Previously titled: Case Report: Odontogenic carcinosarcoma arising from ameloblastic fibrodentinoma with immunohistochemical profile

Odontogenic carcinosarcoma is an extremely rare mixed malignant odontogenic neoplasm in which both the epithelial and the ectomesenchymal components are cytologically malignant. Owing to the scarcity of the reported cases, the clinical behavior of odontogenic carcinosarcoma remains unexplored. The present work reports a case of odontogenic carcinosarcoma of the mandible with the detailed immuno-histochemical profile. A 28-year-old male presented with a left painless swelling, which recurred after hemimandiblectomy of the lesion. Complete excision of the lesion with a wide safety margin was performed. The histopathological examination revealed cytological malignant features in the follicles as well as in the ectomesenchyme. The immuno-histochemistry demonstrated evidence of epithelial mesenchymal transition. After nine months, the patient demonstrated recurrence in the infra-temporal fossa. Odontogenic carcinosarcoma is a rare highly aggressive malignant odontogenic tumor. Positive expression of smooth muscle actin and vimentin in the epithelial component is a useful aid in the diagnosis of odontogenic carcinosarcoma.


Introduction
Malignant odontogenic tumors are rare group of malignant neoplasms that arise from odontogenic remnants 1 . One of these neoplasms is odontogenic carcinosarcoma (OCS), an extremely rare mixed malignant odontogenic neoplasm in which both the epithelial and the ectomesenchymal components are cytologically malignant 2 . In the 1992 World Health Organization (WHO) classification of tumors, OCS was included in the malignant odontogenic tumors after Tanaka and co-workers (1991) first reported an odontogenic tumor with a mixture of malignant epithelial and ectomesenchymal components 3 . In the 2005 WHO classification, the tumor was removed due to an absence of current diagnostic criteria 4 . OCS has been added again in the 2017 edition because of the availability of cases with adequate diagnostic immunohistochemical and molecular criteria 5,6 . Owing to the scarcity of reported cases -only eleven reported cases in the English literature -OCS clinical behavior remains unexplored 7,8 . In the current work, we report a case of OCS with the detailed clinical, radiographic, histopathological and immunohistochemical description.

Case report
In December 2016, a 28-year-old Egyptian male patient working as an accountant was referred to the Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, Cairo University, with a complaint of painless swelling of six months duration in the left side of his face measuring 7 × 6 cm. The patient had a pathological report of an incisional biopsy, performed outside our institute, which was diagnosed as ameloblastoma. There were no palpable lymph nodes. Intra-oral examination revealed an absence of the lower left molars with a fistula opening on the alveolar crest with no oozing pus.
A cone beam computed tomography examination revealed an ill-defined multilocular osteolytic lesion with fine radiopacities extending from the lower left second premolar up to the ramus.
There was prominent cortical expansion with areas of perforation ( Figure 1). A hemi-mandibulectomy was performed based on the incisional biopsy diagnosis and the pathological fracture of the mandible. The ramus was totally destructed and the tumor was invading the masseter muscle ( Figure 2). Differential diagnosis was made based on the clinical examination, radiographic appearance and gross examination as: ghost cell odontogenic carcinoma, ameloblastic carcinoma, ameloblastic fibrosarcoma, ameloblastic carcinosarcoma, calcifying epithelial odontogenic tumor, atypical type of ameloblastic fibro-dentinoma 9 and ameloblastoma.
Histopathological examination revealed hypercellular follicles and strands of odontogenic epithelium, that were lined peripherally by multilayers of tall columnar ameloblast like cells. The center of the follicles was filled by stellate reticulum like cells, which were basaloid in appearance in some follicles. In between the follicles, there was a highly cellular primitive ectomesenchyme resembling dental papilla. Some of the nuclei of the ectomesenchymal cells were hyperchromatic. In some fields, dentinoid matrix was detected around the epithelial strands ( Figure 3A).Based on the histopathological examination and the case report of Giraddi and Garg 2012 9 , and because it was before the publication of the new WHO classification of 2017, a diagnosis was made of ameloblastic fibro-dentinoma (atypical type).

Amendments from Version 1
We changed the title because it didn't follow the nomenclature stated in the new WHO classification.
We tried to improve the histopathological description of the lesion.
We edited the histopathological Figure 3 & Figure 4 to clarify the features of the lesion.
We repeated the ki67 staining and measured the area percent by leica Qwin software Figure 6.
We contacted the surgeon to improve the follow-up information of the patient.
We tried to improve the discussion as recommended by the reviewer.
We wrote the take-home message in the conclusion section.   After nine months and after the publication of 2017 WHO classification, the patient returned with another large painless swelling in the left temporal and infra-temporal fossa of three months duration. There were no palpable lymph nodes. An oral and maxillofacial surgeon used local anesthesia and an intra-oral approach to completely excise the recurrent lesion ( Figure 2D). Augmentin 1gm tablet/12 hours for five days and anti-inflammatory medication were prescribed for the patient after the surgery. Histopathological examination revealed highly cellular neoplasm, which showed cellular atypia and increased nuclear cytoplasmic ratio in both epithelial and ectomesenchymal components. The typical ameloblastic architecture of the epithelial follicles was lost in some areas. The ectomesenchymal component showed large pleomorphic cells ( Figure 4).
The epithelial nests showed strong membranous positivity with AE1/AE3, while vimentin stained the ectomesenchyme diffusely and the epithelial nests patchily. Alpha-smooth muscle actin (α-SMA) showed positivity in the endothelial cells as well as in scattered epithelial cells ( Figure 5). Ki-67 index was measured by Leica Qwin software and was 7.46% in the epithelial component, while in the ectomesenchymal component it was 2.78% ( Figure 6). Based on the histopathological and immunohistochemical findings, the case was diagnosed as OCS. In 2019 and after nearly a year and a half, the patient came to Oral & Maxillofacial Surgery department for the reconstruction surgery and the examination revealed neither a recurrence nor complications.

Discussion
OCS is a rare biphasic malignant odontogenic neoplasm that has the same architecture of ameloblastic fibroma, in which the epithelial and the ectomesenchymal components are cytologically malignant. It can develop de novo from odontogenic remnants or as a transformation from a preexisting odontogenic benign or malignant neoplasm 6 . This transformation may be attributed to multiple surgical procedures or recurrences of the neoplasm 2 . The present case arose from a preexisting immature odontoma which was formerly called ameloblastic fibrodentinoma. Kunkel et al. Our case was a 28-year-old male with the lesion affecting the posterior mandible. In the English literature, there was a male predilection and only one case that occurred in maxilla 4 . The most common radiographic picture of OCS is ill-defined multilocular radiolucency with cortical perforation 6 . The current case showed multiple radiopacities; may be due to the preexisting immature odontoma.
OCS must be distinguished from ameloblastic fibrosarcoma, in which the ectomesenchymal component only shows cellular   Regarding the clinical behavior of OCS, it is a highly aggressive highly recurrent malignant neoplasm 7 . In our case, the patient encountered recurrence of the lesion after 9 months follow up with no evident metastasis. In the English literature, six out of the eleven cases showed recurrence of the lesion and only 4 cases showed metastasis; whether to lungs or lymph nodes 6 . Kunkel et al. (2004) reported the late metastasis of their case; which was evident about 5 years after the first diagnosis 10 . There were five out of the eleven cases encountered death 6 , one of them was actually due to systemic complication after the resection of the lesion 7 .
The principle line of treatment, as with other malignant odontogenic neoplasms, is surgical resection with a wide safety margin along with neck dissection. However, adjunctive radiotherapy is still a matter of question, and it may be helpful in cases with soft tissue invasion 6 .

Conclusion
OCS is an extremely rare odontogenic malignant neoplasm that shows very aggressive clinical behavior with multiple recurrences and possible metastasis. Close long follow-up is recommended; due to the possible late metastasis of the lesion. Immunohistochemical staining with vimentin, α-SMA and Ki-67 is helpful in the diagnosis of OCS.

Data availability
All data underlying the results are available as part of the article and no additional source data are required.

Consent
Written informed consent for publication of their clinical details or clinical images was obtained from the patieMnt.

John Wright
Department of Diagnostic Sciences, Texas A&M College of Dentistry, Dallas, TX, USA I have reviewed the case report of an odontogenic carcinosarcoma arising from a premature odontoma and I do not agree with the diagnostic interpretation rendered. While the description provided is more supportive of the diagnosis, the illustrations provided do not in my opinion confirm the diagnosis. The illustrations are not of the highest quality. In Fig 3, there is nothing to suggest malignancy and I would question the presence of dentinoid. I would have diagnosed this as ameloblastic fibroma. Fig 4 is not obviously malignant to me either, either component. There is some nuclear hyperchromasia in the epithelial component but this does not appear malignant. I admit there is increased cellularity in the ectomesenchymal component along with some limited cytologic atypia, which is concerning but this component is not obviously malignant to me either. The Ki-67 reactivity is more consistent with a benign diagnosis. I don't think the IHC in Fig 5 adds any evidence for a malignant diagnosis. The cytokeratin and vimentin reactivity would be expected with a benign or malignant diagnosis and the SMA illustrated shows only the vessel walls. At best from the illustrations provided, this might be called an atypical ameloblastic fibroma but I am not convinced either component is malignant and must recommend against its indexing.

Are enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes
Is sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? No Is the case presented with sufficient detail to be useful for other practitioners?

João Paulo Silva Servato
University of Uberaba, Uberaba, Brazil 1) Is the background of the case's history and progression described in sufficient detail?
Partly -In reason to understand OCS progression and outcomes, more follow up details should be addressed.
Moreover, the authors should attend to the new WHO classification nomenclature. As stated by Speight PM, and Takata T., 2018: "the ameloblastic fibrodentinoma and ameloblastic fibroodontoma have been removed as distinct entities" 1 . In this way, the nomenclature should be reviewed.
2) Are enough details provided of any physical examination and diagnostic tests, treatment given and outcomes?
No -Please, improve the details in histopathological and immunohistochemical profile.
2.1) High power field images is needed to improve the recognition of the main expected features, as described by El-Mofty, S. K., in the 2017 WHO blue book "The cells in the sarcomatous component are markedly pleomorphic, with enlarged and bizarre nuclei and occasional multinucleation and mitosis. The epithelial component is frankly malignant, with large hyperchromatic nuclei and an increased N:C ratio. The typical ameloblastic features such as peripheral nuclear palisading and inner stellate reticulum may be lost focally." 2 In the same way, Ki-67 images must be improved to clearly demonstrate the high levels of Ki-67 tagging in both epithelial and mesenchymal components.
3) Is sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment?
Partly, the discussion section should be expanded to access the relevance of this case in the understand of OCS main epidemiological characteristics, histopathological features, the biological profile and its prognosis/ behavior. Moreover, as describe in CARE guidelines, this section must describe: a) Strengths and limitations in your approach to this case. b) The rationale for your conclusions. c) The primary "take-away" lessons from this case report. 4) Is the case presented with sufficient detail to be useful for other practitioners?
No, since it do not attended to most recent WHO classification and to CARE guidelines.

No
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Oral and maxillofacial pathology; Oral medicine; Immunology.
I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

Author Response 15 Dec 2020
Sarah Ahmed Mohamed Mahmoud Badawy, Cairo University, 11553, Egypt First, we can't thank you enough for your sincere and detailed review. We made a second version of the article with the revisions you recommended in your review; as follow: We did contact the surgeon to collect more details about the follow-up of the patient.

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We changed the nomenclature of '' ameloblastic fibrodentinoma'' as you recommended, and clarified the reason why we diagnosed it like that.

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We changed and added histopathological figures as you recommended with more detailed interpretation.

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We repeated the ki67 staining and analysed it with the image analyser Leica QWin software.

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We tried to improve the discussion part as you recommended to follow CARE guidelines.

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We hope that our revisions meet your expectations. Again, thank you for your precious time.
Thank you for your time.
Competing Interests: No competing interests were disclosed.
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