ORIGINAL PAPER
CEOT Variants or Entities: Time for a Rethink? A Case Series with Review of the Literature
B. S. M. S. Siriwardena1,2 · Paul M. Speight1 · Christopher D. Franklin1 · Rasha Abdelkarim1 · Syed Ali Khurram1 · Keith D. Hunter1,3
Received: 7 May 2020 / Accepted: 1 July 2020 / Published online: 8 July 2020
© The Author(s) 2020
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
Jens Pindborg described the calcifying epithelial odonto- genic tumor (CEOT), a rare epithelial odontogenic tumor, in detail in 1958 [1]. Many authorities suggest, however, that the first description was by Thoma and Goldman ten years previously, who termed it adenoid-type adamantoblas- toma [2], although earlier descriptions do exist [3]. Vari- ous synonyms have been used to describe this lesion, such
as adamantoblastoma [4], ameloblastoma of unusual type with calcification [5], malignant odontoma [6], and cystic complex odontoma [7]. In 1963, the term ‘Pindborg tumor’
was first used by Shafer and this is a well-recognized epo- nym for this neoplasm [8]. Twenty years after the original CEOT description, Pindborg and Franklin reviewed 113 cases reported in the literature [9].
Since the original descriptions, the number of cases has 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 males and females and the peak age of occurrence of central lesions was in the 3rd and 4th decades, similar to that pre- sented in our recent series of odontogenic tumors [11]. The majority occurred in the body of the mandible, but some were large lesions, extending widely antero-posteriorly and involving the ramus [10, 11]. Most presentations are intra- osseous but in 1966, Pindborg described an extra-osseous/
peripheral CEOT [12].
* Keith D. Hunter k.hunter@sheffield.ac.uk
1 Unit of Oral and Maxillofacial Medicine, Pathology and Surgery, School of Clinical Dentistry, University of Sheffield, 19 Claremont Crescent, Sheffield S10 2TA, UK
2 Department of Oral Pathology, Faculty of Dental Sciences, University of Peradeniya, Peradeniya, Sri Lanka
3 Oral Pathology and Biology, University of Pretoria, Pretoria, South Africa
Radiologically, CEOTs vary from small, unilocular radio- lucent lesions to extensive multilocular, mixed radio-dense lesions often associated with an impacted tooth (in 61% of central cases [10]). Some authors have considered the pres- ence of radio-opaque flecks in the pericoronal tissues of an impacted tooth (as originally described by Pindborg) as char- acteristic for CEOT [13]. Half of the central lesions show evidence of cortical bone perforation whilst 40% of periph- eral CEOTs have subjacent bone erosion [10]. On Computed tomography (CT) scans, there is diffuse high attenuation, suggesting calcification and/or ossification. On magnetic resonance imaging (MRI), CEOT is a hypointense tumor on T1-weighted images and a mixed hyper intense tumor on T2-weighted images [14]. CT scans and 3D reconstructions may be useful in delineating the extent of the lesion, which is essential for surgical treatment planning [15]. Whilst CEOT is considered a benign epithelial neoplasm, evidence
of clinically aggressive behavior, malignant transformation with multiple recurrences and cases with metastasis have been reported [10, 16].
The histological hallmarks of the “classic” CEOT are sheets of polyhedral epithelial cells with distinct cell bor- ders, prominent intercellular bridges, nuclear pleomorphism, and few mitoses (Fig. 1) [1, 9, 12]. Also common are con- centric calcifications (Liesegang rings) and the presence of deposits of amorphous ‘amyloid-like’ eosinophilic material which stains with Congo Red (Fig. 2) and demonstrates apple-green birefringence on polarization. This material is largely PAS negative prior to calcification [9].
It has been suggested that CEOTs originate from remnants of the dental lamina [17] or stratum intermedium [18]. Two cell types have been demonstrated by electron microscopy:
polyhedral epithelial cells and myoepithelial-like cells con- 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)
bodies, and fine lamina dense filaments [19]. Immunohisto- chemically, the polyhedral cells of CEOT express laminins 1 and 5, cytokeratins, fibronectin and vimentin [20]. High levels of alkaline phosphatase and ATPase localization to the cell membrane are significant findings [21]. The amyloid material has been shown to contain a number of ameloblast associated proteins, most consistently Odontogenic Amelo- blast-Associated Protein (ODAM) [22].
Apart from the classic features, a number of CEOT vari- ants have been reported, with various proportions of clear cells, Langerhans cells and some cases without calcification.
Furthermore, hybrid tumors with adenomatoid odontogenic tumor or ameloblastoma [10, 23, 24], and cystic/microcystic variants have been reported [25, 26]. Ai-Ru et al. proposed a sub-classification comprising four histological patterns, indi- cating that some tumors might show a cribriform appearance without clear cell borders; others may contain multinucle- 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 cases and has not been widely adopted or otherwise assessed in a larger study population.
In this case series, we aimed to review all of our diagno- ses of CEOT in the diagnostic archive (either definitive or in differential diagnosis) and review them in light of the three WHO classifications published during this time (1991, 2005 and 2017) and the current literature on this entity.
Materials and Methods
The diagnostic database of the department of Oral and Max- illofacial Pathology, Charles Clifford Dental Hospital/School of Clinical Dentistry, University of Sheffield, was searched for cases either with the diagnostic code of CEOT (as a definitive diagnosis) or by keyword search where CEOT was raised as a differential diagnosis in more challenging cases from 1975 to 2017. Clinical information including age, gender and location of the tumor were recorded, and plain film radiology was reviewed where available. Very limited clinical follow-up data was available, and none of the cases for which this was available recurred.
Given the passage of time since the original diagnoses in the series (a span of 42 years: and three intervening WHO classifications), the original slides were re-evaluated using contemporary diagnostic criteria, with attention to the 2017 WHO classification of odontogenic lesions [28]. Hematoxy- lin and Eosin and Congo Red stained sections of the selected cases from the database were re-evaluated by 3 experienced OMF Pathologists (PMS, KDH and SAK), and consensus diagnoses recorded. Cases with multiple biopsies (incisional and resection) were considered as single cases.
Immunohistochemical analysis of the expression of Ki67 (Rabbit polyclonal Abcam ab16667 at 1:50; to assess the proliferation fraction) and Amelogenin/AMELX (Rabbit monoclonal, Abcam ab129418 at 1:150; to assess ameloblas- tic differentiation) was conducted on 10 and 8 cases respec- tively, where sufficient formalin fixed paraffin embedded (FFPE) material remained. Slides were dewaxed and rehy- drated before quenching of endogenous peroxidase using H2O2. Heat-induced epitope retrieval in 0.01 M sodium cit- rate was undertaken before blocking with normal serum.
After primary antibody incubation, biotinylated secondary antibodies were used and specific staining demonstrated using the Vector Nova Red kit (Vector Laboratories Inc, Burlingame, CA, USA). Ki67 was assessed as % of cells positive and AMELX expression was assessed using a modi- fied quickscore method [29], with a maximum possible score of 24.
Results
Thirty two cases had been coded as CEOT in the diagnostic database from 1975 to 2017. Histological slides (H&E and Congo Red) were available for 26 cases (Table 1). In one additional case, whilst a differential diagnosis of CEOT was suggested in the incisional biopsy, the resection showed an 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 the 26 cases, 18 were referral/consult cases, so the FFPE blocks were not available for further analysis. In 15 cases, a definitive diagnosis of CEOT had been made, whilst in the remaining 11, it was part of a differential diagnosis.
The age range was 23–74 years with a mean age of 42 ± 2.6 (Table 1). There was an equal gender distribu- tion. 62% occurred in the mandible and, of the mandibular tumors, the majority were in the posterior mandible (54%).
Of those in the maxilla, 3/10 (30%) involved the maxillary sinus. The majority of CEOTs were intraosseous (18/26;
69%), whilst 8 were peripheral lesions (31%). Association with unerupted teeth was not consistently recorded.
Histologically, a variety of appearances were seen and many cases met the criteria for diagnosis originally described by Pindborg (13/26; 50%), but a number of other histological appearances were also observed. Clear cell clus- ters (of varying extent) were observed in 46% (12/26), more commonly in peripheral tumors (6/8; 75%). Out of the total sample, 10 cases had no identifiable calcifications (Table 2).
Three of the cases (7, 24 and 26) contained dentin-like mate- rial (dentinoid).
The relationship of the review diagnoses to the origi- nal diagnoses is presented in Table 2. Of the 26 cases, 14
were confirmed as CEOT (12 “classic” CEOT, and 2 of the clear cell variant of CEOT). In 6 cases, CEOT was part of a differential diagnosis, which variably included 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- noma, and one ameloblastoma with clear cells), and four were odontogenic tumors which were difficult to classify with no consensus achieved.
Immunohistochemistry for Ki67 expression was avail- able for 10 of the cases with a mean of 2.1% of positive cells (SEM = 0.18; range 1–6%; Fig. 3a). This reinforces the concept that despite frequent nuclear and pleomor- phism, the proliferation rate is low. There was no discern- ible pattern of ki67 expression with regard to histological subtype, nor in those cases where a malignant diagnosis was considered. The lowest (1%) and highest (6%) Ki67 expression were both found in “classic” subtypes. AMELX (amelogenin) was expressed in the epithelium in all 8 cases tested, with the histoscore varying between 5 and
18 (Fig. 3b), indicating that this may be of use, similar to ODAM, in demonstrating ameloblastic differentiation in the epithelial cells.
Discussion
A summary of the main histological variants of CEOT, which have been described in the literature, is presented in Table 3 and a summary of the histochemical and immu- nohistochemical staining characteristics of these different cell types is presented in Table 4. In addition to these main variants, others, such as melanin-containing lesions have also been described [24, 30]. The reported variation in clinical outcomes may represent a spectrum of biological behavior in CEOT, but conversely may merely represent a group of heterogeneous entities which have, for various reasons discussed below, been classified together as “vari- ants” of CEOT, which are briefly reviewed below.
Table 1 Demographic and histological data of the cohort of 26 CEOTs
Dx diagnosis
Case no Year of Dx Age Sex Site Central/peripheral
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
Table 2 Histological features of the cohort of 26 CEOTs Case noEpithelium descriptionDistinct cellular outline
Prominent intercellular bridges
Eosino-
philic cytoplasm
Nuclear/cel- lular pleomor- phism
Mitotic figures
Calcifications/ Lieseg
ang rings
AmyloidClear cellsOriginal diagnosisReview consensus diagnosisIHC 1NestsYYYYNYYNTypical CEOTCEOT 2NestsYNYYNNYNTypical CEOTCEOTki67 6% 3Small nests and thin strandsYNYYNNYY (focal)Unusual, maybe CEOT or OD hamartoma
CEOT vs OdF 4NestsYYYYNYYNTypical CEOTCEOT 5Small nests and thin strandsNNYYNY (focal)NY (focal)Unusual, CEOT (preferred) vs OdF
CEOT vs CCOCki67 5% 6Small nests and thin strandsYNYYNNYY (focal)CEOTCEOT vs OdF 7Sheets and thin strandsYNYYNY*NYCEOT, clear cell variantNo consensuski67 < 1% 8Sheets and small nestsYYYYNYYNTypical CEOTCEOTki67 2% 9Sheets and thin strandsNNYYNNYNUnusual OT, CEOT vs OFNo consensus 10Sheets and thin strandsYNYYNYYY (focal)CEOTCEOT, clear cell variantki67 < 1% 11Small nests and thin strandsYNYYNNYNCEOTCEOT vs OdF/ SOCki67 2% 12Small nests and thin strandsYYNYNYYYCEOTCEOT 13Small nestsYNYYN
Y (min)
YYCEOTCEOTki67 < 1% 14Small nestsYYYYNYYY (focal)CEOT, maybe arising from dentigerous cyst
CEOTki67 1% 15Small nestsYYYYNYYNTypical CEOTCEOT 16Sheets and thin strandsYNYYNNNYUnusual, maybe CEOT variantAmeloblastoma with clear cells 17Small nests and thin strandsYFew?YNNNNUnusual, perhaps
non-calcifying CEO
T
No consensus 18Small nests and thin strandsYIn some areasYYNYEquivY (most)Clear cell CEOTCEOT, clear cell variant 19Small nests and thin strandsYYYYNNYNCEOTCEOT 20Small nests
Y (feNYYN w)
Y (feYY (focal)CEOTCEOT vs OdF w)
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,
OT odontogenic tumor, OdF odontogenic fibroma, OC odontogenic carcinoma, CCOC clear cell odontogenic carcinoma, SOC sclerosing odontogenic carcinoma, equiv equivocal *Calcifications assessed as “dentinoid”
Table 2 (continued) Case noEpithelium descriptionDistinct cellular outline
Prominent intercellular bridges
Eosino-
philic cytoplasm
Nuclear/cel- lular pleomor- phism
Mitotic figures
Calcifications/ Lieseg
ang rings
AmyloidClear cellsOriginal diagnosisReview consensus diagnosisIHC 21SheetsYN (few)YYNYYYUnusual, perhaps CEOTNo consensuski67 < 1% 22SheetsYN (few)YYNYYNCEOTCEOT 23Small nests and thin strandsYNYYNNYNCEOTCEOT 24Sheets and thin strandsNNYYN
Y* (feEquivnCEOTCEOT vs OC w)with dentinoid 25Small nests and YNYYNYYNCEOTCEOT thin strands 26Small nests and YNYYNY*YY (most)OT, perhaps CEOT, Clear Cell thin strandsCEOTvariant vs OC with dentinoid
Ki67 < 1%
Fig. 3 Photomicrograph of Ki67 (a) and AMELX expression (b) in a selected CEOT case from the cohort
Table 3 A summary of reported cases and case series of variants of CEOT AuthorsAge/sexLocationRadiographic featuresHistopathological findingsC/P CEOT cystic variant Gopalakrishnan et al. [26]15MLeft posterior maxillaUnilocular radiolucency with radiopacitiesCyst lining varying from NKSSE to thick- ened epithelium with characteristics of CEOT
C Channappa et al. [48]30MLeft posterior maxillaUnilocular radiolucency with calcifications in association with impacted tooth #13Cyst lined by odontogenic epithelium, major- ity with uniform thickness, with classic features of CEOT
C Urias Barreras et al. [49]31MLeft Posterior mandibleUnilocular radiopaque/lucent areaLining of odontogenic epithelium with necrosis, featuring clear cells (PASD posi- tive and osteodentin
C Dantas et al. [50]22MRight posterior mandibleUnilocular, mixed radiodensity lesion, root resorptionMicrocystic lined by typical CEOT with abundant clear cellsC Sánchez-Romero et al. [25]42FRight posterior mandibleWell-defined mixed radiodense lesion in rela- tion to an un-erupted third molarMicrocystic compartments of varying size and occasional clear cells with classic features of CEOT
C CEOT clear cell variant Abrams and Howell [31]50MPosterior mandibleUnilocular mixed radiodense/radiolucentProminent clear cells with classic features of CEOTC Anderson et al. [37]68FLeft mandible molar areaUnilocular radiolucent/radiopacityProminent clear cells with classic features of CEOTC Oikarinen et al. [59]36FMandible Left molar to right premolarMultiloculated radiolucent with radiopaque central regionProminent clear cells with classic features of CEOT. Amyloid diagnosed under electron microscopy
C Yamaguchi et al. [60]36MRight mandible from anterior to premolar regionUnilocular radiolucencyProminent clear cells with classic features of CEOT. PAS positive granules in clear cellsC Ai-Ru et al. [27]64FAnterior mandibleNot recordedProminent clear cells with classic features of CEOTC Asano et al. [43]44FRight maxillaUnilocular, radiolucent area with root resorp- tionIslands that frequently contained clear cells with typical features of CEOTC Schmidt-Westhausen et al. [36]38MRight premolar to left incisor region in mandibleRadiolucency with diffuse radiopacities in part of the lesionCentral necrosis of large epithelial islands and clusters of clear cellsC Hicks et al. [61]59FRight posterior mandibleUnilocular mixed radiolucency and radiopac- ityProminent clear cells with classic features of CEOTC Kumamoto et al. [62]14FRight maxillary 3rd molar regionUnilocular radiolucency impacted upper right 3rd molarProminent clear cells, few mitotic figures and typical features of CEOTC Anavi et al. [33]27MLeft mandibular canine and first premolarUnilocular well-circumscribed radiolucencySheets of clear cells, amyloid and few small oval calcificationsC Germanier et al. [63]44FRight angle of the mandible enclosing the 3rd molarMultiloculated radiolucency with calcifica- tionsClear cells in some places and with typical CEOTC Mohtasham et al. [64]18MRight anterior maxillaRadiolucency with calcificationScattered clear cells with typical features of CEOTC
Table 3 (continued) AuthorsAge/sexLocationRadiographic featuresHistopathological findingsC/P Rangel et al. [32]65MRight mandible between lateral incisor and canineUnilocular radiolucency with radio-opacitiesSignificant portion of cells are clear and other areas with typical features of CEOTC Sahni et al. [65]52MRight maxillaMixed radiodensity lesionAreas of clear cells within epithelial islands and with typical features of CEOTC Chen et al. [66]59FPosterior mandible/ ramusUnilocular radiolucencyNests of clear cells in a pseudoglandular pattern. Other areas with typical features of CEOT
C Turatti et al [67]25FLeft mandibleUnilocular radiolucency with root displace- mentSheets and nests of clear cells with areas of calcifications and amyloidC Rydin et al. [68]40FLeft mandibleUnilocular radiolucency with scattered calcificationsCentral portion of the tumor composed of clear cells and periphery with typical CEOT
C Chatterjee et al. [69]73FLeft maxillary molar regionA large mixed radiodense/RL area spearing maxillary antrumTypical CEOT with Clear cells. PAS positiveC Sabir et al. [70]63FAngle of the mandibleRadiolucent lesion in ramus distal to 3rd molarAlmost all islands are clear cells amyloid in betweenC Júnior et al. [71]42MMandibular symphysial regionUnilocular radiolucency with patchy radio densityMost clusters with clear cells and abundant small calcifications and amyloidC Wertheimer et al. [35]20MRight maxillary gingivaPremolar region cup-shaped areaTypical areas of CEOT with some areas with clear cellsP Ai-Ru et al. [27]
32F 47F
mandibular gingivaNo signs of bone involvementTypical areas of CEOT with some areas with clusters of clear cellsP Houston and Fowler [72]27MGingiva of right posterior mandibleUnderlying bone was normalProminent clear cells with classic features of CEOTP Orsini et al. [73]32MMaxillary gingivaNATypical areas of CEOT with some areas with clusters of clear cellsP Mesquita et al. [74]48FRight maxilla, canine regionNAPolyhedral and clear epithelial cells associ- ated with amyloid-like depositionP Anavi et al. [33]27MLeft mandibleAlveolar crest resorptionSheets of clear cells, focal mild atypia with amyloid in between cells and clustersP de Oliveira et al. [75]43FLesion 1: Left mandible Lesion 2: Left maxillaSuperficial cupping in canine areasome clusters are composed with clear cells with typical features of CEOTP Habibi et al. [76]70FLeft maxillaNormal underlying alveolar boneTypical areas of CEOT with some areas with clusters of clear cellsP Gadodia et al. [77]18MLeft mandibleAlveolar crest resorptionScattered clear cells with classic features of CEOTP CEOT Non-calcified with Langerhans cells Asano et al. [43]44FRight maxillaUnilocular radiolucencyLess cellular, clear cells within polyhedral cell clusters. Birbeck granules seen. No calcification
C
Table 3 (continued) AuthorsAge/sexLocationRadiographic featuresHistopathological findingsC/P Takata et al. [44]58MLeft maxillary canine premolar regionUnilocular radiolucencyScattered small islands of epithelial cells. Within islands many spherical bodies seen. Amyloid present. S-100 positive. Birbeck granules identified
C Wang et al. [78]52FRight maxilla, central incisor canine regionUnilocular radiolucencySmall nests of polyhedral cells and amyloid deposition. Clear cells present. CD1a posi- tive cells are frequent. No calcification
C Wang et al. [79]
38M 39F
Right mandible LefUnilocular radiolucency with patchy radi-Small nests and cords of epithelial cells. t maxillaopacitiesFew clear cells. Amyloid present. CD1a + , Birbeck granules identified. No calcifica- tion
C P
Afroz et al. [40]20FRight maxilla, lateral incisor areaNormal underlying alveolar boneScattered small islands of polygonal cells and occasional clear cells. Amyloid present. No calcifications. Clear cells confirmed as Langerhans cells (S100)
P Chen et al. [45]
40F 58M
MaxillaUnilocular radiolucency with root resorption Multilocular radiolucency with root resorp- tion
Small nests and cords of epithelial islands with some clear cells. Amyloid present, CD1a + , langerin + , No calcification
Both C Tseng et al. [80]24MLeft maxilla, canine premolar areaUnilocular radiolucency with root resorption in canine and premolarStrands and island of epithelial cells and some clear cells. Scant amyloid, CD1a + , No calcification
C Santosh et al. [81]44MLeft anterior maxillaLarge unilocular radiolucencyBland epithelial islands with admixed amyloid. CD1a + cells. No calcification was present
C Combined epithelial odontogenic tumor. CEOT /AOT Damm et al. [18]
18M 15F
MandibleUnilocular predominantly radiolucent, one case with radiopacitiesA cystic tumor lined with areas of typical AOT. And some CEOT-like areasC Bingham et al. [82]14FRight mandibleUnilocular radiolucent lesion related to impacted first premolar toothCystic tumor with multiple intraluminal nodules. Some typical AOT and others are CEOT. Amyloid positive. Calcifications noted
C Takeda and Kudo [83]17FRight maxilla between incisorsUnilocular radiolucent lesion with flakes of radio densitiesEncapsulated solid tumor with areas of typi- cal AOT and CEOT. Amyloid positiveC Siar and Ng [51]
13–28 2M, 3F
3 in maxilla, 2 in mandibleRadiolucent lesionThick walled cystic tumor lined with areas of typical AOT and variable amounts of CEOT-like areas
All C Ledesma et al. [84]
10–21 10F
, 2M9 in maxilla (most canine region), 2 mandibleRadiolucent lesion most related to impacted canine tooth. Some have radiopacitiesTypical AOT areas with CEOT-like areas of variable sizes
11 C 1 P
Miyake et al. [85]16FLeft maxilla, canine regionRadiolucent lesion related to impacted canine toothEncapsulated solid tumor composed with areas of typical AOT and CEOT. Amyloid positive
C
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
Table 3 (continued) AuthorsAge/sexLocationRadiographic featuresHistopathological findingsC/P Rosa et al. [86]17Anterior mandibleUnilocular radiolucent lesion with radio- opacity centrallyA cystic tumor with solid mural nodules with typical AOT and CEOT areas. Amyloid positive
C C Central, P Peripheral
a “poorly differentiated non-calcified CEOT” [41]. Others contained Langerhans cells. Takata et al. reported a case with a histologic appearance consistent with “pattern four”
in the Ai-Ru subtypes of conventional CEOT [44]. It was suggested by Kaushal et al. that the non-calcified variant of CEOT behaves more aggressively than calcified CEOTs [39]. However, this contrasted with suggestions made in previous studies that most non-calcified CEOTs contain Langerhans cells, which may indicate a less aggressive lesion. More research in non-calcified CEOT cases with and without LCs is required to address this issue. There has been recent discussion regarding the nature of these non- calcifying, Langerhans cell-rich lesions [47]. This issue will be explored further later.
Cystic/Microcystic Variant
Recently, a number of reports of cystic and microcystic vari- ants of CEOT have been published. The initial report was of a large cystic lesion in a 15 year-old male, in which the lining demonstrated CEOT features [26]. The lesion was enucleated. A number of similar cases have been reported [48–50], and subsequently, a microcystic variant has also been described [25]. In this lesion, a pseudo-glandular appearance was reported in association with otherwise rather conventional CEOT histology. The natural history of these lesions is not known, but there have been no reports of recur- rences so far.
Table 4 Histochemical and immunohistochemical stains in CEOT
The information has been gathered from references [9, 20–22, 35, 67, 87, 42]
a If not calcified
Epithelial cells Amyloid- like
material Calcification Clear cells Langerhans cell Stromal cells Histochemical stains
Congo red ✔
Thioflavin T ✔
PAS ✔a
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 ✔ ✔ ✔
Combined CEOT‑Adenomatoid Odontogenic Tumor Although it is not a variant of CEOT, Adenomatoid odonto- genic tumor (AOT) is worth mentioning in this context, as some contain CEOT-like areas. AOT is a separate odonto- genic tumor with its own distinctive histological features. In 1983 Damm et al. reported an AOT that contained CEOT- like features and named it ‘combined epithelial odontogenic tumor’ [18]. Philipsen and Reichart reported 24 AOTs with some areas of CEOT-like components [23]. None of these combined AOTs /CEOT were dominated by CEOT- like areas. According to Ng and Siar, the behavior of these forms of AOT was no different from that of the conventional AOT and suggested they were benign hamartomas without any evidence of CEOT-like aggressive behavior, and none recurred [51]. Thus, combined CEOT-AOTs should be man- aged as conventional AOTs.
The designation of these cases as variants of CEOT has resulted in a dramatic widening of the histological spectrum of appearances that fall under the diagnostic umbrella of CEOT, far beyond the original histological description [1].
Furthermore, there are some odontogenic tumors that do not fit very well into the diagnostic criteria of the existing classification. This includes a number of lesions containing dentinoid and dispersed nests of tumor cells within a hyalin- ized stroma, which can share some histological features of CEOT. This raises an important issue as to the usefulness of tumor sub-classifications that develop incrementally, without periodic review of the variations in histological appearances in other tumors and integration of new insights from other molecular features including genomic analyses. It also raises questions regarding the usefulness of historical surveys of variants of this tumor, as, given progress in knowledge of the biology of odontogenic tumors, some variants which have been labelled as part of the CEOT family, may not be so.
In the present report, 26 sequentially accessioned cases from a single Oral and Maxillofacial Pathology Diagnostic Service from 1975 to 2017 have been analyzed. In these cases, diverse histomorphology was seen, but the index diag- nosis was of a CEOT, or CEOT was included in the differen- tial diagnosis. The whole cohort has been reviewed taking into account a number of other entities which have been described since the original diagnoses were made, particu- larly those in the early years of the cohort. In one case the resection specimen showed an odontogenic malignancy, with necrosis, a high mitotic rate and areas of de-differentiation.
We excluded this as there was limited evidence of CEOT in the biopsy or resection. However, this does raise the issue of malignant CEOT, which we did not identify in the review of our diagnostic archive. A small number of individual case reports have been published, most of which show areas of conventional CEOT with associated malignant transforma- tion [16, 52]. A detailed discussion of diagnostic features is
beyond the scope of this review, however, as with ameloblas- tic carcinoma, this is fraught with difficulty. A combination of the use of a proliferation marker, such as Ki67, with his- tological features of malignancy may be useful, but this has not been assessed in a cohort of these lesions.
In our cohort, the “classic” appearance, as described in the initial Pindborg paper [1], was found in only 13/26 cases (50%). In our series, we defined this as a tumor demonstrat- ing the described epithelial features (polyhedral cells with clear boundaries), and containing amyloid, in keeping with the WHO 2017 classification [28]. Other features, such as calcification and nuclear pleomorphism were variably pre- sent. Tumors with these histological features present little difficulty in diagnosis. Two other tumors were diagnosed as clear cell CEOT as, although they were dominated by a clear cell population, they also contained areas of “classic”
CEOT, with amyloid.
The main differential diagnosis to be considered in the tumors with a significant clear cell component is Clear Cell Odontogenic Carcinoma (CCOC). CCOC is an intraosseous malignant neoplasm consisting of sheets, nests and cords of polygonal to round clear cells, usually separated by fibrous septa and often showing peripheral palisading [53]. The lesional clear cells are usually PAS positive, diastase sen- sitive and negative for mucicarmine (mucin). Congo Red (amyloid) is also negative. Histologically, CC-CEOTs that contain few epithelial islands with clear cells in an eosino- philic homogenous stroma need careful investigations in order to confirm them as CEOT. It is mandatory to identify the presence of amyloid for confirmation. Metastatic tumors that contain clear cells are most likely renal cell carcinoma, clear cell breast carcinoma or thyroid carcinoma and, there- fore, immunomarkers such as RCC, CD10, PAX8, ER/PR, TTF-1 are useful [54].
In difficult cases or small biopsies, fluorescence in situ hybridization (FISH) for EWSR1 gene rearrangement can be used to resolve this dilemma. EWSR1 gene rearrange- ment is absent in CEOT, clearly separating CC-CEOT from CCOC. Bilodeau et al. analyzed 12 CCCa and 8 CCOCs for EWSR-ATF1 FISH with 92% and 63% posi- tive respectively. Subsequent Congo Red staining revealed that two of the CCOC that were negative for EWSR1 rear- rangement contained amyloid; therefore these were more likely to be hypocellular CEOTs rather than CCOC with hyalinized stroma [55]. A key element in this analysis is the availability of tissue which has not been decalcified.
Unfortunately, a combination of unavailability of FFPE blocks, very old tissue and a high frequency of decalcifi- cation in our cohort meant that EWRSR1 rearrangement studies were either not possible, or failed, in our cohort.
In cases where a differential diagnosis was agreed after review, four included odontogenic fibroma (OdF) and scle- rosing odontogenic carcinoma as differential diagnoses.
