Ameloblastic fibrosarcoma: clinicopathological and molecular analysis of seven cases highlighting frequent BRAF and occasional NRAS mutations

Ameloblastic fibrosarcoma (AFS) is an aggressive odontogenic neoplasm featuring malignant mesenchymal stroma in addition to an ameloblastic epithelial component, and is hence considered to be the malignant counterpart of ameloblastic fibroma (AF). AFS is exceedingly rare, with <110 cases having been reported so far. Although BRAF mutations are recognised driver mutations in ameloblastoma, the molecular pathogenesis of AFS remains elusive.

Ameloblastic fibrosarcoma: clinicopathological and molecular analysis of seven cases highlighting frequent BRAF and occasional NRAS mutations Aims: Ameloblastic fibrosarcoma (AFS) is an aggressive odontogenic neoplasm featuring malignant mesenchymal stroma in addition to an ameloblastic epithelial component, and is hence considered to be the malignant counterpart of ameloblastic fibroma (AF). AFS is exceedingly rare, with <110 cases having been reported so far. Although BRAF mutations are recognised driver mutations in ameloblastoma, the molecular pathogenesis of AFS remains elusive. Methods and results: We herein describe seven AFSs that were analysed, for the first time, for mutations in the BRAF-NRAS pathway. The patients were four females and three males aged 23-57 years (median, 26 years). Three tumours developed after one or multiple recurrences of AF (4-20 years after initial diagnosis), two showed transition from AF-like bland areas, and two developed de novo. All patients were treated with surgery; adjuvant chemotherapy was given to one patient. At the last follow-up, five patients were alive and well (19-344 months). The remainder were lost to follow-up. Histological examination showed variable sarcomatous overgrowth with varying degrees of atypia and increased mitotic activity. The epithelial component varied greatly according to the degree of sarcomatous overgrowth. Molecular testing revealed BRAF V600E mutations in five cases and NRAS p.Gln61Lys mutation in one case. One tumour was wild-type. Conclusion: To our knowledge, this is the first study on BRAF/NRAS mutations in AFS. Given the activity of RAF and MEK inhibitors across different cancers harbouring V600E mutations, our data strongly suggest that all AFS cases should be genetically tested, and that targeted treatment approaches for this extremely rare sarcoma subtype should be clinically investigated.
Keywords: ameloblastic fibroma, ameloblastic fibrosarcoma, BRAF, molecular pathogenesis, NRAS, targeted therapy Introduction Ameloblastic fibrosarcoma (AFS) is an aggressive neoplasm of odontogenic origin that features malignant mesenchymal spindle cell stroma in addition to an ameloblastic (ameloblastoma-like) epithelial component. 1 By definition, the epithelial component is histologically benign, in contrast to the exceedingly rare ameloblastic carcinosarcoma. 2 AFS is exceedingly rare with <110 cases reported so far. 1,3 AFS represents 0.3% of all odontogenic tumours and 24% of malignant odontogenic tumours. 4 AFS is considered to be the malignant counterpart of ameloblastic fibroma (AF). Indeed, sarcomatous overgrowth and a frankly malignant stromal appearance are the only distinguishing features between AF and AFS. 1 Although frequent BRAF (and rarely also NRAS and KRAS) mutations have been recognised as driver mutations in ameloblastoma, 5-7 the molecular pathogenesis of AFS has remained elusive. In this study, we describe a series of seven previously unreported AFS cases with an emphasis on the mutation status of the RAF-RAS pathway.

Materials and methods
The seven cases were retrieved from the consultation files of the authors (A.A., A.F., A.S., and D.B.) and from the surgical pathology files of the University Dental Hospital, Riyadh, Saudi Arabia. Samples were used in accordance with ethical guidelines for the use of retrospective tissue samples provided by the local ethics committee of the Friedrich-Alexander University Erlangen-Nuremberg (ethics committee statements 24.01.2005 and 18.01.2012). Histological diagnosis was based on criteria defined in the most recent World Health Organization classification of head and neck tumours. 1 Assessment of the proliferation fraction was performed with Ki67 (mouse monoclonal anti-human Ki67 antibody; clone MIB-1, dilution 1:100; Dako, Hamburg, Germany). Only unequivocal nuclear staining was considered to be positive. The proliferation fraction (Ki67 index) was counted separately for the bland-looking component

C L I N I C A L F E A T U R E S
All tumours presented as progressively enlarging masses (Table 1). One tumour presented during pregnancy (at 2 months of gestation) and was resected postpartum. Two tumours developed following recurrent AF; one 6 years after initial resection of AF, and the other after two recurrences of AF (47 months). One patient (27 years old) developed AFS after three recurrences of an unspecified ameloblastic tumor since age 7. Four of the six tumours with detailed information were located in the mandible, and two were located in the maxilla. All patients underwent surgical resection with clear margins. One patient received unspecified adjuvant chemotherapy. Followup was available for five patients, and ranged from 19 months to 344 months (median, 37 months); all were alive without recurrence or metastases. Two patients were lost to follow-up after surgery.  Figure 1C). A variable, but no more than mild, degree of atypia with occasional mitoses was seen in the epithelial component, but overt features of malignancy were lacking ( Figure 1C). By contrast, the stromal component was predominant and showed overt features of malignancy, including significant cellularity, at least moderate nuclear atypia, and high mitotic activity, and was in some areas indistinguishable from undifferentiated spindle cell sarcoma (Figures 1D-F and 2A-C). Focally, a more rounded cell morphology and variable myxoid stromal changes were noted ( Figure 2C,D). Mitotic activity averaged >5 mitoses in 10 high-power fields in all cases. The Ki67 fraction could be determined in the benign-looking and sarcomatous components in four tumours. It showed a striking difference between the AF-like (range, 1-10%; mean, 4%) and the sarcomatous (range, 45-90%; mean, 67%) areas ( Figure 2E,F).
BRAF mutations were detected in five cases, and all represented the known V600E mutation. One case was analysed separately for stromal and epithelial components, and both components showed V600E mutation. The BRAF V600E mutation-specific antibody VE1 was tried on all cases, but, probably because of the decalcification process, staining was assessable in only one case (case 2); it revealed specific staining only in the stromal component (Figure 3), confirming BRAF mutation as driver of the malignant stromal component, and suggesting that the mutation detected in the epithelial component might be due to contamination with the malignant stroma: this needs verification by the use of laser-microdissected samples in future studies. One case showed the NRAS p.Gln61Lys mutation. The seventh case (the only paediatric one in this series) was wild-type. BRAF and NRAS mutations were found to be mutually exclusive in this study.

Discussion
The precise subtyping of mixed epithelial-mesenchymal odontogenic neoplasms is mainly based on the absence (AF) or presence (odontogenic sarcomas) of morphological features of malignancy in the stromal component. Odontogenic sarcomas are further subdivided on the basis of the absence (AFS) or presence of specialised hard substance (fibrodentinosarcoma and ameloblastic fibro-odontosarcoma). 1 AFS is considered to be the malignant counterpart of AF. 1 AF is rare accounting for 2% of odontogenic tumours. It is composed of odontogenic ectomesenchyme (recapitulating the dental papilla) admixed with ameloblastic epithelial nests and strands. AFS is distinguished from AF by an overtly malignant stromal component defined by high cellularity, unequivocal nuclear atypia, and increased mitoses. 1 Approximately 25% of AFS cases originate from recurrent AF. 3 According to a recent review the mean interval between treatment of AF and the development of AFS was 55.1 months. 3 However, it is possible that the literature overestimates the rates of recurrence and/or malignant transformation of AF, as unusual and, particularly, more aggressive cases are more likely to be reported.
Owing to its rarity, AFS remains a poorly characterised orphan disease. AFS is defined by the presence of malignant mesenchymal overgrowth of the stromal component. 1 By definition, the epithelial component of AFS lacks features of malignancy, thus allowing AFS to be distinguished from ameloblastic carcinosarcoma, in which both the epithelial and the mesenchymal components are histologically malignant. 1 However, rarely, mild atypia and mitotic activity may be encountered in the ameloblastic epithelial component of AFS without justifying a diagnosis of ameloblastic carcinosarcoma. 9 A thorough literature review from 2017 uncovered a total of 289 AFs (279 central and 10 peripheral) and 103 AFSs. 3 The authors of that review recognised significant differences between AF and AFS with regard to the mean age of affected patients, expansion and cortical perforation of involved bone, and size of the tumour. 3 Local recurrence rates were 19.2% and 12.5% for central and peripheral AF, respectively. For AFS, local recurrence rates were 35% for all cases, 28.8% for primary (de-novo) AFS, and 50% for secondary AFS occurring following previous AF. 3 The mandible, and mainly its posterior part, is a more common site for AFS than the maxilla (4:1).
Larger lesions and older patients were more often treated with surgical resection for central AF. Segmental resection resulted in the lowest rate of recurrence for most of the lesion types. 3 The local recurrence rate was lower for those AFS patients treated with radical segmental resection than for those who received marginal resection. Because of its aggressive clinical course, with an overall death rate related to disease complications of 21.3%, radical segmental resection with free margins is recommended for AFS. 3 In this study, which is the first to address the molecular pathogenesis of AFS, we found a high rate of oncogenic BRAF mutations (five of seven cases) in AFS. All BRAF mutations were the classic BRAF V600E mutation. The presence of the BRAF mutation in both components of the tumour on separate analysis, and the unequivocal immunoexpression of the mutant BRAF VE1 in the stromal component of one case, confirm the presence of BRAF mutation in the sarcomatous mesenchymal component of AFS. The question of whether BRAF mutation is present in both components of a single tumour could not be conclusively addressed in this study, given the scarcity of the epithelial component in some cases, which precluded reliable separate molecular testing without laser microdissection. These findings make targeted therapy using available BRAF inhibitors an option that merits clinical testing in patients with this aggressive rare disease.
Very sparse data are available on the genetic features of AF. Muller et al. analysed the DNA ploidy status in AF as compared with that in AFS. They found diploid status in all three AFs, but also in 80% of AFSs. 10 Brunner et al. detected BRAF V600E mutations in six of 18 ameloblastic fibro-odontomas and in two of five AFs. 11 However, microdissected stromal components of four ameloblastic fibro-odontomas and one AF with a known BRAF V600E mutation were BRAF wild-type. 11 The study by Brown et al. identified a BRAF V600E mutation in one ameloblastic fibro-dentinomas and two AFs. 12 Although the authors reported 100% concordance with the VE1 IHC, there was no mention of the presence or absence of reactivity in the stromal components of the two AFs. A recent study published in Chinese (only abstract reviewed) reported the BRAF V600E mutation in 16 of 16 AFs (100%). 13 According to defined morphological criteria, our cases are consistent with previous observations that at least a subset of AFSs do originate from pre-existing recurring AFs or contain AF-like bland-looking areas indicating development within a background of AF. In our series, five of the seven cases had either AF-like areas (n = 2) or developed in a background of previously treated odontogenic tumour (reported as AF in one case, as ameloblastoma versus adenomatoid odontogenic tumour in another case, and as unspecified ameloblastic tumour in the third case).
From a differential diagnostic point of view, AFS should be distinguished from the stroma-rich variant of AF, recurrent AF, and undifferentiated sarcomas. AFS differs from cellular AF by the presence of unequivocal malignant features such as significant   Figure 3. In the one case with successful testing, the VE1 immunohistochemistry showed positive staining in the bland-looking less cellular stroma (A) and the highly cellular malignant stroma (B), but no specific staining in the epithelial component.

A B
In summary, this is the first study of the molecular features of AFS highlighting frequent BRAF and occasional NRAS mutations. There is no difference in the distribution of BRAF mutations among tumours originating from AF or de novo. AFS should be tested for these mutations, given the availability of approved promising drugs targeting mutant BRAF.