A Case of Ectopic Odontogenic Ghost Cell Tumor: Histogenetic Features of a New Entity

Background: Odontogenic tumors arising from extra-alveolar sites are extremerly rare. Dentinogenic ghost cell tumor (DGCT) is an uncommon odontogenic neoplasm characterized by CTNNB1 mutation, ghost cell appearance, and dentinoid-like calcication. We present a case of an ectopic DGCT arising from a calcifying odontogenic cyst in the oor of the mouth. Case presentation: A 72-year-old man presented with a painless sublingual swelling. Imaging revealed a multi-lobulated, solid-cyst mass on the oor of the mouth. Cytology showed folded epithelial clusters composed of basaloid cells, keratinized material, and dentinoid matrix. Histology also revealed a multi-cystic, cribriform to solid nest. Immunohistochemically, CK19, CK5/6, bcl-2, and p63 were diffuse positive. CTNTTB1 mutation was detected, leading to the nal diagnosis of an ectopic DGCT. There was no recurrence during a 6-month follow-up. Conclusion: This is the rst report to comprehensively describe the clinicopathological features of an ectopic DGCT of odontogenic origin, developing similarly to that of a true odontogenic DGCT. Accurate diagnosis of this rare entity is necessary to avoid overtreatment.

We present a case of ectopic DGCT arising from a COC on the oor of the mouth. This is the rst report of an ectopic DGCT having an odontogenic origin, with a development pathway and precursor lesion similar to that of a true odontogenic DGCT. We believe this report could promote accurate diagnosis of this rare entity.

Case Presentation
A 72-year-old Japanese man with no remarkable medical or family history presented with a painless sublingual swelling discovered during follow-up for myocardial infarction. Clinical examination revealed an elastic mass in the sublingual area covered by normal mucosa. Magnetic resonance imaging (MRI) showed a well-circumscribed lobulated, multi-cystic solid mass located on the oor of the mouth (Fig.  1a). There was no connection between the mass and the gingiva and jaw bone (Fig. 1b). Fine needle aspiration showed folded epithelial clusters with duct-like formation (Fig. 2a). These clusters consisted of basaloid cells lacking prominent nuclear atypia and admixed orange G positive, round material lacking nuclei. Peripheral palisading and some hyalinized dentin-like contents were observed (Fig. 2b, c). Cytologically, a basaloid tumor was suspected, and a diagnosis of "atypia of undetermined signi cance" was considered. Based on the location and cytological features, a sublingual tumor was suspected, and tumor excision was performed. Intraoperative ndings revealed a circumscribed mass with no connection to the alveolar bone and oral oor mucosa.
The surgical specimen was a tan-white, elastic, lobulated solid mass, including multi-small cystic spaces ( Fig. 3a). Histological examination revealed a multi-cystic, solid mass surrounded by a thin brous capsule. The cyst lining, of variable thickness, was composed of squamous-to-polygonal epithelial cells, which translated to the plexiform and cribriform component adjacent to the dentinoid material deposition ( Fig. 3b-d). Both the cyst and solid components had anuclear eosinophilic cells (Fig. 3e). The tumor nest contained basaloid cell proliferation with peripheral palisading (Fig. 3e). Tumor cells showed hyperchromatic nuclei with mild atypia and mitoses (2/10HPF). There was no invasion of the adjacent salivary gland, adipose tissue, lymphovascular, or perineural structures. In the gland-like structure, alcian blue staining showed focal positivity, whereas d-PAS was negative. Immunohistochemically, CK19, CK5/6, bcl-2, and p63 were diffusely positive. Nuclear accumulation of β-catenin was detected (Fig. 3f) and Ki-67 index was 5%. Myoepithelial cell markers, such as S-100, GCDFP, and WT1, were absent.
Immunostaining for ductal markers, such as CK7, was positive in the cyst wall, whereas that for CEA was negative. There were no true ducts composed of ductal cells and myoepithelial or basaloid cells. Nextgeneration sequencing (AmpliSeq Cancer Hotspot Panel V2) revealed missense point mutation in CTNNB1 (p.Ile35Ser, c.104T > G). A nal diagnosis of DGCT associated with COC on the oor of the mouth was established. The resection margin was tumor-free, and no additional treatment was performed postoperatively. The patient was followed up for 6 months with no sign of recurrence on MRI.

Discussion And Conclusion
Odontogenic ghost cell lesions, originally described by Gorlin et al. in 1962 [3], comprise COC, DGCT and GCOC [1,2]. DGCT is considered the solid counterpart of COC and is occasionally associated with it.
These lesions can be classi ed as central (intraosseous) or peripheral (gingival or alveolar mucosal) based on their clinical presentation, and an extraoral or ectopic DGCT is not yet an established entity [1,2].
Clinically, most DGCTs occur in the jaw bone (maxilla:mandible = 1:1) and show benign but locally in ltrating behavior [1,2]. They are more common in men (M:F = 2:1), especially at a younger age (range 11-79 years, mean: 39.7) [1]. The patients usually complain of progressive or slow-growing nodules with swelling, with or without pain [1,6]. Radiologically, DGCT shows a cystic or solid mass with calci cation [7]. In the present case, although the patient was older than the mean age for DGCT occurrence, the imaging ndings were consistent with those reported previously [1,2,7].
To our knowledge, this the rst report to meticulously describe the cytological ndings of DGCT. The cell cluster chie y consisted of basaloid cell proliferation with peripheral palisading. These ndings are consistent with those of basal cell adenoma/carcinoma, and we also suspected salivary gland tumors.
However, calci cation and admixed orange G positive structures without nuclei, similar to ghost cells, are a differential feature and therefore an important cytological feature of DGCT.
The histological features of DGCT include basaloid cell proliferation with ameloblatoma-like epithelial nests resembling the stellate reticulum. Aberrant keratinization was seen with ghost cells having enlarged, polygonal eosinophilic cytoplasm, with or without nuclei, and immature to mature dentinoid or dentino-osteoid structures [1,2]. Findings indicating the odontogenic nature of the tumor and its transition from a COC are important. The neoplastic cells have been shown to be strongly positive for cytokeratin AE1/3, 5, 7, 14, and 19, but negative for vimentin, desmin, SMA, and CD34. The Ki-67 index has been reported to be < 5% [1,2]. These histological ndings are consistent with those of the present case.
Considering the anatomical site and histogenetic features, the most important differential diagnosis in the present case is of basal cell adenoma/carcinoma. However, basal cell adenoma/carcinoma exhibits two-cell morphology consisting of CK7-positive ductal structures and p63, SMA, CK5/6, WT-1, or podoplanin positive myoepithelial/basal cell components, unlike the ndings of the present case. Moreover, all above-mentioned basaloid tumors with ghost cell differentiation lack histological ndings of dentinoid material and precursor COC-like cystic components.
To date, only two cases of ectopic dentinogenic ghost cell-like lesions have been reported. One was a DGCT-like lesion in the ethmoid sinus of an 8-year-old boy [4], and the other was a GCOC-like carcinoma on the oor of the mouth of a 54-year-old man [5]. Both exhibited characteristic odontogenic epithelium proliferation with ghost cells but lacked the anatomic association to the oral and alveolar mucosa and bone on radiological, intraoperative, and pathological examinations. Further, CTNNB1 mutation was detected in the latter case. The clinicopathological features of the present case were similar to those reported previously. Moreover, the characteristic precursor lesion, COC, was detected, with no history of an odontogenic tumor, trauma, or surgery that could have caused tumor dissemination or metastasis.
Based on this clinical, histological, and genetic evidence, a nal diagnosis of extraosseous DGCT arising from a COC in the oor of mouth was con rmed.
The development of DGCT occurs through two major pathways: de novo or from a preceding COC. However, the true etiology of an extraosseous DGCT remains unclear [4,5,15]. Peripheral DGCT can originate from oral epithelium following trauma or exposure to an irritating agent [6,15]. In the present case, these factors were absent, and the lesion had no connection with the oral mucosa. Therefore, ectopic odontogenic epithelium may have been associated with the tumor's development.
The recurrence rates of central and peripheral DGCT are 73% and 0%, respectively [1]. While segmental resection is indicated for central DGCT, simple excision is recommended for peripheral DGCT. As an ectopic DGCT is extremely rare, the tumor aggressiveness and optimal treatment are unknown. Liu et al. [4] reported no recurrence of an ectopic DGCT arising from the ethmoid sinus after endoscopic sinus surgery, during a 2-year follow-up. Similar to our ndings, they observed that the Ki-67 labeling index was not high, and there was no invasion of the adjacent tissue, vascular, and perineural structures, suggestive of low malignant potential of ectopic DGCT. In our opinion, simple excision of the tumor is therefore justi ed, and further studies are needed to clarify the nature of the tumor.
This report described a case of DGCT occurring as an ectopic lesion. Despite characteristic histological features, its diagnosis is di cult. Comprehensive clinicopathological examination is important to accurately identify this rare entity to avoid misdiagnosis and overtreatment.

List Of Abbreviations
COC, calcifying odontogenic cyst; DGCT, dentinogenic ghost cell tumor; GCOC, ghost cell odontogenic carcinoma; HE, hematoxylin and eosin; MRI, magentic resonance imaging Declarations Ethics approval and consent to participate This brief report was conducted in accordance with the Declaration of Helsinki, and the study protocol was approved by the Institutional Review Board of Kansai Medical University Hospital (Approval no.: 160954). Written informed consent was obtained from the patient.

Consent for publication
Written informed consent was obtained from the patient for the publication of this report.

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Competing interests
The authors declare that they have no competing interests.