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Targeted Next-Generation Sequencing and Allele-Specific Quantitative PCR of Laser Capture Microdissected Samples Uncover Molecular Differences in Mixed Odontogenic Tumors
2020; Elsevier BV; Volume: 22; Issue: 12 Linguagem: Inglês
10.1016/j.jmoldx.2020.08.005
ISSN1943-7811
AutoresBruna Pizziolo Coura, Vanessa Fátima Bernardes, Sílvia Ferreira de Sousa, Marina Gonçalves Diniz, Rennan Garcias Moreira, Bruno Augusto Benevenuto de Andrade, Mário José Romañach, Hélder Antônio Rebelo Pontes, Ricardo Santiago Gomez, Edward Odell, Carolina Cavaliéri Gomes,
Tópico(s)dental development and anomalies
ResumoThe molecular pathogenesis of mixed odontogenic tumors has not been established, and understanding their genetic basis could refine their classification and help define molecular markers for diagnostic purposes. Potentially pathogenic mutations in the component tissues of 28 cases of mixed odontogenic tumors were assessed. Laser capture microdissected tissue from 10 ameloblastic fibromas (AF), 4 ameloblastic fibrodentinomas (AFD), 6 ameloblastic fibro-odontomas (AFO), 3 ameloblastic fibrosarcomas (AFS), and 5 odontomas (OD) were screened by next-generation sequencing and results confirmed by TaqMan allele-specific quantitative PCR. BRAF p.V600E mutation in the mesenchymal component was shown in 4 of 10 AF (40%), 2 of 4 AFD (50%), 2 of 6 AFO (33%), and 2 of 3 AFS (67%), whereas all 5 OD were wild type for BRAF p.V600E. Mutation in the epithelial component was only observed in one AF and one AFO. One AFS contained an area of benign AF, and the mesenchymal component of both (AFS and AF) contained BRAF p.V600E, supporting the concept of malignant progression from a benign AF precursor. KDR, TP53, KIT, and PIK3CA single-nucleotide polymorphisms are reported. In conclusion, AF, AFD, AFO, and AFS show BRAF p.V600E in their mesenchymal component, unlike OD, which are BRAF wild type, suggesting that at least a subset of AF, AFD, and AFO are molecularly distinct from OD, and may represent distinct entities and be neoplastic. The molecular pathogenesis of mixed odontogenic tumors has not been established, and understanding their genetic basis could refine their classification and help define molecular markers for diagnostic purposes. Potentially pathogenic mutations in the component tissues of 28 cases of mixed odontogenic tumors were assessed. Laser capture microdissected tissue from 10 ameloblastic fibromas (AF), 4 ameloblastic fibrodentinomas (AFD), 6 ameloblastic fibro-odontomas (AFO), 3 ameloblastic fibrosarcomas (AFS), and 5 odontomas (OD) were screened by next-generation sequencing and results confirmed by TaqMan allele-specific quantitative PCR. BRAF p.V600E mutation in the mesenchymal component was shown in 4 of 10 AF (40%), 2 of 4 AFD (50%), 2 of 6 AFO (33%), and 2 of 3 AFS (67%), whereas all 5 OD were wild type for BRAF p.V600E. Mutation in the epithelial component was only observed in one AF and one AFO. One AFS contained an area of benign AF, and the mesenchymal component of both (AFS and AF) contained BRAF p.V600E, supporting the concept of malignant progression from a benign AF precursor. KDR, TP53, KIT, and PIK3CA single-nucleotide polymorphisms are reported. In conclusion, AF, AFD, AFO, and AFS show BRAF p.V600E in their mesenchymal component, unlike OD, which are BRAF wild type, suggesting that at least a subset of AF, AFD, and AFO are molecularly distinct from OD, and may represent distinct entities and be neoplastic. Odontogenic tumors are difficult to classify, and there are few molecular data to aid diagnosis or help categorize them. They show a broad range of appearances linked to the complex developmental processes in normal teeth, during which iterative epithelial–mesenchymal interactions control tissue differentiation, induce hard tissue formation, and determine the shape and size of teeth.1Lumsden A.G. Spatial organization of the epithelium and the role of neural crest cells in the initiation of the mammalian tooth germ.Development. 1988; 103 Suppl: 155-169PubMed Google Scholar,2Miletich I. Sharpe P.T. Normal and abnormal dental development.Hum Mol Genet. 2003; 12 Spec No 1: R69-R73Crossref PubMed Google Scholar Odontogenic tumors include malignant and benign neoplasms and hamartomas. Ameloblastic fibroma (AF) is considered a benign neoplasm of both its epithelial and mesenchymal components that is sometimes a precursor to ameloblastic fibrosarcoma.3Lai J. Blanas N. Higgins K. Klieb H. Ameloblastic fibrosarcoma: report of a case, study of immunophenotype, and comprehensive review of the literature.J Oral Maxillofac Surg. 2012; 70: 2007-2012Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar By contrast, odontomas (OD) are hamartomas. Histologically intermediate between these defined entities is a group of poorly characterized tumors, the ameloblastic fibrodentinoma (AFD) and ameloblastic fibro-odontoma (AFO),4Takata T. Slootweg P.J. Benign mixed epithelial and mesenchymal odontogenic tumors.in: El-Naggar A.K. Chan J.K.C. Grandis J.R. Takata T. Slootweg P.J. WHO Classification of Head and Neck Tumors, ed 4. IARC, Lyon, France2017: 222-226Google Scholar,5Slootweg P.J. An analysis of the interrelationship of the mixed odontogenic tumors-ameloblastic fibroma, ameloblastic fibro-odontoma, and the odontomas.Oral Surg Oral Med Oral Pathol. 1981; 51: 266-276Abstract Full Text PDF PubMed Scopus (175) Google Scholar whose status has been long debated.5Slootweg P.J. An analysis of the interrelationship of the mixed odontogenic tumors-ameloblastic fibroma, ameloblastic fibro-odontoma, and the odontomas.Oral Surg Oral Med Oral Pathol. 1981; 51: 266-276Abstract Full Text PDF PubMed Scopus (175) Google Scholar, 6Philipsen H.P. Reichart P.A. Praetorius F. Mixed odontogenic tumours and odontomas. Considerations on interrelationship. Review of the literature and presentation of 134 new cases of odontomas.Oral Oncol. 1997; 33: 86-99Crossref PubMed Scopus (220) Google Scholar, 7Tomich C.E. Benign mixed odontogenic tumors.Semin Diagn Pathol. 1999; 16: 308-316PubMed Google Scholar, 8Chen Y. Li T.-J. Gao Y. Yu S.-F. Ameloblastic fibroma and related lesions: a clinicopathologic study with reference to their nature and interrelationship.J Oral Pathol Med. 2005; 34: 588-595Crossref PubMed Scopus (53) Google Scholar, 9Buchner A. Kaffe I. Vered M. Clinical and radiological profile of ameloblastic fibro-odontoma: an update on an uncommon odontogenic tumor based on a critical analysis of 114 cases.Head Neck Pathol. 2013; 7: 54-63Crossref PubMed Scopus (31) Google Scholar, 10Chrcanovic B.R. Gomez R.S. Ameloblastic fibrodentinoma and ameloblastic fibro-odontoma: an updated systematic review of cases reported in the literature.J Oral Maxillofac Surg. 2017; 75: 1425-1437Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar The 2017 World Health Organization Classification of Head and Neck Tumors considered that AFD and AFO should be classified as the early stages of OD4Takata T. Slootweg P.J. Benign mixed epithelial and mesenchymal odontogenic tumors.in: El-Naggar A.K. Chan J.K.C. Grandis J.R. Takata T. Slootweg P.J. WHO Classification of Head and Neck Tumors, ed 4. IARC, Lyon, France2017: 222-226Google Scholar,11Speight P.M. Takata T. New tumour entities in the 4th edition of the World Health Organization Classification of Head and Neck tumours: odontogenic and maxillofacial bone tumours.Virchows Arch. 2018; 472: 331-339Crossref PubMed Scopus (116) Google Scholar,12Mosqueda-Taylor A. New findings and controversies in odontogenic tumors.Med Oral Patol Oral Cir Bucal. 2008; 13: E555-E558PubMed Google Scholar to prevent overtreatment. Recently, the BRAF p.V600E mutation was reported in AF,13Brown N.A. Rolland D. Mchugh J.B. Weigelin H.C. Zhao L. Lim M.S.1 Elenitoba-Johnson K.S. Betz B.L. Activating FGFR2-RAS-BRAF mutations in ameloblastoma.Clin Cancer Res. 2014; 20: 5517-5526Crossref PubMed Scopus (123) Google Scholar, 14Brunner P. Bihl M. Jundt G. Baumhoer D. Hoeller S. BRAF p.V600E mutations are not unique to ameloblastoma and are shared by other odontogenic tumors with ameloblastic morphology.Oral Oncol. 2015; 51: e77-e78Crossref PubMed Scopus (34) Google Scholar, 15You Z. Xu L.L. Li X.F. Zhang J.Y. DU J. Sun L.S. [BRAF gene mutations in ameloblastic fibromas].Beijing Da Xue Xue Bao Yi Xue Ban. 2019; 51 (Chinese): 4-8PubMed Google Scholar AFO,14Brunner P. Bihl M. Jundt G. Baumhoer D. Hoeller S. BRAF p.V600E mutations are not unique to ameloblastoma and are shared by other odontogenic tumors with ameloblastic morphology.Oral Oncol. 2015; 51: e77-e78Crossref PubMed Scopus (34) Google Scholar one case of AFD,13Brown N.A. Rolland D. Mchugh J.B. Weigelin H.C. Zhao L. Lim M.S.1 Elenitoba-Johnson K.S. Betz B.L. Activating FGFR2-RAS-BRAF mutations in ameloblastoma.Clin Cancer Res. 2014; 20: 5517-5526Crossref PubMed Scopus (123) Google Scholar and in five of seven AFS,16Agaimy A. Skalova A. Franchi A. Alshagroud R. Gill A.J. Stoehr R. Baumhoer D. Bauer S. Ameloblastic fibrosarcoma: clinicopathological and molecular analysis of seven cases highlighting frequent BRAF and occasional NRAS mutations.Histopathology. 2020; 76: 814-821Crossref PubMed Scopus (5) Google Scholar but it is not known whether epithelium or mesenchyme are altered in each tumor type and whether all share the same molecular profile. Only one study to date has used laser capture microdissection (LCM) to dissect the mesenchymal component in equivocal cases of a total of three AFO and one AF,14Brunner P. Bihl M. Jundt G. Baumhoer D. Hoeller S. BRAF p.V600E mutations are not unique to ameloblastoma and are shared by other odontogenic tumors with ameloblastic morphology.Oral Oncol. 2015; 51: e77-e78Crossref PubMed Scopus (34) Google Scholar and it remains unknown whether there may be other genetic drivers in these lesions, as next-generation sequencing (NGS) assays have only been applied to AFS.16Agaimy A. Skalova A. Franchi A. Alshagroud R. Gill A.J. Stoehr R. Baumhoer D. Bauer S. Ameloblastic fibrosarcoma: clinicopathological and molecular analysis of seven cases highlighting frequent BRAF and occasional NRAS mutations.Histopathology. 2020; 76: 814-821Crossref PubMed Scopus (5) Google Scholar The study aimed to interrogate a large discovery cohort of mixed odontogenic tumors for hotspot mutations in 50 commonly mutated tumor suppressor genes and oncogenes by NGS. The BRAF p.V600E mutational status in LCM separated epithelial and mesenchymal components was then assessed by quantitative PCR (qPCR) in a validation cohort. This study was approved by the Research Ethics Committee of Universidade Federal de Minas Gerais (protocol number CAAE 30556120.0.0000.5149). Formalin-fixed, paraffin-embedded (FFPE) samples were selected from the oral pathology files of the authors' institutions (Universidade Federal de Minas Gerais, Universidade Federal do Rio de Janeiro, and Universidade Federal do Pará, Brazil; and King's College London, UK), with the exception of AF1, which was freshly collected and snap frozen at Universidade Federal de Minas Gerais, Brazil. Cases were selected and diagnoses were reviewed on the basis of pathologic, clinical, and radiological features. The diagnosis of the cases of each institution was reviewed and confirmed by oral pathologist coauthors of this study (B.A.B.d.A., M.J.R., H.A.R.P., R.S.G., and E.W.O.), and all diagnoses were additionally reviewed by an oral pathologist of the institution where the experiments were performed (R.S.G. and C.C.G.). The histopathologic features of the mixed odontogenic tumors included in the study are illustrated in Figures 1 and 2. Although previous studies have been able to detect BRAF p.V600E mutation in decalcified ameloblastomas and AFS,16Agaimy A. Skalova A. Franchi A. Alshagroud R. Gill A.J. Stoehr R. Baumhoer D. Bauer S. Ameloblastic fibrosarcoma: clinicopathological and molecular analysis of seven cases highlighting frequent BRAF and occasional NRAS mutations.Histopathology. 2020; 76: 814-821Crossref PubMed Scopus (5) Google Scholar,17Kurppa K.J. Catón J. Morgan P.R. Ristimäki A. Ruhin B. Kellokoski J. Elenius K. Heikinheimo K. High frequency of BRAF V600E mutations in ameloblastoma.J Pathol. 2014; 232: 492-498Crossref PubMed Scopus (139) Google Scholar undecalcified tissue blocks were selected for all cases, including all OD, for NGS and qPCR.Figure 2Histopathologic features of an ameloblastic fibrosarcoma. A: Sample AFS1 showed a residual area of ameloblastic fibroma (AF) (asterisk) in addition to the malignant tumor area (double asterisk). B and C: The benign area reveals epithelium and mesenchymal components consistent with AF-like area. D and E: The sarcomatous part of the tumor showed high cellularity with hyperchromatic nuclei, cellular atypia, and mitotic figures. Hematoxylin and eosin staining was used. Original magnification: ×50 (A and B); ×100 (C); ×200 (D); ×400 (E).View Large Image Figure ViewerDownload Hi-res image Download (PPT) A convenience sample of 11 mixed odontogenic tumors was obtained and used as a discovery cohort for molecular assessment by NGS. The DNA and libraries of four mixed odontogenic tumors, including three AF samples, and one AFD tumor sample, passed the quality control, and their sequencing results were included in the study. Genomic DNA was isolated using a commercially available kit, QIAamp DNA FFPE Tissue kit (Qiagen, Hilden, Germany) in FFPE samples, and DNeasy Blood and Tissue Kit (Qiagen) in fresh tissue, according to the manufacturer's recommendations. Genomic DNA quality was quantified and assessed using a Qubit 3.0 Fluorometer (Life Technologies, Carlsbad, CA) and spectrophotometry (NanoDrop 2000; Thermo Fisher Scientific, Wilmington, DE). Ion AmpliSeq Cancer Hotspot Panel v2 (Thermo Fisher Scientific, Waltham, MA) was used to interrogate 2856 COSMIC (Catalog of Somatic Mutations in Cancer, https://cancer.sanger.ac.uk/cosmic, last accessed July 15, 2020) hotspot mutations in a panel of 50 oncogenes and tumor suppressor genes, with successful results in three AF and one AFD samples. This panel includes several MAPK pathway genes, among others listed hereafter: ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, CDKN2A, CSF1R, CTNNB1, EGFR, ERBB2, ERBB4, EZH2, FBXW7, FGFR1, FGFR2, FGFR3, FLT3, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, IDH2, JAK2, JAK3, KDR, KIT, KRAS, MET, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, SRC, STK11, TP53, and VHL. The DNA libraries were prepared using Ion AmpliSeq Library Kit 2.0 (Thermo Fisher Scientific) and Ion Xpress Barcode Adapters 1-16 Kit (Thermo Fisher Scientific) was used to pool samples in the same sequencing run. Each library was individually purified using Agencourt AMPure XP beads (Beckman Coulter, Indianapolis, IN) and quantified by qPCR with the Ion Library TaqMan Quantitation Kit (Thermo Fisher Scientific). The libraries underwent emulsion PCR and Ion Sphere enrichment using the Ion OneTouch 2 System (Thermo Fisher Scientific). Sequencing was performed on Ion 316 Chip Kit v2 (Thermo Fisher Scientific) using the Ion Torrent PGM system (Thermo Fisher Scientific). The sequences were aligned to the human genome reference (hg19) and Ion Reporter Software version 5.2 (Thermo Fisher Scientific) was used to filter and report variants. Missense and INDEL variants with sequencing depth >300 and frequency >5% were filtered. The software Integrative Genomics Viewer version 2.3 (Broad Institute, Cambridge, MA) was used to exclude false variants by checking the alignments manually. In silico analysis to predict the variants pathogenicity/effect on protein was performed using ClinVar (ClinVar, https://www.ncbi.nlm.nih.gov/clinvar, last accessed July 15, 2020), SIFT (SIFT, http://sift-dna.org, last accessed July 15, 2020), and PolyPhen-2 (PolyPhen-2, http://genetics.bwh.harvard.edu/pph2, last accessed July 15, 2020). The variants selected for further validation were those that fit the criteria described above and that were reported as pathogenic at ClinVar or classified as damaging and nontolerated by SIFT and PolyPhen-2 scores. To validate the NGS results by an independent method, the four successfully sequenced samples of the discovery cohort were also submitted to allele-specific qPCR (validation cohort n = 4) and an additional 28 mixed odontogenic tumor cases were selected from the same sources following the same diagnostic criteria specified for the discovery cohort section. These expanded the validation cohort to a total of 32 samples. Four expansion cohort samples failed qPCR amplification, so that the final validation cohort comprised 28 cases, 10 AF, 4 AFD, 6 AFO, 3 AFS, and 5 OD (Table 1).Table 1Clinical and Molecular Information of Mixed Odontogenic Tumor CasesSampleClinical informationBRAF p.V600E status/tumor histopathologic componentAge, yearsSexLocationBulk, not LCMEpithelialMesenchymalMalignant mesenchymalAmeloblastic fibroma AF1∗Samples were bulk sequenced by next-generation sequencing, and the mutation was assessed/confirmed by quantitative PCR.23FMandible, posteriorMutant∗Samples were bulk sequenced by next-generation sequencing, and the mutation was assessed/confirmed by quantitative PCR.NANANA AF2∗Samples were bulk sequenced by next-generation sequencing, and the mutation was assessed/confirmed by quantitative PCR.5MMaxilla, posteriorMutant∗Samples were bulk sequenced by next-generation sequencing, and the mutation was assessed/confirmed by quantitative PCR.MutantMutantNA AF314FMandible, anteriorNAUnMutantNA AF46FMandible, body and ramusNAUnMutantNA AF512FMandible, body and ramusNAWild-typeWild-typeNA AF612MMandible, posteriorNAWild-typeWild-typeNA AF79MMandible, body and ramusNAUnWild-typeNA AF83MMandible, body and ramusNAUnWild-typeNA AF9∗Samples were bulk sequenced by next-generation sequencing, and the mutation was assessed/confirmed by quantitative PCR.7FMandible, body and ramusWild-type∗Samples were bulk sequenced by next-generation sequencing, and the mutation was assessed/confirmed by quantitative PCR.Wild-typeWild-typeNA AF1014FMandible, posteriorNAWild-typeUnNAAmeloblastic fibrodentinoma AFD1†Incisional and excisional biopsies of these cases have been evaluated with concordant results.23FMandible, body and ramusNAWild-typeMutantNA AFD28FMandible, posteriorNAWild-typeMutantNA AFD311MMaxilla, posteriorNAWild-typeUnNA AFD4∗Samples were bulk sequenced by next-generation sequencing, and the mutation was assessed/confirmed by quantitative PCR.9MMaxilla, anteriorWild-type∗Samples were bulk sequenced by next-generation sequencing, and the mutation was assessed/confirmed by quantitative PCR.Wild-typeWild-typeNAAmeloblastic fibro-odontoma AFO19MMandible, premolar regionNAWild-typeMutantNA AFO210FMaxilla, posteriorNAMutantMutantNA AFO323MMaxilla, posteriorNAWild-typeWild-typeNA AFO49MMandible, posteriorNAWild-typeWild-typeNA AFO516MMandible, body and ramusNAWild-typeWild-typeNA AFO6†Incisional and excisional biopsies of these cases have been evaluated with concordant results.14MMandible, posteriorNAWild-typeWild-typeNAAmeloblastic fibrosarcoma AFS114FMandible, anterior and bodyNAWild-typeMutantMutant AFS232FMandible, posteriorNAWild-typeNAMutant AFS325MMandible, posteriorNAUnNAWild-typeOdontoma OD114MMaxillaWild-typeNANANA OD214FMandibleWild-typeNANANA OD312MMandibleWild-typeNANANA OD415FMandibleWild-typeNANANA OD514FMandibleWild-typeNANANAF, female; M, male; NA, not applicable or not tested; Un, uninterpretable, the amplification either occurred in a high threshold cycle or there was no amplification.∗ Samples were bulk sequenced by next-generation sequencing, and the mutation was assessed/confirmed by quantitative PCR.† Incisional and excisional biopsies of these cases have been evaluated with concordant results. Open table in a new tab F, female; M, male; NA, not applicable or not tested; Un, uninterpretable, the amplification either occurred in a high threshold cycle or there was no amplification. LCM was performed on 22 of the 28 validation samples, two AF and one AFD (n = 3) from the validation cohort and seven AF, three AFD, six AFO, and three AFS from the expansion cohort (n = 19) (Table 1). LCM was performed on a MMI CellCut system with MMI CellTools control software version 4.0.7 RC2 (MMI Molecular Machines & Industries, Tokyo, Japan) and an optical microscope. By using LCM, the epithelial and mesenchymal components were separated for analysis (Figure 3) and collected in separate sterile plastic tube caps. Notably, one AFS sample (AFS1) contained a precursor area of AF in addition to the malignant tumor area (Figure 2) and had three different components isolated by LCM: epithelial and mesenchymal tissues of the AF area and the mesenchymal sarcomatous component. Genomic DNA of all materials submitted to LCM was isolated separately for further molecular analyses. The only pathogenic variant detected by NGS was BRAF p.V600E, and this mutation was interrogated in the LCM mixed odontogenic tumor tissue samples as well as in OD cases. The BRAF p.V600E mutation was screened using BRAF_476_mu and BRAF_rf assays (Applied Biosystems, Foster City, CA), following the manufacturer's instructions. This assay is highly specific and sensitive (sensitivity of 0.1%) and can detect very low levels of mutated DNA in a background of wild-type DNA. Reactions were run on a StepOne Plus instrument (Applied Biosystems), and the mutation status was determined using TaqMan Mutation Detector Software version 2.0 (Life Technologies). Sample components (epithelial or mesenchymal) that failed amplification twice or amplified at a threshold cycle above the cutoff were excluded from analysis. Twenty-eight mixed odontogenic tumors were included in the final analyses, because genomic DNA from one AF, one AFD, and two OD cases failed qPCR amplification twice and were excluded. There was an equal number of female and male patients (n = 14 each), and tumors arose most often in the mandible (n = 22), with a few cases in the maxilla (n = 6). Clinicopathologic data for each sample and the molecular results are shown in Table 1 and Figure 4. Of the 11 cases included in the discovery cohort, 4 were successfully sequenced by NGS, and 7 failed. In the four samples successfully sequenced, a total of five missense single-nucleotide variants sequenced at high depth (>300×) with a frequency >5% was identified. BRAF c.1799T>A leading to p.V600E, an activating mutation in exon 15, predicted in silico as a pathogenic mutation, was reported in two of three AF (samples AF1 and AF2) (Table 1 and Figure 4). The other four single-nucleotide variants reported in this cohort were single-nucleotide polymorphisms: KDR p.Gln472His was reported in one of three AF and in the AFD; TP53 p.Pro72Arg was reported in one of three AF and in the AFD; KIT p.Met541Leu and PIK3CA p.Ile391Met were reported in the AFD. Table 1 shows the mutation status for each individual sample and for each tissue component of the mixed odontogenic tumor cases. In the validation cohort, the presence of the BRAF p.V600E mutation was assessed by allele-specific qPCR in the four samples successfully sequenced by NGS and confirmed in the two AF samples in which the mutation was originally detected (samples AF1 and AF2). Considering the results of the final validation cohort, BRAF p.V600E mutation was detected in a total of 4 of 10 AF (40%), 2 of 4 AFD (50%), 2 of 6 AFO (33%), and 2 of 3 AFS (67%) cases, whereas all 5 OD samples were wild type for the BRAF p.V600E mutation. The mutation was detected in the mesenchymal component of all mutant cases, but it was also detected in the epithelial component of one case of AF and one case of AFO. The incisional and excisional biopsies of one AFD case (AFD1) and one AFO (AFO6) were tested and showed concordant results. Interestingly, the AFS1 case presenting an AF-like area revealed BRAF p.V600E positivity in the mesenchymal component of both the sarcomatous and benign regions of the tumor. The mean ages of the mutant and wild-type cases were similar. The molecular pathogenesis of mixed odontogenic tumors has been poorly explored13Brown N.A. Rolland D. Mchugh J.B. Weigelin H.C. Zhao L. Lim M.S.1 Elenitoba-Johnson K.S. Betz B.L. Activating FGFR2-RAS-BRAF mutations in ameloblastoma.Clin Cancer Res. 2014; 20: 5517-5526Crossref PubMed Scopus (123) Google Scholar, 14Brunner P. Bihl M. Jundt G. Baumhoer D. Hoeller S. BRAF p.V600E mutations are not unique to ameloblastoma and are shared by other odontogenic tumors with ameloblastic morphology.Oral Oncol. 2015; 51: e77-e78Crossref PubMed Scopus (34) Google Scholar, 15You Z. Xu L.L. Li X.F. Zhang J.Y. DU J. Sun L.S. [BRAF gene mutations in ameloblastic fibromas].Beijing Da Xue Xue Bao Yi Xue Ban. 2019; 51 (Chinese): 4-8PubMed Google Scholar, 16Agaimy A. Skalova A. Franchi A. Alshagroud R. Gill A.J. Stoehr R. Baumhoer D. Bauer S. Ameloblastic fibrosarcoma: clinicopathological and molecular analysis of seven cases highlighting frequent BRAF and occasional NRAS mutations.Histopathology. 2020; 76: 814-821Crossref PubMed Scopus (5) Google Scholar,18Bernardes V.F. Gomes C.C. Gomez R.S. Molecular investigation of ameloblastic fibroma: how far have we gone?.Head Neck Oncol. 2012; 4: 45Google Scholar,19Sousa S.F. Gomez R.S. Diniz M.G. Bernardes V.F. Soares F.F. Brito J.A. Liu S. Pontes H.A. Stratakis C.A. Gomes C.C. Defects of the Carney complex gene (PRKAR1A) in odontogenic tumors.Endocr Relat Cancer. 2015; 22: 399-408Crossref PubMed Scopus (5) Google Scholar A high frequency of BRAF p.V600E pathogenic mutation has been reported in mixed odontogenic tumors, in 57% (4/7) of AF from two studies,13Brown N.A. Rolland D. Mchugh J.B. Weigelin H.C. Zhao L. Lim M.S.1 Elenitoba-Johnson K.S. Betz B.L. Activating FGFR2-RAS-BRAF mutations in ameloblastoma.Clin Cancer Res. 2014; 20: 5517-5526Crossref PubMed Scopus (123) Google Scholar,14Brunner P. Bihl M. Jundt G. Baumhoer D. Hoeller S. BRAF p.V600E mutations are not unique to ameloblastoma and are shared by other odontogenic tumors with ameloblastic morphology.Oral Oncol. 2015; 51: e77-e78Crossref PubMed Scopus (34) Google Scholar and a third study written in Chinese reported mutation in 16 of 16 AF cases in the abstract.15You Z. Xu L.L. Li X.F. Zhang J.Y. DU J. Sun L.S. [BRAF gene mutations in ameloblastic fibromas].Beijing Da Xue Xue Bao Yi Xue Ban. 2019; 51 (Chinese): 4-8PubMed Google Scholar Also, 33% (6/18) of AFO14Brunner P. Bihl M. Jundt G. Baumhoer D. Hoeller S. BRAF p.V600E mutations are not unique to ameloblastoma and are shared by other odontogenic tumors with ameloblastic morphology.Oral Oncol. 2015; 51: e77-e78Crossref PubMed Scopus (34) Google Scholar and 71% (5/7) of AFS16Agaimy A. Skalova A. Franchi A. Alshagroud R. Gill A.J. Stoehr R. Baumhoer D. Bauer S. Ameloblastic fibrosarcoma: clinicopathological and molecular analysis of seven cases highlighting frequent BRAF and occasional NRAS mutations.Histopathology. 2020; 76: 814-821Crossref PubMed Scopus (5) Google Scholar cases have been reported to harbor the BRAF mutation. Notably, the NRAS p.Q61K mutation was detected in one AFS wild-type for BRAF p.V600E.16Agaimy A. Skalova A. Franchi A. Alshagroud R. Gill A.J. Stoehr R. Baumhoer D. Bauer S. Ameloblastic fibrosarcoma: clinicopathological and molecular analysis of seven cases highlighting frequent BRAF and occasional NRAS mutations.Histopathology. 2020; 76: 814-821Crossref PubMed Scopus (5) Google Scholar Mixed odontogenic tumors have epithelial and mesenchymal components, and LCM is necessary to identify the component bearing the mutation. However, it has been used on only a few samples and in only one study.14Brunner P. Bihl M. Jundt G. Baumhoer D. Hoeller S. BRAF p.V600E mutations are not unique to ameloblastoma and are shared by other odontogenic tumors with ameloblastic morphology.Oral Oncol. 2015; 51: e77-e78Crossref PubMed Scopus (34) Google Scholar The results of the discovery cohort in the current study suggest that BRAF p.V600E may be the only potential driver mutation and is present in the same tissue component within each tumor type. The BRAF p.V600E mutation was found in 40% (4/10) AF, 33% (2/6) AFO, and 67% (2/3) AFS, in line with the results of previous studies.13Brown N.A. Rolland D. Mchugh J.B. Weigelin H.C. Zhao L. Lim M.S.1 Elenitoba-Johnson K.S. Betz B.L. Activating FGFR2-RAS-BRAF mutations in ameloblastoma.Clin Cancer Res. 2014; 20: 5517-5526Crossref PubMed Scopus (123) Google Scholar, 14Brunner P. Bihl M. Jundt G. Baumhoer D. Hoeller S. BRAF p.V600E mutations are not unique to ameloblastoma and are shared by other odontogenic tumors with ameloblastic morphology.Oral Oncol. 2015; 51: e77-e78Crossref PubMed Scopus (34) Google Scholar, 15You Z. Xu L.L. Li X.F. Zhang J.Y. DU J. Sun L.S. [BRAF gene mutations in ameloblastic fibromas].Beijing Da Xue Xue Bao Yi Xue Ban. 2019; 51 (Chinese): 4-8PubMed Google Scholar, 16Agaimy A. Skalova A. Franchi A. Alshagroud R. Gill A.J. Stoehr R. Baumhoer D. Bauer S. Ameloblastic fibrosarcoma: clinicopathological and molecular analysis of seven cases highlighting frequent BRAF and occasional NRAS mutations.Histopathology. 2020; 76: 814-821Crossref PubMed Scopus (5) Google Scholar Additionally, the BRAF mutation has been previously investigated in only one case of AFD,13Brown N.A. Rolland D. Mchugh J.B. Weigelin H.C. Zhao L. Lim M.S.1 Elenitoba-Johnson K.S. Betz B.L. Activating FGFR2-RAS-BRAF mutations in ameloblastoma.Clin Cancer Res. 2014; 20: 5517-5526Crossref PubMed Scopus (123) Google Scholar and in the current study, the authors report such mutation in 50% (2/4) of AFD cases studied. The authors have found BRAF mutation to be limited to the mesenchymal component of seven of the nine mutation-positive tumors subjected to LCM. The mutation in the epithelial component was limited to one AF and one AFO cases. It appears probable that the apparent epithelial mutation result could be accounted for by contamination with mesenchymal tissue and the high sensitivity of the qPCR assay. In both of these cases, the mutation was also found in the mesenchymal component. These results are different from those of Brunner et al,14Brunner P. Bihl M. Jundt G. Baumhoer D. Hoeller S. BRAF p.V600E mutations are not unique to ameloblastoma and are shared by other odontogenic tumors with ameloblastic morphology.Oral Oncol. 2015; 51: e77-e78Crossref PubMed Scopus (34) Google Scholar who reported that the mesenchymal component of one AF and three AFO cases with known positivity for BRAF p.V600E was wild type for BRAF. The authors attribute this difference to the fact that LCM was used in association with a highly sensitive mutation detection assay and that not all cases in the previous study were subject to LCM. Notably, both LCM AFS mutation-positive samples had the BRAF p.V600E mutation in only the mesenchymal component. Case AFS1 showed the mutation in both the benign mesenchymal component of the AF-like area and in the sarcomatous areas of the tumor. This study's results reinforce the reported malignant transformation from a pre-existing AF.4Takata T. Slootweg P.J. Benign mixed epithelial and mesenchymal odontogenic tumors.in: El-Naggar A.K. Chan J.K.C. Grandis J.R. Takata T. Slootweg P.J. WHO Classification of Head and Neck Tumors, ed 4. IARC, Lyon, France2017: 222-226Google Scholar,20Chrcanovic B.R. Brennan P.A. Rahimi S. Gomez R.S. Ameloblastic fibroma and ameloblastic fibrosarcoma: a systematic review.J Oral Pathol Med. 2018; 47: 315-325Crossref PubMed Scopus (19) Google Scholar Recently, BRAF p.V600E mutation was detected in both tumor components of one AFS, but immunohistochemistry using VE1 antibody showed mutation in the mesenchymal component only, Agaimy et al16Agaimy A. Skalova A. Franchi A. Alshagroud R. Gill A.J. Stoehr R. Baumhoer D. Bauer S. Ameloblastic fibrosarcoma: clinicopathological and molecular analysis of seven cases highlighting frequent BRAF and occasional NRAS mutations.Histopathology. 2020; 76: 814-821Crossref PubMed Scopus (5) Google Scholar speculating that there may have been cross contamination between tissue component samples because LCM was not used. This question has been answered by our LCM experiments which support their immunohistochemical results16Agaimy A. Skalova A. Franchi A. Alshagroud R. Gill A.J. Stoehr R. Baumhoer D. Bauer S. Ameloblastic fibrosarcoma: clinicopathological and molecular analysis of seven cases highlighting frequent BRAF and occasional NRAS mutations.Histopathology. 2020; 76: 814-821Crossref PubMed Scopus (5) Google Scholar suggesting that only the mesenchymal component harbours BRAF mutation. Additionally, this study's results confirm the long-held view that mixed odontogenic tumor growth is driven by the mesenchymal component and is concordant with the histopathologic features of the AFS, in which only the mesenchymal component appears malignant. Because both AF and AFS harbored the BRAF p.V600E mutation in the mesenchymal component, malignant transformation must be driven by other oncogenic stimuli. The histologic appearances of AFO and AFD are indistinguishable from those of odontomas in their early stages, so that their status as a separate tumor entity has been debated over the years.4Takata T. Slootweg P.J. Benign mixed epithelial and mesenchymal odontogenic tumors.in: El-Naggar A.K. Chan J.K.C. Grandis J.R. Takata T. Slootweg P.J. WHO Classification of Head and Neck Tumors, ed 4. IARC, Lyon, France2017: 222-226Google Scholar, 5Slootweg P.J. An analysis of the interrelationship of the mixed odontogenic tumors-ameloblastic fibroma, ameloblastic fibro-odontoma, and the odontomas.Oral Surg Oral Med Oral Pathol. 1981; 51: 266-276Abstract Full Text PDF PubMed Scopus (175) Google Scholar, 6Philipsen H.P. Reichart P.A. Praetorius F. Mixed odontogenic tumours and odontomas. Considerations on interrelationship. Review of the literature and presentation of 134 new cases of odontomas.Oral Oncol. 1997; 33: 86-99Crossref PubMed Scopus (220) Google Scholar, 7Tomich C.E. Benign mixed odontogenic tumors.Semin Diagn Pathol. 1999; 16: 308-316PubMed Google Scholar, 8Chen Y. Li T.-J. Gao Y. Yu S.-F. Ameloblastic fibroma and related lesions: a clinicopathologic study with reference to their nature and interrelationship.J Oral Pathol Med. 2005; 34: 588-595Crossref PubMed Scopus (53) Google Scholar, 9Buchner A. Kaffe I. Vered M. Clinical and radiological profile of ameloblastic fibro-odontoma: an update on an uncommon odontogenic tumor based on a critical analysis of 114 cases.Head Neck Pathol. 2013; 7: 54-63Crossref PubMed Scopus (31) Google Scholar, 10Chrcanovic B.R. Gomez R.S. Ameloblastic fibrodentinoma and ameloblastic fibro-odontoma: an updated systematic review of cases reported in the literature.J Oral Maxillofac Surg. 2017; 75: 1425-1437Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 11Speight P.M. Takata T. New tumour entities in the 4th edition of the World Health Organization Classification of Head and Neck tumours: odontogenic and maxillofacial bone tumours.Virchows Arch. 2018; 472: 331-339Crossref PubMed Scopus (116) Google Scholar In the last World Health Organization Classification of Head and Neck Tumors, AFD and AFO were not included as separate entities.4Takata T. Slootweg P.J. Benign mixed epithelial and mesenchymal odontogenic tumors.in: El-Naggar A.K. Chan J.K.C. Grandis J.R. Takata T. Slootweg P.J. WHO Classification of Head and Neck Tumors, ed 4. IARC, Lyon, France2017: 222-226Google Scholar Retrospective studies analyzing the clinical data of mixed odontogenic tumors do not support the progressive maturation theory of mixed odontogenic tumors to OD.8Chen Y. Li T.-J. Gao Y. Yu S.-F. Ameloblastic fibroma and related lesions: a clinicopathologic study with reference to their nature and interrelationship.J Oral Pathol Med. 2005; 34: 588-595Crossref PubMed Scopus (53) Google Scholar, 9Buchner A. Kaffe I. Vered M. Clinical and radiological profile of ameloblastic fibro-odontoma: an update on an uncommon odontogenic tumor based on a critical analysis of 114 cases.Head Neck Pathol. 2013; 7: 54-63Crossref PubMed Scopus (31) Google Scholar, 10Chrcanovic B.R. Gomez R.S. Ameloblastic fibrodentinoma and ameloblastic fibro-odontoma: an updated systematic review of cases reported in the literature.J Oral Maxillofac Surg. 2017; 75: 1425-1437Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar However, coming to a conclusion on this issue is challenging, and there is no definitive evidence to support this assumption. In the present study, the included cases had clinicoradiological features more suggestive of the diagnosis of true mixed odontogenic tumors, reaching a significant size. The BRAF mutation was detected in these mixed odontogenic tumors but was absent in all the OD cases studied. In a previous study by the authors' group,21Pereira N.B. de Souza J.C. Bastos V.C. Fonseca F.P. de Avelar G.F. Castro W.H. Dias A.A.M. Mosqueda-Taylor A. Gomez R.S. Gomes C.C. Patient-derived xenografts of a case of ameloblastic fibrodentinoma.Oral Dis. 2019; 25: 1229-1233Crossref PubMed Scopus (3) Google Scholar the development of a recurrent AFD case (that did not previously show maturation after 6 years) was evaluated using a patient-derived xenograft model. The patient-derived xenograft model was followed for 250 days, and it was observed to be tumorigenic and maintain morphologic characteristics of the primary tumor and MAPK pathway activation, together with cell proliferation activity similar to the human tumor.21Pereira N.B. de Souza J.C. Bastos V.C. Fonseca F.P. de Avelar G.F. Castro W.H. Dias A.A.M. Mosqueda-Taylor A. Gomez R.S. Gomes C.C. Patient-derived xenografts of a case of ameloblastic fibrodentinoma.Oral Dis. 2019; 25: 1229-1233Crossref PubMed Scopus (3) Google Scholar Taking together the molecular data and the above described results, the authors conclude that at least a subset of those lesions diagnosed as AF, AFD, and AFO are molecularly distinct from OD, which does not support the assumption that these subsets represent progressive stages of odontoma development and suggests they should not be grouped together. Though the study data appear consistent, the study has limitations that should be appreciated. First, several discovery cohort samples, which were from FFPE tissue, did not pass sequencing quality control, resulting in only four samples being successfully sequenced by NGS sequencing. The targeted NGS panel included 50 oncogenes and tumor suppressor genes. Though these are commonly mutated in human neoplasms, mutations in other genes could have been missed, and the number of samples is relatively low. In conclusion, although OD during their development mimic mixed odontogenic tumors histologically, at least a subset of AF, AFD, and AFO show a clear molecular profile with BRAF p.V600E in their mesenchymal components. This is distinct from OD, which are BRAF wild type. This suggests that at least a subset of AF, AFD, and AFO may be distinct pathologic entities and that at least some AFD and AFO may be neoplastic. Additionally, the molecular findings of this study strengthen the notion that malignant transformation of AF may be predisposed by the presence of the BRAF p.V600E mutation in the mesenchymal component and support the long-held view that mixed odontogenic tumor growth is driven by the mesenchymal component. We thank the Genomics Multi-User Laboratory (Centro de Laboratórios Multiusuários, ICB/Universidade Federal de Minas Gerais) for providing support on the next-generation sequencing, and Centro de Aquisição e Processamento de Imagens (CAPI – ICB/Universidade Federal de Minas Gerais) for the scanner service of histologic slides and for providing the equipment to perform laser capture microdissection. R.S.G., E.W.O., and C.C.G. conceived the study; B.P.C., V.F.B., S.F.d.S., M.G.D., R.G.M., and C.C.G. performed the experiments and analyzed the data; B.A.B.d.A., M.J.R., H.A.R.P., R.S.G., and E.W.O. obtained samples and data; B.P.C., V.F.B., M.G.D., R.S.G., and C.C.G. prepared the figures; B.P.C., V.F.B., and C.C.G., wrote the manuscript; R.S.G. and E.W.O. critically revised the manuscript; all authors have read and approved the final version of the manuscript; all authors gave their final approval and agreed to be accountable for all aspects of the work.
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