ameloblastoma is a rare tumour of odontogenic epithelial origin , which accounts for approximately 1% of all mandibular tumours and cysts [ 1 - 2 ] .
it has high propensity for local recurrence if not adequately removed and although the tumour may appear microscopically benign , the development of distant metastasis is possible .
classification as a benign or malignant lesion has generated much debate because ameloblastoma has different histopathologic patterns and displays a wide spectrum of biologic behaviours . in the who classification , a clear distinction between ameloblastoma , metastasizing ameloblastoma and ameloblastic carcinoma was made . here ,
metastasizing ameloblastoma is defined as an ameloblastoma that metastasize in spite of a benign histology .
this occurrence is estimated in approximately 2% of the cases , and the most common site for metastasis is the lung ( 75 - 80% ) , followed by the cervical lymph nodes , the diaphragm , the liver and the brain . on the other hand , ameloblastic carcinoma shows combined histologic features of ameloblastoma with cytologic atypia , regardless of the presence of metastasis .
this report describes a patient who presented a long history of local recurrence and distant metastasis of ameloblastoma .
molecular analysis was performed with the aim to better characterize this neoplasm and its peculiar behaviour .
a 29-year - old male presented a swelling of the oral cavity without any symptoms .
the radiographic examination showed diffuse opacity of the right nasal fossa and maxillary sinus , with destruction of the medial and inferior walls of the maxillary sinus , and thinning of the pavement of the orbita ( figure 1a ) .
he underwent extended right maxillectomy with resection of a portion of the hard palate with a histologic diagnosis of ameloblastoma . in the following years , his medical history was significant for an adenocarcinoma of the colon and an acute myocardial infarction .
after a disease - free interval of 20 years , the tumour recurred locally in the right maxillary sinus ( figure 1b ) , and the diagnosis of ameloblastoma was confirmed histologically .
one year later a computed tomography highlighted a recurrence involving the right orbita ( figure 1c ) and the hard palate ( figure 1d ) .
after 10 months , twenty - seven years after the onset of the primary lesion , the patient referred frontal headache , postural instability , disorientation and bewilderment with impaired short - term memory .
magnetic resonance imaging revealed a recurrence in the infratemporal fossa ( figure 1e ) and a single metastasis in the right temporal area of the brain ( figure 1f ) .
these were surgically removed , and the sphenoidal - orbital region and the right temporal lobe were also treated with radiation therapy ( 60 gray total dose ) .
nine months later the patient developed a further metastatic deposit in the right neck , which was surgically removed .
a = diffuse opacity of the right nasal fossa and maxillary sinus , with destruction of the medial and inferior walls of the maxillary sinus , and thinning of the pavement of the orbita . 
 

b = axial computed tomography showing the first recurrence in the maxillary sinus , which occurred 20 years after the surgical treatment of the tumour . 
 

f = single metastasis in the right temporal area of the brain . 
  the histological appearance of the tumour was the same in the primary lesion , in the recurrence and in the metastases .
the tumour mainly presented a plexiform pattern , characterized by a proliferation of basal cells organized in anastomosing strands with an inconspicuous stellate reticulum ( figure 2 ) .
the basal cells were columnar and hyperchromatic , with nuclei displaced away from the basement membrane . in the primary lesion ,
focal areas of the tumour displayed cystic changes ( figure 2 ) . there were no cytologic atypia , mitotic activity and areas of necrosis in any of the specimens . 
 a = low power view of the primary lesion , showing a plexiform pattern , with cystic areas . 
 

b = high power view of the primary lesion , showing anastomosing strands of epithelium in fibrous stroma , with tall columnar at the periphery . 
 

c = metastatic lesion of the neck , showing infiltration of the salivary tissue of the parotid gland . 
 

staining for epidermal growth factor receptor ( egfr ) conducted on the infratemporal recurrence and on the brain metastasis , showed strong membrane and cytoplasmic positivity of neoplastic cells .
tumour protein p53 ( tp53 ) nuclear immunostaining was detected in the majority of neoplastic cells .
conversely , human epidermal growth factor receptor 2 ( her2 ) was negative in all samples tested . 
 a = immunohistochemical staining showing positivity for egfr . 
 

fluorescent in situ hybridization ( fish ) analysis the analysis was conducted on the infratemporal recurrence .
neoplastic cells showed disomy , with an average ratio of egfr gene to chromosome 7 centromere signals ( cep7 ) signals per cell of 0.8 .
direct dna sequencing of tp53 gene exons 5 - 9 was carried out in tumour samples from the infratemporal recurrence and brain metastasis , with no mutational alteration detected .
similarly , sequencing analysis of b - raf proto - oncogene ( braf ) exon 15 ( v600 ) and egfr showed wild type results in all samples tested .
according to the current who classification , metastasizingameloblastoma shows the same histological features of non - metastasizing ameloblastoma , and therefore the diagnosis can be made only retrospectively , when metastasis has occurred .
thus , it is difficult to predict the behaviour of this neoplasm , which has always an uncertain malignant potential . generally accepted risk factors for the development of metastasis include large primary lesion , late diagnosis of the initial tumour , multiple local recurrences , inadequate surgical treatment , radio - chemotherapy treatment and plexiform pattern , even if the exact nature and the role played by each factor is not clear . in addition
, long - term follow - up is recommended , because metastasis may occur after a very long disease - free interval , which may be up to 42 years after the treatment of the primary lesion , with an average time to metastasis of 18 years .
indeed , in the present case the first recurrence occurred 20 years after the primary lesion , and 7 years later two metastases were detected .
, in the absence of malignant cytologic transformation of the metastatic lesion , the clinical course of the metastatic tumour is relatively indolent , with an average survival of 10 years after the diagnosis of metastasis . in the recent past , several studies attempted to elucidate the complex aspects of ameloblastoma biology , in order to improve treatment and outcome of the patients .
in addition , patients must be followed strictly with instrumental exams and punctual follow - up to prevent recurrence .
recently , several molecular studies have been undertaken in order to improve our understanding of the pathogenesis of this neoplasm , and to identify possible targets for alternative treatments , to potentially reduce the need for extensive and repeated surgery .
the status of different genes has been evaluated , including transmembrane tyrosine kinase egfr , braf , her2 and tp53 .
epidermal growth factor and egfr participate in the differentiation of ameloblasts and in the control of their cellular function .
egfr regulates the proliferation of both normal and neoplastic cells , it is expressed by normal odontogenic epithelium and the presence of alterations of this gene has been investigated both with immunohistochemical [ 11 - 13 ] and molecular analysis .
immunohistochemical studies have shown positivity for egfr both in primary lesions and in metastases , although this marker did not predict recurrence . in agreement , in the present case we observed egfr cytoplasmic and membranous immunostaining in both the recurrence and the brain metastasis . in the molecular study performed by kurppa et al .
, an over - expression of egfr was observed in ameloblastomas studied with real - time reverse transcription polymerase chain reaction ( rt - pcr ) , while in the present case , fish analysis did not demonstrate gene amplification .
therefore , data from literature indicate that anti - egfr targeted therapy could be potentially used in the treatment of recurrent disease , especially in cases where the surgical approach is limited . indeed , as reported by vered et al .
, egfr treatment is recommended in aggressive ameloblastomas not only in monotherapy , but also in combination with conventional radiotherapy .
the administration of anti - egfr agents could be also intralesional , to avoid the adverse events of the treatment .
recently , new anti - egfr monoclonal antibodies were developed , and the target therapy with these drugs showed clinically successful results , underlying the possibility to control the disease also in advanced cases .
braf is a potent activator of the mitogen activated protein kinase ( mapk ) cascade , which in turn phosphorylate and activate extracellular signal - regulated kinases 1 and 2 ( erk1/erk2 ) .
erk regulates the expression of several genes through the phosphorylation of nuclear transcription factors or by targeting other intracellular signalling molecules .
thus , through the stimulation of erk signalling , braf is capable to induce proliferation and to promote transformation .
more than 40 different mutations are identified in braf gene human cancer . ninety percent of b - raf mutations are represented by a missense mutation at residue 600 that substitutes a glutamine with a valine ( v600e ) , resulting in constitutively activation of the gene . in a study of braf v600e mutation in 24 ameloblastomas , 15 tumours ( 63% ) resulted positive for the mutation , without significant association with specific ethnicity , sex or age of the patients , or tumour histology .
this mutation has been associated with resistance to egfr - targeted drugs in primary ameloblastoma cells , similar to what happens in colorectal cancer .
her2 , also known as c - erb or her2/neu , is a human epidermal growth factor receptor belonging to the same family of egfr and its protein overexpression has been found in several carcinomas .
previous studies showed that immunoreactivity for her2 in ameloblastoma is significantly lower than in normal dental follicles   and , albeit with some differences , all variants of ameloblastoma resulted negative or weakly positive for her2 .
this is in agreement with the present case , which showed no immunohistochemical expression of her2 protein , neither in the primary or metastatic lesions .
tp53 suppressor gene is situated on chromosome 17p13 and is one of the most frequently altered genes in tumours . mutation or loss of heterozygosity in the p53 gene or accumulation of its protein is associated with increased cellular proliferation and malignant transformation .
ameloblastoma shows variable reactivity to p53 depending on the histological types , with plexiform variant being more frequently positive than follicular variant . in our study , direct sequencing of tp53 did not show any alteration of the gene , while immunohistochemistry showed positive nuclear staining of neoplastic cells .
this is in agreement with the results of the study by kumamoto et al . , who observed no tp53 gene alteration in a series of 11 ameloblastomas , including a metastasizing example .
however , these authors found a higher expression of p53 , mdm2 and p14 in benign and malignant ameloblastomas than in tooth germs , supporting the hypothesis that these factors may be involved in the malignant transformation of odontogenic epithelium , while tp53 gene mutation is likely to play a minor role .
we report a case of ameloblastoma with metastatic spread to the brain and the neck after repeated local recurrences .
further studies are needed to identify molecular pathways that may provide an opportunity of alternative treatments and/or new potential predictive markers of local and distant spread of this rare tumour .
 abstractbackgroundameloblastoma is a benign odontogenic tumour that may exhibit aggressive biological behaviour with local recurrence and metastasis following initial surgical resection . 
 surgery is the most acceptable modality of treatment , even if a biological approach is currently on study . 
 we report a case of maxillary ameloblastoma with development of neck and brain metastases after repeated local recurrences . 
 molecular analysis was performed with the aim to better characterize this neoplasm and its peculiar behaviour.methodswe investigated the status of tumour protein p53 ( tp53 ) , epidermal growth factor receptor ( egfr ) , b - raf proto - oncogene ( braf ) and human epidermal growth factor receptor 2 ( her2 ) genes with immunohistochemical , fluorescent in situ hybridization and/or direct sequencing in order to clarify their possible role in the development of this neoplasm and the possibility of a targeted treatment.resultsthe histological appearance of the tumour was the same in the primary lesion , in the recurrence and in the metastases . 
 egfr positivity was present in the recurrence and the brain metastasis , while her2 was negative in all samples tested . 
 fluorescent in situ hybridization analysis for egfr showed disomy of neoplastic cells . 
 direct dna sequencing of tp53 gene exons 5 - 9 was carried out in tumour samples from the infratemporal recurrence and brain metastasis , with no mutational alteration detected . 
 similarly , sequencing analysis of braf exon 15 ( v600 ) and egfr gene showed wild type results in all samples tested.conclusionsfurther studies are needed to identify molecular pathways that may provide an opportunity of alternative treatments and/or new potential predictive markers of local and distant spread of this rare tumour . 