusters. Birbeck granules seen. No calcification 194 Head and Neck Pathology (2021) 15:186–201 1 3 Table 3 (continued) Authors Age/sex Location Radiographic features Histopathological findings C/P Takata et al. [44] 58M Left maxillary canine premolar region Unilocular radiolucency Scattered small islands of epithelial cells. C Within islands many spherical bodies seen. Amyloid present. S-100 positive. Birbeck granules identified Wang et al. [78] 52F Right maxilla, central incisor canine region Unilocular radiolucency Small nests of polyhedral cells and amyloid C deposition. Clear cells present. CD1a posi- tive cells are frequent. No calcification Wang et al. [79] 38M Right mandible Unilocular radiolucency with patchy radi- Small nests and cords of epithelial cells. C 39F Left maxilla opacities Few clear cells. Amyloid present. CD1a + , P Birbeck granules identified. No calcifica- tion Afroz et al. [40] 20F Right maxilla, lateral incisor area Normal underlying alveolar bone Scattered small islands of polygonal cells and P occasional clear cells. Amyloid present. No calcifications. Clear cells confirmed as Langerhans cells (S100) Chen et al. [45] 40F Maxilla Unilocular radiolucency with root resorption Small nests and cords of epithelial islands Both C 58M Multilocular radiolucency with root resorp- with some clear cells. Amyloid present, tion CD1a + , langerin + , No calcification Tseng et al. [80] 24M Left maxilla, canine premolar area Unilocular radiolucency with root resorption Strands and island of epithelial cells and C in canine and premolar some clear cells. Scant amyloid, CD1a + , No calcification Santosh et al. [81] 44M Left anterior maxilla Large unilocular radiolucency Bland epithelial islands with admixed C amyloid. CD1a + cells. No calcification was present Combined epithelial odontogenic tumor. CEOT /AOT Damm et al. [18] 18M Mandible Unilocular predominantly radiolucent, one A cystic tumor lined with areas of typical C 15F case with radiopacities AOT. And some CEOT-like areas Bingham et al. [82] 14F Right mandible Unilocular radiolucent lesion related to Cystic tumor with multiple intraluminal C impacted first premolar tooth nodules. Some typical AOT and others are CEOT. Amyloid positive. Calcifications noted Takeda and Kudo [83] 17F Right maxilla between incisors Unilocular radiolucent lesion with flakes of Encapsulated solid tumor with areas of typi- C radio densities cal AOT and CEOT. Amyloid positive Siar and Ng [51] 13–28 3 in maxilla, 2 in mandible Radiolucent lesion Thick walled cystic tumor lined with areas All C 2M, 3F of typical AOT and variable amounts of CEOT-like areas Ledesma et al. [84] 10–21 9 in maxilla (most canine region), 2 mandible Radiolucent lesion most related to impacted Typical AOT areas with CEOT-like areas of 11 C 10F, 2M canine tooth. Some have radiopacities variable sizes 1 P Miyake et al. [85] 16F Left maxilla, canine region Radiolucent lesion related to impacted canine Encapsulated solid tumor composed with C tooth areas of typical AOT and CEOT. Amyloid positive Head and Neck Pathology (2021) 15:186–201 195 consistent with glycogen, and does not stain with Alcian Blue [35]. This finding is consistent with suggestions that the clear cells form by epithelial cell degradation [36, 37]. Although the presence of typical areas of conventional CEOT, with minor cellular atypia and absence of mitoses helps in diagnosis, special stains and cytogenetics may be helpful in arriving at a final diagnosis. CEOTs with promi- nent clear cells must be diagnosed with caution, as many clear cell neoplasms are malignant and further investigations are needed to exclude clear cell malignancies such as CCOC and other carcinomas with a clear cell component (for exam- ple, of renal or salivary origin) [38]. It is unclear to what extent difficulties in distinguishing clear cell CEOTs from CCOC has contributed to the reported apparent increased aggressiveness of clear cell CEOT. Non‑Calcified and Langerhans Cell‑Rich Variants of CEOT The non-calcified variant of CEOT is the least reported variant (Table  3). To date, eight intraosseous cases and two extraosseous cases of non-calcified CEOT have been reported [39, 40]. The absence of calcification in CEOT may be due to the relative immaturity of the lesion, as long- standing tumors tend to have more calcifications than young, underdeveloped ones [41]. In a study of 19 patients with CEOT by Azevedo et al., the age of patients at the time of diagnosis was linked to the amount of calcification; older patients showing more calcifications [42]. This variant of CEOT usually appears as a radiolucent area on radiographs that may be misdiagnosed as an odontogenic cyst. Many of these cases contain Langerhans cells (LC), which are antigen-presenting immune cells that are normally found in oral epithelium but have also been described in conventional CEOT in small numbers. If abundant, LC-rich lesions are considered a variant of CEOT [43, 44]. They appear histologically as clear cells, which contain Birbeck granules, within the tumor’s conventional pattern of poly- hedral sheets of epithelial cells and amyloid-like material. Five of the cases reported so far were without associated calcification, all of whom presented in patients of Asian origin [45]. However, a Langerhans cell–rich case with cal- cification has been reported in one black individual [46], challenging the concept that ‘all CEOTs with a Langerhans cell component are non-calcified variants’. Diagnosis of this variant is based on either electron microscopic examination of the LC structure or positive staining of LCs for S100 and CD1a [46]. The natural history of this variant is not well described. Histological examination was important in all of the reported cases of non-calcified CEOT, in order to evalu- ate the presence of the classic features of epithelial sheets and amyloid-like material. In one reported case there was 1 3 Table 3 (continued) Authors Age/sex Location Radiographic features Histopathological findings C/P Rosa et al. [86] 17 Anterior mandible Unilocular radiolucent lesion with radio- A cystic tumor with solid mural nodules with C opacity centrally typical AOT and CEOT areas. Amyloid positive C Central, P Peripheral 196 Head and Neck Pathology (2021) 15:186–201 Table 4 Histochemical and immunohistochemical stains in CEOT Epithelial cells Amyloid- like Calcification Clear cells Langerhans cell Stromal cells material Histochemical stains  Congo red ✔  Thioflavin T ✔  PAS ✔  Tryptophan ✔ IHC stains  Pan-cytokeratin ✔ ✔  Cytokeratin cocktail ✔  EGFR ✔  p63 ✔ ✔  CK7 ✔ ✔  CK14 ✔ ✔  CK8 ✔ ✔  CK13 ✔ ✔  CK19 ✔ ✔  Vimentin ✔  Ameloblast-associated protein ✔ ✔ ✔  Amelotin ✔  Ameloblastin ✔  Amelogenin ✔ ✔  S100 protein ✔  CD1a ✔  Langerin ✔  Enamelin ✔  Syndecan-1 ✔ ✔ ✔ (CD138)  E-Cadherin ✔  Amyloid A ✔ ✔ ✔ The information has been gathered from references [9, 20–22, 35, 67, 87, 42] If not calcified a “poorly differentiated non-calcified CEOT” [41]. Others Cystic/Microcystic Variant contained Langerhans cells. Takata et al. reported a case with a histologic appearance consistent with “pattern four” Recently, a number of reports of cystic and microcystic vari- in the Ai-Ru subtypes of conventional CEOT [44]. It was ants of CEOT have been published. The initial report was suggested by Kaushal et al. that the non-calcified variant of a large cystic lesion in a 15 year-old male, in which the of CEOT behaves more aggressively than calcified CEOTs lining demonstrated CEOT features [26]. The lesion was [39]. However, this contrasted with suggestions made in enucleated. A number of similar cases have been reported previous studies that most non-calcified CEOTs contain [48–50], and subsequently, a microcystic variant has also Langerhans cells, which may indicate a less aggressive been described [25]. In this lesion, a pseudo-glandular lesion. More research in non-calcified CEOT cases with appearance was reported in association with otherwise rather and without LCs is required to address this issue. There has conventional CEOT histology. The natural history of these been recent discussion regarding the nature of these non- lesions is not known, but there have been no reports of recur- calcifying, Langerhans cell-rich lesions [47]. This issue will rences so far. be explored further later. 1 3 Head and Neck Pathology (2021) 15:186–201 197 beyond the scope of this review, however, as with ameloblas- Combined CEOTA ‑ denomatoid Odontogenic Tumor tic carcinoma, this is fraught with difficulty. A combination of the use of a proliferation marker, such as Ki67, with his- Although it is not a variant of CEOT, Adenomatoid odonto- genic tumor (AOT) is worth mentioning in this context, as tological features of malignancy may be useful, but this has not been assessed in a cohort of these lesions. some contain CEOT-like areas. AOT is a separate odonto- genic tumor with its own distinctive histological features. In In our cohort, the “classic” appearance, as described in the initial Pindborg paper [1], was found in only 13/26 cases 1983 Damm et al. reported an AOT that contained CEOT- like features and named it ‘combined epithelial odontogenic (50%). In our series, we defined this as a tumor demonstrat- ing the described epithelial features (polyhedral cells with tumor’ [18]. Philipsen and Reichart reported 24 AOTs with some areas of CEOT-like components [23]. None of clear boundaries), and containing amyloid, in keeping with the WHO 2017 classification [28]. Other features, such as these combined AOTs /CEOT were dominated by CEOT- like areas. According to Ng and Siar, the behavior of these calcification and nuclear pleomorphism were variably pre- sent. Tumors with these histological features present little forms of AOT was no different from that of the conventional AOT and suggested they were benign hamartomas without difficulty in diagnosis. Two other tumors were diagnosed as clear cell CEOT as, although they were dominated by a any evidence of CEOT-like aggressive behavior, and none recurred [51]. Thus, combined CEOT-AOTs should be man- clear cell population, they also contained areas of “classic” CEOT, with amyloid. aged as conventional AOTs. The designation of these cases as variants of CEOT has The main differential diagnosis to be considered in the tumors with a significant clear cell component is Clear Cell resulted in a dramatic widening of the histological spectrum of appearances that fall under the diagnostic umbrella of Odontogenic Carcinoma (CCOC). CCOC is an intraosseous malignant neoplasm consisting of sheets, nests and cords of CEOT, far beyond the original histological description [1]. Furthermore, there are some odontogenic tumors that do polygonal to round clear cells, usually separated by fibrous septa and often showing peripheral palisading [53]. The not fit very well into the diagnostic criteria of the existing classification. This includes a number of lesions containing lesional clear cells are usually PAS positive, diastase sen- sitive and negative for mucicarmine (mucin). Congo Red dentinoid and dispersed nests of tumor cells within a hyalin- ized stroma, which can share some histological features of (amyloid) is also negative. Histologically, CC-CEOTs that contain few epithelial islands with clear cells in an eosino- CEOT. This raises an important issue as to the usefulness of tumor sub-classifications that develop incrementally, without philic homogenous stroma need careful investigations in order to confirm them as CEOT. It is mandatory to identify periodic review of the variations in histological appearances in other tumors and integration of new insights from other the presence of amyloid for conr fi mation. Metastatic tumors that contain clear cells are most likely renal cell carcinoma, molecular features including genomic analyses. It also raises questions regarding the usefulness of historical surveys of clear cell breast carcinoma or thyroid carcinoma and, there- fore, immunomarkers such as RCC, CD10, PAX8, ER/PR, variants of this tumor, as, given progress in knowledge of the biology of odontogenic tumors, some variants which have TTF-1 are useful [54]. In difficult cases or small biopsies, fluorescence in situ been labelled as part of the CEOT family, may not be so. In the present report, 26 sequentially accessioned cases hybridization (FISH) for EWSR1 gene rearrangement can be used to resolve this dilemma. EWSR1 gene rearrange- from a single Oral and Maxillofacial Pathology Diagnostic Service from 1975 to 2017 have been analyzed. In these ment is absent in CEOT, clearly separating CC-CEOT from CCOC. Bilodeau et  al. analyzed 12 CCCa and 8 cases, diverse histomorphology was seen, but the index diag- nosis was of a CEOT, or CEOT was included in the differen- CCOCs for EWSR-ATF1 FISH with 92% and 63% posi- tive respectively. Subsequent Congo Red staining revealed tial diagnosis. The whole cohort has been reviewed taking into account a number of other entities which have been that two of the CCOC that were negative for EWSR1 rear- rangement contained amyloid; therefore these were more described since the original diagnoses were made, particu- larly those in the early years of the cohort. In one case the likely to be hypocellular CEOTs rather than CCOC with hyalinized stroma [55]. A key element in this analysis is resection specimen showed an odontogenic malignancy, with necrosis, a high mitotic rate and areas of de-differentiation. the availability of tissue which has not been decalcified. Unfortunately, a combination of unavailability of FFPE We excluded this as there was limited evidence of CEOT in the biopsy or resection. However, this does raise the issue of blocks, very old tissue and a high frequency of decalcifi- cation in our cohort meant that EWRSR1 rearrangement malignant CEOT, which we did not identify in the review of our diagnostic archive. A small number of individual case studies were either not possible, or failed, in our cohort. In cases where a differential diagnosis was agreed after reports have been published, most of which show areas of conventional CEOT with associated malignant transforma- review, four included odontogenic fibroma (OdF) and scle - rosing odontogenic carcinoma as differential diagnoses. tion [16, 52]. A detailed discussion of diagnostic features is 1 3 198 Head and Neck Pathology (2021) 15:186–201 On H&E, these cases resemble “pattern 4” in the subtypes likely require a collaborative international approach to col- described by Ai-Ru et al. [27], with dominance of a fibrous lect sufficiently large cohorts of these cases allow a more stroma component. The difficulties in distinguishing these comprehensive molecular characterization of this group of entities have been recently discussed in the literature and lesions. In this way, more variants may be defined as other are very relevant to addressing the issues of the uncer- entities, whilst the true spectrum of CEOT is established. tain nature of the non-calcifying CEOT variants. As high- Such analysis may also aid in defining the histogenesis of lighted recently by Ide et al. [47], differential diagnosis these lesions. of odontogenic fibroma (OdF) has been raised in these This will not be without its challenges: many of the cases lesions and, indeed, there is much to suggest (including a of CEOT are decalcified, which may significantly com- lack of recurrence) that they may represent odontogenic promise the quality of genomic information which can be fibromas, rather than non-calcifying CEOTs. This is rein- obtained from these specimens. To this end, careful con- forced in the case series reported by Eversole [56], where sideration will have to be given to a concerted international a small number of the 65 OdFs described contained both effort to collect samples which have been optimally col- ODAM positive amyloid and Langerhans cells. It is worth lected, stored and processed. The development of an inter- noting that this issue was raised in the 1971 WHO clas- national prospective database, with associated availability of sification, in relation to the differential diagnosis of non- both fixed and fresh material, which has not undergone harsh calcifying CEOT and cellular OdF [57]. decalcification will be needed, and this could be coordinated We considered sclerosing odontogenic carcinoma as a via various international specialist societies. This will then differential diagnosis in some cases (Table  1). This tumor allow for a program of translational research, which can has now been added to the WHO classification [28], but include multi-omics analyses of these tumors. is somewhat controversial, and clear diagnostic criteria have not been established. Perineural invasion was not Author Contributions KH, PS and CF contributed to the study concep- seen in any of these cases where this was considered as a tion and design. The literature review was undertaken by BSMSS and diagnosis. RA. Material preparation, data collection and analysis were performed Three of these cases contained dentinoid. The signifi- by BSMSS, PS, RA, SAH and KH. The first draft of the manuscript was written by BSMSS and KH and all authors commented on previ- cance of this is unclear, but in two cases, we included ous versions of the manuscript. All authors read and approved the final odontogenic carcinoma with dentinoid in the differential manuscript. diagnosis, as these tumors presented some features similar to the case reports of this entity [58]. In particular, this Funding No external funding was received for this project. was considered in cases where the original diagnosis was rather uncertain, where CEOT was a suggested diagnosis Compliance with Ethical Standards whilst acknowledging the tumor was difficult to classify. This indicates that the classification, and what may be con - Conflict of interest None of the authors have any conflict of interest to declare. sidered to fall within the diagnostic remit of CEOT, may further evolve as other odontogenic entities are described Ethics Approval Approval for the project was granted by West Glasgow and their diagnostic criteria established. Research Ethics Committee (Reference: 08/S0709/70). All cases were pseudoanonymised before analysis. Open Access This article is licensed under a Creative Commons Attri- Conclusion bution 4.0 International License, which permits use, sharing, adapta- tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, The development of diagnostic criteria for a tumor is an provide a link to the Creative Commons licence, and indicate if changes iterative process and the description and acceptance of were made. The images or other third party material in this article are tumor variants is limited to some degree by the lack of included in the article’s Creative Commons licence, unless indicated appropriate molecular tools to confirm or refute the plac- otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not ing of a particular tumor into its place on the classifica- permitted by statutory regulation or exceeds the permitted use, you will tion. The description of a number of the variants of CEOT need to obtain permission directly from the copyright holder. To view a very much falls into this trap. Whilst some of the vari- copy of this licence, visit http://creativ ecommons .or g/licenses/b y/4.0/. ants are most likely true variants of CEOT, it is becoming increasingly apparent that others are most likely a part of the spectrum of other odontogenic entities. 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