成釉细胞纤维瘤中BRAF突变基因的检测
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摘要
目的:检测BRAF基因在成釉细胞纤维瘤(ameloblastic fibroma,AF)中的突变情况,进一步分析该突变与AF的临床表现之间的关系,以期为寻求AF的靶向治疗提供新方法。方法:收集2002年1月至2015年12月期间北京大学口腔医院诊断为AF的病例16例。AF石蜡包埋组织经切片后进行苏木精伊红染色,由两名病理科专家确认病例选取的准确性,按照DNA提取试剂盒使用说明提取组织DNA。对BRAF V600E位点进行PCR扩增,采用直接测序的方法进行BRAF V600E的突变检测,并进一步分析16例AF的临床资料。结果:16例AF患者中包括7例男性,9例女性; 3例发生于上颌,13例发生于下颌;入院年龄为2～67岁(中位年龄14. 5岁)。病变常表现为无痛性肿胀,生长缓慢。16例AF均携带BRAF基因突变,突变率为100%(16/16),均为15号外显子上V600E突变型(c. 1799T> A),导致在氨基酸水平上由缬氨酸变为谷氨酸,从而使胸腺嘧啶转化为腺苷酸。现有病例表明BRAF突变与AF的年龄、性别、发病部位和复发等无相关性。结论:AF中存在BRAF V600E位点的高突变率,提示BRAF V600E突变可能成为AF发生发展的关键事件,同时为AF患者应用BRAF抑制剂进行靶向治疗提供了一定的理论支持,其病理学意义有待进一步研究。
Objective: To investigate the BRAF gene mutations in ameloblastic fibroma(AF),and to further analyze the relationship between the BRAF mutation and clinical characteristics so as to provide new reference to the study of AF's molecular pathology. Methods: Sixteen cases diagnosed as AF at the Department of Oral Pathology,Peking University School of Stomatology between January 1990 and December 2017 were collected. Genomic DNA was extracted from formalin-fixed,paraffin embedded tissues using the QIAamp DNA Mini Kit(Qiagen,Germany) according to the manufacturer's instructions.Polymerase chain reaction(PCR) and direct sequencings were used to detect the BRAF gene mutations.The clinicopathological data,such as the age,location of the lesion,symptoms and treatments were retrospectively analyzed. Results: The sixteen cases of AF involved nine women and seven men aged 2-67 years. Three lesions occurred in the maxilla and thirteen in the mandible. The most common presenting symptom of AF was a painless slowly enlarging mass with swelling. Ten patients received conservative treatment and the other six patients received radical surgery. Three cases relapsed during the study period. BRAF gene mutation was found in sixteen of all the sixteen samples analyzed(100%). The BRAF mutation was a point mutation with a thymine-adenine transversion at nucleotide 1 799 of 15 exons,resulting in a change at residue 600 that substituted glutamine for valine. This mutation was the strongest activator of the downstream RAS/RAF/MEK/ERK-MAPK signaling pathway. This helped to bring about a gain-of-function mutation due to a V600 E substitution. Many studies identified that BRAF regulated survival,apoptosis,and proliferation of cells by inducing MAPK pathways activation. For the existing cases,none of the age,sex,location,recurrence and treatments had a statistically significant correlation with BRAF mutation. Conclusion: Our findings demonstrated high prevalence of BRAF V600 E mutation in AF. The pathogenic role remains to be clarified.
Objective: To investigate the BRAF gene mutations in ameloblastic fibroma(AF),and to further analyze the relationship between the BRAF mutation and clinical characteristics so as to provide new reference to the study of AF's molecular pathology. Methods: Sixteen cases diagnosed as AF at the Department of Oral Pathology,Peking University School of Stomatology between January 1990 and December 2017 were collected. Genomic DNA was extracted from formalin-fixed,paraffin embedded tissues using the QIAamp DNA Mini Kit(Qiagen,Germany) according to the manufacturer's instructions.Polymerase chain reaction(PCR) and direct sequencings were used to detect the BRAF gene mutations.The clinicopathological data,such as the age,location of the lesion,symptoms and treatments were retrospectively analyzed. Results: The sixteen cases of AF involved nine women and seven men aged 2-67 years. Three lesions occurred in the maxilla and thirteen in the mandible. The most common presenting symptom of AF was a painless slowly enlarging mass with swelling. Ten patients received conservative treatment and the other six patients received radical surgery. Three cases relapsed during the study period. BRAF gene mutation was found in sixteen of all the sixteen samples analyzed(100%). The BRAF mutation was a point mutation with a thymine-adenine transversion at nucleotide 1 799 of 15 exons,resulting in a change at residue 600 that substituted glutamine for valine. This mutation was the strongest activator of the downstream RAS/RAF/MEK/ERK-MAPK signaling pathway. This helped to bring about a gain-of-function mutation due to a V600 E substitution. Many studies identified that BRAF regulated survival,apoptosis,and proliferation of cells by inducing MAPK pathways activation. For the existing cases,none of the age,sex,location,recurrence and treatments had a statistically significant correlation with BRAF mutation. Conclusion: Our findings demonstrated high prevalence of BRAF V600 E mutation in AF. The pathogenic role remains to be clarified.
引文
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[2] Slootweg PJ. An analysis of the interrelationship of the mixed odontogenic tumors:ameloblastic fibroma, ameloblastic fibroodontoma,and the odontomas[J]. Oral Surg Oral Med Oral Pathol,1981,51(3):266-276.
[3] Davies H,Bignell GR,Cox C,et al. Mutations of the BRAF gene in human cancer[J]. Nature,2002,417(6892):949-954.
[4] Curtin JA,Fridlyand J,Kageshita T,et al. Distinct sets of genetic alterationsin melanoma[J]. N Engl J Med,2005,353(20):2135-2147.
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[7] Puxeddu E,Moretti S,Elisei R,et al. BRAF(V599E)mutation is the leading genetic event in adult sporadic papillary thyroid carcinomas[J]. J Clin Endocrinol Metab,2004,89(5):2414-2420.
[8] Philipsen HP,Reichart PA,Praetorius F. Mixed odontogenic tumours and odontomas. Considerations on interrelationship. Review of the literature and presentation of 134 new cases of odontomas[J]. Oral Oncol,1997,33(2):86-99.
[9] Chen Y,Wang JM,Li TJ. Ameloblastic fibroma:a review of published studies with special reference to its nature and biological behavior[J]. Oral Oncol,2007,43(10):960-969.
[10] Buchner A,Vered M. Ameloblastic fibroma:a stage in the development of a hamartomatous odontoma or a true neoplasm? Critical analysis of 162 previously reported cases plus 10 new cases[J].Oral Surg Oral Med Oral Pathol Oral Radiol,2013,116(5):598-606.
[11] Trodahl JN. Ameloblastic fibroma. A survey of cases from the Armed Forces Institute of Pathology[J]. Oral Surg Oral Med Oral Pathol,1972,33(4):547-558.
[12] Kim SG,Jang HS. Ameloblastic fibroma:report of a case[J]. J Oral Maxillofac Surg,2002,60(2):216-218.
[13] Hansen LS,Ficarra G. Mixed odontogenic tumors:an analysis of23 new cases[J]. Head Neck Surg,1988,10(5):330-343.
[14] Bertoni F,Del CG,Bacchini P,et al. Ameloblastic fibrosarcoma of the mandible evolving from a prior ameloblastic fibroma after two years:an unusual finding[J]. Int J Surg Pathol,2016,24(7):656-659.
[15] Abughazaleh K,Andrus KM,Katsnelson A,et al. Peripheral ameloblastic fibroma of the maxilla:report of a case and review of the literature[J]. Oral Surg Oral Med Oral Pathol Oral Radiol Endod,2008,105(5):e46-e48.
[16] Kale SG,Shetty A,Balakrishnan J,et al. Ameloblastic fibroodontoma with a predominant radiopaque component[J]. Ann Maxillofac Surg,2017,7(2):304-307.
[17] Kobayashi K,Murakami R,Fujii T,et al. Malignant transformation of ameloblastic fibroma to ameloblastic fibrosarcoma:case report and review of the literature[J]. J Craniomaxillofac Surg,2005,33(5):352-355.
[18] Vasconcelos BC,Andrade ES,Rocha NS,et al. Treatment of large ameloblastic fibroma:a case report[J]. J Oral Sci,2009,51(2):293-296.
[19] Yaktapour N,Meiss F,Mastroianni J,et al. BRAF inhibitor-associated ERK activation drives development of chronic lymphocytic leukemia[J]. J Clin Invest,2014,124(11):5074-5084.
[20] Hou P,Liu D,Xing M. Functional characterization of the T1799-1801del and A1799-1816ins BRAF mutations in papillary thyroid cancer[J]. Cell Cycle,2007,6(3):377-379.
[21] Kurppa KJ,Caton J,Morgan PR,et al. High frequency of BRAF V600E mutations in ameloblastoma[J]. J Pathol,2014,232(5):492-498.
[22] Sweeney RT,Mcclary AC,Myers BR,et al. Identification of recurrent SMO and BRAF mutations in ameloblastomas[J]. Nat Genet,2014,46(7):722-725.
[23] Brown NA,Rolland D,Mchugh JB,et al. Activating FGFR2-RAS-BRAF mutations in ameloblastoma[J]. Clin Cancer Res,2014,20(21):5517-5526.
[24] Brunner P,Bihl M,Jundt G,et al. BRAF p. V600E mutations are not unique to ameloblastoma and are shared by other odontogenic tumors with ameloblastic morphology[J]. Oral Oncol,2015,51(10):e77-e78.
[25] Diniz MG,Gomes CC,Guimaraes BV,et al. Assessment of BRAFV600E and SMOF412E mutations in epithelial odontogenic tumours[J]. Tumour Biol,2015,36(7):5649-5653.
[26] Soltani M,Tabatabaiefar MA,Mohsenifar Z,et al. Genetic study of the BRAF gene reveals new variants and high frequency of the V600E mutation among Iranian ameloblastoma patients[J]. J Oral Pathol Med,2018,47(1):86-90.
[27] Gultekin SE,Aziz R,Heydt C,et al. The landscape of genetic alterations in ameloblastomas relates to clinical features[J]. Virchows Arch,2018,472(5):807-814.
[28] Kaye FJ,Ivey AM,Drane WE,et al. Clinical and radiographic response with combined BRAF-targeted therapy in stage 4 ameloblastoma[J]. J Natl Cancer Inst,2015,107(1):378.
[29] Faden DL,Algazi A. Durable treatment of ameloblastoma with single agent BRAFi Re:Clinical and radiographic response with combined BRAF-targeted therapy in stage 4 ameloblastoma[J]. J Natl Cancer Inst,2017,109(1):dwj190
[30] Tan S,Pollack JR,Kaplan MJ,et al. BRAF inhibitor treatment of primary BRAF-mutant ameloblastoma with pathologic assessment of response[J]. Oral Surg Oral Med Oral Pathol Oral Radiol,2016,122(1):e5-e7.
[2] Slootweg PJ. An analysis of the interrelationship of the mixed odontogenic tumors:ameloblastic fibroma, ameloblastic fibroodontoma,and the odontomas[J]. Oral Surg Oral Med Oral Pathol,1981,51(3):266-276.
[3] Davies H,Bignell GR,Cox C,et al. Mutations of the BRAF gene in human cancer[J]. Nature,2002,417(6892):949-954.
[4] Curtin JA,Fridlyand J,Kageshita T,et al. Distinct sets of genetic alterationsin melanoma[J]. N Engl J Med,2005,353(20):2135-2147.
[5] Itamura H,Ide M,Sato A,et al. Identification of the BRAF V600E mutation in Japanese patients with hairy cell leukemia and related diseases using a quenching probe method[J]. Int J Hematol,2018,108(4):416-422.
[6] Maria MD,Adamaki M,Vlahopoulos S,et al. Synchronous and metachronous thyroid cancer in relation to Langerhans cell histiocytosis; involvement of V600E BRAF mutation?[J]. Pediatr Blood Cancer,2015,62(1):173-174.
[7] Puxeddu E,Moretti S,Elisei R,et al. BRAF(V599E)mutation is the leading genetic event in adult sporadic papillary thyroid carcinomas[J]. J Clin Endocrinol Metab,2004,89(5):2414-2420.
[8] Philipsen HP,Reichart PA,Praetorius F. Mixed odontogenic tumours and odontomas. Considerations on interrelationship. Review of the literature and presentation of 134 new cases of odontomas[J]. Oral Oncol,1997,33(2):86-99.
[9] Chen Y,Wang JM,Li TJ. Ameloblastic fibroma:a review of published studies with special reference to its nature and biological behavior[J]. Oral Oncol,2007,43(10):960-969.
[10] Buchner A,Vered M. Ameloblastic fibroma:a stage in the development of a hamartomatous odontoma or a true neoplasm? Critical analysis of 162 previously reported cases plus 10 new cases[J].Oral Surg Oral Med Oral Pathol Oral Radiol,2013,116(5):598-606.
[11] Trodahl JN. Ameloblastic fibroma. A survey of cases from the Armed Forces Institute of Pathology[J]. Oral Surg Oral Med Oral Pathol,1972,33(4):547-558.
[12] Kim SG,Jang HS. Ameloblastic fibroma:report of a case[J]. J Oral Maxillofac Surg,2002,60(2):216-218.
[13] Hansen LS,Ficarra G. Mixed odontogenic tumors:an analysis of23 new cases[J]. Head Neck Surg,1988,10(5):330-343.
[14] Bertoni F,Del CG,Bacchini P,et al. Ameloblastic fibrosarcoma of the mandible evolving from a prior ameloblastic fibroma after two years:an unusual finding[J]. Int J Surg Pathol,2016,24(7):656-659.
[15] Abughazaleh K,Andrus KM,Katsnelson A,et al. Peripheral ameloblastic fibroma of the maxilla:report of a case and review of the literature[J]. Oral Surg Oral Med Oral Pathol Oral Radiol Endod,2008,105(5):e46-e48.
[16] Kale SG,Shetty A,Balakrishnan J,et al. Ameloblastic fibroodontoma with a predominant radiopaque component[J]. Ann Maxillofac Surg,2017,7(2):304-307.
[17] Kobayashi K,Murakami R,Fujii T,et al. Malignant transformation of ameloblastic fibroma to ameloblastic fibrosarcoma:case report and review of the literature[J]. J Craniomaxillofac Surg,2005,33(5):352-355.
[18] Vasconcelos BC,Andrade ES,Rocha NS,et al. Treatment of large ameloblastic fibroma:a case report[J]. J Oral Sci,2009,51(2):293-296.
[19] Yaktapour N,Meiss F,Mastroianni J,et al. BRAF inhibitor-associated ERK activation drives development of chronic lymphocytic leukemia[J]. J Clin Invest,2014,124(11):5074-5084.
[20] Hou P,Liu D,Xing M. Functional characterization of the T1799-1801del and A1799-1816ins BRAF mutations in papillary thyroid cancer[J]. Cell Cycle,2007,6(3):377-379.
[21] Kurppa KJ,Caton J,Morgan PR,et al. High frequency of BRAF V600E mutations in ameloblastoma[J]. J Pathol,2014,232(5):492-498.
[22] Sweeney RT,Mcclary AC,Myers BR,et al. Identification of recurrent SMO and BRAF mutations in ameloblastomas[J]. Nat Genet,2014,46(7):722-725.
[23] Brown NA,Rolland D,Mchugh JB,et al. Activating FGFR2-RAS-BRAF mutations in ameloblastoma[J]. Clin Cancer Res,2014,20(21):5517-5526.
[24] Brunner P,Bihl M,Jundt G,et al. BRAF p. V600E mutations are not unique to ameloblastoma and are shared by other odontogenic tumors with ameloblastic morphology[J]. Oral Oncol,2015,51(10):e77-e78.
[25] Diniz MG,Gomes CC,Guimaraes BV,et al. Assessment of BRAFV600E and SMOF412E mutations in epithelial odontogenic tumours[J]. Tumour Biol,2015,36(7):5649-5653.
[26] Soltani M,Tabatabaiefar MA,Mohsenifar Z,et al. Genetic study of the BRAF gene reveals new variants and high frequency of the V600E mutation among Iranian ameloblastoma patients[J]. J Oral Pathol Med,2018,47(1):86-90.
[27] Gultekin SE,Aziz R,Heydt C,et al. The landscape of genetic alterations in ameloblastomas relates to clinical features[J]. Virchows Arch,2018,472(5):807-814.
[28] Kaye FJ,Ivey AM,Drane WE,et al. Clinical and radiographic response with combined BRAF-targeted therapy in stage 4 ameloblastoma[J]. J Natl Cancer Inst,2015,107(1):378.
[29] Faden DL,Algazi A. Durable treatment of ameloblastoma with single agent BRAFi Re:Clinical and radiographic response with combined BRAF-targeted therapy in stage 4 ameloblastoma[J]. J Natl Cancer Inst,2017,109(1):dwj190
[30] Tan S,Pollack JR,Kaplan MJ,et al. BRAF inhibitor treatment of primary BRAF-mutant ameloblastoma with pathologic assessment of response[J]. Oral Surg Oral Med Oral Pathol Oral Radiol,2016,122(1):e5-e7.