Ameloblastic Fibrosarcoma : A Case Report and Literature Review

of a woman who was treated surgically and recovered without signs of recurrence or metastasis after 12 years of follow-up. Tumor sections were immunostained for cell cycle, epithelial and mesenchymal markers. Immunohistochemical analysis evidenced high Ki-67 positivity in stromal cells (mean of 20.9 cells/High power field). Epithelial cells displayed strong positivity for p53, p63 and cytokeratin 19. In addition to the case report, a systematic review of current knowledge is presented on the AFS’s clinical-demographic features and prognostic factors. Based on the review, 88/99 cases were diagnosed as AFS, 9/99 as ameloblastic fibro-odontosarcoma and 2/99 as ameloblastic fibrodentinosarcoma. All these lesions displayed very similar clinical-demographic and prognostic features. Moreover, the review provided evidence that first treatment, regional metastasis, distant metastasis and local recurrence were significant prognostic values for malignant odontogenic mesenchymal lesions. Based on the findings, segregation among ameloblastic fibrosarcoma, ameloblastic fibrodentinosarcoma and ameloblastic fibro-odontosarcoma seems illogical, considering all these lesions have similar predilections and outcomes. Ameloblastic Fibrosarcoma: A Case Report and Literature Review


Introduction
Among the malignant odontogenic tumors (MOTs), ameloblastic fibrosarcoma (AFS) is the most common.It is defined as an odontogenic tumor composed of malignant ectomesenchyme in which variable quantities of a benign epithelial component can be seen.It is regarded as the malignant counterpart of ameloblastic fibroma (AF).However, most cases were diagnosed as primary malignant processes (1)(2)(3)(4)(5).
Heath et al. (6) first described AFS in 1887.Since then, there were about 100 cases of similar microarchitectural features described in the literature.The World Health Organization's (WHO's) classification of odontogenic tumors (OTs) designated AFS a distinctive neoplasm since the inaugural "Blue Book" edition, published in 1972 (7).The essence of this view has remained unaltered since that time (1,8).
To diagnose AFS histologically, as proposed by the WHO (1), it is important the identification of columnar and/ or cuboidal benign ameloblastic epithelial cells arranged in budding and branching cords, admixed with islands and knots.All these components are included in a highly cellular malignant connective stromal component, with cells showing variable degrees of anaplasia (1).Although AFS was first described 120 years ago, information about its epidemiology, treatment, predictive factors and expected outcomes is very limited, and is based mainly on case reports (9)(10)(11)(12).Some MOTs showing inductive phenomena in an AFS background have been described.In those cases, when the inductive process resulted only in the deposition of dentine, the lesions were called ameloblastic fibrodentinosarcomas (AFDSs); when dentine and enamel were identified concurrently, the term ameloblastic fibro-odontosarcoma (AFOS) was used (1,13).Discussions about the pathogenetic and clinicopathological relationships among AFS, AFDS, and AFOS were presented in the literature, but no consensus has been attained to date.
Moreover, there are few reports on the proliferative potential of AFS, based on cell cycle markers.There is also a lack of data on the effects of the sarcomatous component of the lesion on proliferative activity and differentiation aspects of the ameloblastic epithelium present in AFS.Taken together, these data could improve the understanding of tumor pathogenesis and progression and aid in the differentiation of subtle malignant transformations of AF.
The aim of this report was to describe a case of AFS with an immunohistochemical evaluation.There is also a discussion of the clinicopathological characteristics of AFS and the nosological relationships of AFS to AFDS and AFOS based on a systematic review of literature.

Case Report
A 32-year-old non-white woman was referred for evaluation and treatment of a large and persistent malignant swelling in the left mandible.The patient complained of pain and dysphagia for 4 months.An extraoral examination disclosed a large, firm swelling, involving the left mandibular ramus.Examination of other head and neck structures and a general physical evaluation yielded unremarkable findings.An intraoral examination revealed lingual and buccal cortical bone expansion affecting the mandibular body, with no clinical or imaging evidence of cortical perforation.Oral mucosa and mandibular nerve functions were normal and intact.Plain radiographic examinations showed an ill-demarcated and partially corticated multilocular radiolucency affecting the left posterior mandible.An incisional biopsy was performed, followed by routine histopathology, resulting in the diagnosis of primary AFS.En bloc surgical resection from the second premolar to the anterior ramus region was performed under general anesthesia.The resected specimen measured 8×6 cm, and showed clear margins, clinically and histologically.Immediate reconstruction was performed using an autogenous right iliac bone graft, stabilized with a titanium plate.The patient did not undergo any other treatment.Histopathological examination of the resected specimen showed evidence of scant cords and nests of odontogenic epithelium scattered within the mesenchymal stromal tissue (Fig. 1).Stromal components contained hyperchromatic and pleomorphic cells, as well as numerous mitotic structures (Fig. 1A-C).There were foci of necrosis near the epithelial islands.Odontogenic epithelial islands, in a follicular or trabecular aspect, showed hyperplasia of peripheral columnar cells; these cells showed hyperchromatic and discrete anisokaryosis (Fig. 1A-C).In some places, dentinoid material of variable dimensions was associated closely with the epithelial islands.
Three of the authors (AML, JPSS, and CVR) evaluated immunohistochemical staining using the Quick-Score method, according to Detre et al (15).The products are multiplicative quick-score and score values, ranging from 0 to 18.For Ki-67 analysis, the mean number of positive cell nuclei in 10 consecutive high-power fields (HPFs) was determined.
The atypical mesenchymal cells showed strong diffuse nuclear and cytoplasmic reactivity for p16, whereas the epithelial component showed only weak staining for this marker (Fig. 2C).However, the epithelial cells showed strong diffuse nuclear and cytoplasmic reactivity for p53 (Fig. 2B) and p63 (Fig. 2D) and the stromal cells were weakly positive.The benign ameloblastomatous component showed weak diffuse cytoplasmic reactivity for CK8 and CK14 (Fig. 2E and F, respectively), but strong CK19 cytoplasmic staining was apparent (Fig. 2G).The stromal malignant component was positive for vimentin and SMA (Fig. 2H).There was no reactivity for CK7, CK18, calretinin, fibronectin or desmin.The patient's healing process was uneventful and no sign of recurrence or metastasis has been observed during   12 years of follow-up.
Descriptive statistics were used to assess the clinicopathological data.All collected information was considered in the overall survival analysis.Curves for different clinicopathological factors were traced using the Kaplan-Meier method and then compared using univariate analyses (log-rank test).The chi-square test was used to analyze any association between clinical and histomorphological factors.The Spearman rank correlation test was used to determine whether lesion size was correlated with evolution time.The significance level was set at 5%.All analyses were performed using the GraphPad Prism software (v.5.01; San Diego, CA, USA).
For the cases diagnosed as AFS, age ranged widely from 0.33 to 89 (mean, 28.1±16) years.Lesions usually affected the posterior mandible (59 cases, 67.1%), without predilection for gender or ethnicity (male:female ratio, 1:0.8; white:non-white ratio, 1:0.8).Most of the lesions presented large (mean, 5.8±3.3cm), painful (40/79, 50.6%)The 9 cases diagnosed as AFOS affected the posterior mandible (100.0%),mostly in female patients (5/8, 62.5%), with a mean age of 21.3±11.8years.Swelling was present in all cases and pain was the chief complaint in six of eight (75.0%)cases.Six of the eight lesions were considered as de novo malignancies; the remaining two were considered as malignant transformations of one AF and one ameloblastic fibroodontoma.Most of these patients were managed surgically (8/9, 88.9%); from them, initial aggressive surgical treatment was performed in five of eight (62.5%)cases.Only three patients received complementary therapy (2/8 chemotherapy and 1/8 radiotherapy).There were recurrent primary tumors in four of nine (44.4%) cases.Regional and distant metastatic foci were diagnosed in only one patient (46).After a mean follow-up period of 19.9±12.4months, only 2/8 patients died of the disease; the remaining patients (6/8) had no evidence of disease at the last recorded appointment.Only two cases were diagnosed with AFDS, (13,45) and both involved the posterior mandibles of non-white male patients in the third decade of life (mean, 26±1.4 years).Both patients complained of swelling and pain.One case was diagnosed as de novo and the other was diagnosed as malignant transformation of a previous AF.The patients received initial aggressive surgical management.None of these cases evolved with relapse or metastatic disease.Treatment was prescribed for only one case and the patient was disease-free for 18 months after diagnosis.
An overall survival analysis was performed for all 99 cases.The median overall survival time was 228 months, and the overall 5-, 10-and 20-year survival rates were 71.3%, 53.3%, and 26.6%, respectively (Fig. 3A).Results of a univariate overall survival analysis are in Table 3. Statistically significant associations were identified for the following parameters: age (Fig. 3B), regional metastasis (Fig. 3C), distant metastasis (Fig. 3D), local recurrence (Fig. 3E) and first surgical treatment (Fig. 3F).Patients treated with adjuvant therapies (radiotherapy and/or chemotherapy) had a very low overall median survival ratio (Table 3).

Discussion
According to a more extensive series of OTs, MOTs accounted for 0.1-6.0% of these tumors; most were carcinomas and odontogenic sarcomas, including AFS, AFDS and AFOS, represented less than 0.2% of cases.AFS is the most common of them (66,(114)(115)(116)(117).In Brazilian series, the reported frequency of AFS is up to 1.0% of OTs (3,(117)(118)(119)(120) The WHO's odontogenic tumor classification describes AFS as being of primary (de novo lesion) and secondary (from benign AF) types (1,4,5).Because of the secondary types (88), initial diagnosis of a malignant phenotype is not always easy.As anaplasia is not distributed uniformly throughout the tumor, definitive diagnosis can be made only from surgically removed pieces, as in the present case.This information is required for the detailed evaluation of the clinical history and radiographic aspects to identify preoperatively aspects that may help the surgeon choose a more representative area to be biopsied or even to make multiple biopsies.However, in the case of clinicoradiographic evidence of an aggressive tumor, serial sampling of surgical specimens should be performed to look for a histologically malignant phenotype.
Clinically, most AFS are similar to AF, but characteristics of aggressive behavior are always identified, as seen in the present case.Based on the present review, AFS is a locally invasive neoplasm predominantly affecting the mandibles of male individuals with a wide age range (4 months to 89 years).The most common clinical features of AFS are facial swelling, often accompanied by pain.Paresthesia, dysesthesia and ulcers are sometimes reported (5,37,47,67).
Radiologically, AFS appears as a uni-or multilocular radiolucent mass with an ill-defined border, frequently causing gross expansion, thinning or even rupture of the cortical bone (88).However, cases with well-defined radiolucency are uncommon (72).Maxillary lesions tend to show antral involvement with apparent erosion of the sinus walls and a propensity to invade adjacent soft tissues, the base of the cranium and intracranial and orbital tissues (12,62,78).The gold standard for the treatment of AFS is radical surgery with clear margins (5,89).Neck dissection is not usually indicated, because regional lymph node metastases are seldom identified.Postoperative radiotherapy at a dose of 50-60 Gy (10,65) was sometimes used, with no recurrence during the follow-up period (4).In a few described cases, adjuvant chemotherapy was used, with inconclusive results (4).
In described case, the patient was treated by mandibulectomy, with no adjuvant therapy.The postoperative period was uneventful and the patient is disease-free, with no metastasis, after 12 years of follow-up.Because of such outcomes, some authors are doubtful of the prognostic benefit related to complementary chemoand/or radiotherapy for these lesions (71,74).Cases first treated with a conservative approach have demonstrated lower overall survival, mainly because of multiple relapses and the involvement of other structures (Table 3 and Fig. 3).The present case clearly demonstrates the importance of aggressive initial treatment, as the patient remained disease-free after only one surgical procedure.
Immunohistochemical studies may facilitate diagnosis of these lesions, given that Ki-67, PCNA and p53 were found expressed at higher levels within the sarcomatous component of the AFS, whereas they are absent or expressed at lower levels in AF (5,12,62,69,79,89,101,113,123).Results obtained from several papers (4,5,69,79,82,88,89,113) revealed that Ki-67 was overexpressed (highly diffuse positivity) in the malignant mesenchymal portion of AFS, compared with AF.When Ki-67 was quantified, positivity rates ranged from 13.5% to 60.0% for the malignant mesenchymal component.Although no established cutoff value for Ki-67 positivity has been reported in the literature, the present case is within the interval of Ki-67 expressed in malignant lesions (~20.0%),suggesting that proliferative factors, in association with histopathological features, may be useful markers for the identification of malignant cases (4,5,69,79,88,89,113).
Reports have also indicated that the epithelial component is not associated with AF or AFS growth.However, evidence of epithelial proliferative activity has been described in AFS (4,5,69,79,88,89,113).In this study case, for example, marked hyperplasia of ameloblasticlike cells was observed; however, positivity for Ki-67 was found to be less than in the mesenchymal component.Change in embryonic mesenchymal density is an important phenomenon that induces epithelial modifications during odontogenesis.Epithelial proliferation in AFS may represent an atavistic behavior of the epithelium in front of a dense neoplastic stroma.The presence of differences in proliferative potential between epithelial and mesenchymal AFS components is relevant for the differentiation of AFS from odontogenic carcinosarcoma, in which the epithelial and mesenchymal components are similarly proliferative and anaplastic (101,121,122).
Disruption in the mechanisms of control cell growth is a hallmark of cancer, which occur mainly due to derangement of the cell cycle checkpoints.A family of cyclins, cyclindependent kinases (CDKs) and their inhibitors (CDKIs) controls the CELL cycle.These proteins have been used as biological behavior and prognosis for different neoplasia.p53 was the most studied tumor suppression protein, which binds DNA and activates expression of several genes, that culminates with inhibition of cell cycle.Recent studies about AF malignant transformation linked such evolution to the acquisition of oncogenic aberrations in TP53.These alterations have been shown by loss of heterozygosity and immunohistochemistry studies and the present data reinforce such evidences (75,123).
Other important regulatory cell cycle proteins are p63 and p16.p63 protein has been recognized as a member of the p53 family and is also responsible for cell cycle control.p16 protein is a tumor suppressor gene protein, which is a CDKI that regulates the G1-S phase of the cell cycle.Studies regarding p63 and p16 on the pathogenesis of AFS are apparently elusive.In this study, p16 and p63 were also found highly expressed, which confirms that deregulation in cell cycle controlling proteins are common events in AFS pathogenesis (1,88,105).
Absence of CK-7, CK-8 and CK-18, and high expression CK-14 and CK-19 support the odontogenic origin of the epithelial counterpart.Although cell proliferation is up-regulated in the sarcomatous component, it does not appear to affect the differentiation status of the ameloblastic epithelium in AFS.The malignant sarcomatous component showed positivity only for vimentin, demonstrating no apparent transdifferentiation signal.More studies based on a comparative approach are required to improve the significance of the present findings on the pathogenesis and progression of AFS.
According to the WHO's classification, (1) AFS, AFDS and AFOS represent distinct lesions (1,88,105).However, these lesions appear to represent different stages of tumor parenchyma differentiation, reflecting the complex inductive phenomena involving odontogenic epithelium and ectomesenchyme.Variations in tumor parenchyma differentiation seem to have no influence on clinical behavior, as the lesions show very similar clinicdemographic aspects and are associated with similar overall median survival rates, as described in this review.Data from the present review argue against the influence of the tumor odontogenic matrix or hard-tissue deposition on the nature of odontogenic sarcomatous lesions.It thus agrees with other reports that AFS, AFDS and AFOS should not be classified as separate nosological entities (4,9,13,3 0, 37,43,62,69,104,124).
In conclusion, the data compiled to date demonstrate that AFS is a low-grade mesenchymal odontogenic malignant neoplasia with a predilection for the posterior mandible, occurring mainly in the third decade of life.The histopathological features of AFS suggest that this entity has pathogenetic relationships with AFDS and AFOS.There was some suggestive morphological and behavioral evidence of similarities among these lesions.For the authors, segregation of these lesions based on their mesenchymal inductive potentials seems to be inappropriate.This study reinforces the necessity of treating AFS with an aggressive surgical approach, with no need for other complementary therapies.

Figure 3 .
Figure 3. Overall Kaplan-Meier survival curve for patients with AFS (A) and stratified according to age (B), regional metastasis (C), distant metastasis (D), local recurrence (E) and the employed first surgery (F).

Table 1 .
Immunohistochemical data of the present case, Quickscores are presented inside brackets Tables.Clinic-pathological and treatment data of the sample about ameloblastic fibrosarcoma, retrieved from international literature AWD: alive with disease; ChT: chemotherapy; DOD: Died of disease; NED: no evidence of disease; RxT: radiotherapy; ST: surgical treatment.

Table 3 .
Ameloblastic fibrosarcoma: comparison of median survival based on demographics, tumor characteristics and applied therapy, related with the case reports retrieved from English literature a Only valid information; b Log-rank test.