Abstract
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。
Keywords: 成釉细胞纤维瘤, BRAF基因突变, 靶向治疗
Abstract
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 V600E 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 V600E mutation in AF. The pathogenic role remains to be clarified.
Keywords: Ameloblastic fibroma, BRAF gene mutation, Targeted therapy
成釉细胞纤维瘤(ameloblastic fibroma,AF)是一种良性牙源性肿瘤,发生于儿童者属于发育中的混合性牙瘤、错构瘤,应保守治疗,但保守治疗可能导致约16%的患者复发,且有报道复发性AF有向肉瘤转变的可能[1,2];对大于22岁(即发育结束后)的AF患者应适当扩大切除范围,根治性治疗往往给患者带来巨大的影响,因此,寻求AF的无创靶向治疗新方法,从而最大程度地避免手术治疗给患者带来的颜面部畸形、心理障碍等影响显得尤为重要。
BRAF基因是RAF家族中的一种原癌基因,已发现大约有8%的肿瘤发生BRAF突变,包括黑色素瘤、乳头状甲状腺癌、毛细胞白血病、朗格汉斯细胞增生症、结直肠癌和胆管癌等[3,4,5,6,7]。BRAF突变导致丝裂原活化蛋白激酶信号通路持续活化,引发细胞异常增殖,进而导致肿瘤的发生。
近年来,BRAF 突变基因被证明是成釉细胞瘤中最显著的突变类型,但在AF中BRAF的突变情况尚无定论。本研究旨在探究AF中BRAF的突变情况,BRAF在AF中的极高突变率提示了临床上应用BRAF靶向抑制剂治疗该疾病的潜在价值。
1. 资料与方法
1.1. 研究对象
本研究经北京大学口腔医院生物医学伦理委员会批准,批准号为PKUSSIRB-201840167,所有研究对象均签署知情同意书。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,所有石蜡包埋组织经苏木精/伊红染色后由两名高年资口腔病理学专家检查确保病例选择的准确性。对患者的临床资料,如年龄、性别、部位、症状、手术方式和复发情况等进行回顾性分析。
1.2. 基因组DNA提取
选取10%甲醛固定的AF石蜡包埋组织,按照QIAamp DNA提取试剂盒(QIAamp DNA Mini Kit,Qiagen公司,德国)操作说明进行基因组DNA的提取。取2 μL DNA提取物于ND-8000紫外分光光度计(赛默飞世尔公司,美国)的检测小孔中进行DNA纯度和浓度的检测,检测结果保证DNA纯度值不小于1.6、浓度不小于200 mg/L方满足实验要求。DNA提取物随即进行PCR扩增或分装后于-20 ℃低温保存。
1.3. PCR反应
从美国国家生物技术信息中心的基因库中找出BRAF基因的mRNA序列(参考序列:NM_004333.4), 按照引物合成原则设计引物,交由北京华大基因科技有限公司进行引物合成。BRAF基因的15号外显子包含V600E突变位点,其特异性引物正向序列为5'-TGCTTGCTCTGATAGGAAAATG-3',反向序列为 5'-CCACAAAATGGATCCAGACA-3',产物长度为300 bp。PCR反应采用50 μL体系,含有 25 μL Ex Taq酶(Takara公司,日本)、正反引物各0.5 μL(每种引物约10 pmol)、22 μL无核酸酶水和100 ng DNA模板。使用以下条件进行PCR扩增: 94 ℃热启动5 min后进入循环,94 ℃变性30 s,60 ℃退火30 s,72 ℃延伸30 s,35个循环后72 ℃延伸10 min。
1.4. 测序分析
使用凝胶成像与分析系统(Vilber Lourmat公司,法国)观察DNA条带并记录图像,对PCR产物进行质量控制后进行纯化(GelExtraction Kit,Sigma公司,美国)。测序在AB 3730xl遗传分析仪(Applied Biosystems公司,美国)进行,使用两次独立正反测序确保检测结果的准确性。使用Chromas软件对测序峰图进行分析,同时采用基本局部比对搜索工具(http://blast.ncbi.nih.gov/Blast.cgi)对测序序列进行对比,确定是否发生突变及突变所在位置。
2. 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),22岁之前发病者共11例。病变常表现为无痛性肿胀,生长缓慢,随着时间推移可导致面部不对称。10例采取保守治疗方法,6例采取根治性治疗方法,共有3例复发。对患者的临床资料,如年龄、性别、部位、症状、手术方式和复发情况等进行了回顾性分析(表1)。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), 氨基酸由缬氨酸变为谷氨酸,从而导致胸腺嘧啶转化为腺苷酸(图1)。
1.
16例成釉细胞纤维瘤的临床资料
Clinical features of the 16 studied ameloblastic fibroma cases
| Case No. | Age/ years | Gender | Site | Symptoms | Treatment | Recurrence |
| M, male; F, female. | ||||||
| 1 | 23 | F | Left mandible | Swelling | Curettage | Recurrent, 14 years |
| 2 | 28 | F | Right mandible | Swelling | Lost to follow-up | Lost to follow up |
| 3 | 26 | M | Right mandible | Chin swelling and numbness |
Right mandibular segmental osteo- tomy and right iliac crest bone graft |
Recurrent, 1 month |
| 4 | 13 | F | Right mandible | Swelling | Right mandibular segmental osteotomy and right fibula flap repair |
Lost to follow up |
| 5 | 20 | F | Left mandible | Swelling, firm | Left mandibular segmental osteotomy and left fibula flap repair |
Lost to follow up |
| 6 | 1 | M | Right maxilla | Swelling | Curettage | Non recurrent, 3 years |
| 7 | 6 | M | Right maxilla | Swelling | Curettage | Lost to follow up |
| 8 | 13 | F | Right mandible | Swelling, firm | Curettage | Non recurrent, 3 years |
| 9 | 2 | M | Left mandible | Swelling | Curettage | Non recurrent,1 year |
| 10 | 3 | F | Right maxilla | Swelling | Curettage | Non recurrent, 7 months |
| 11 | 12 | F | Right mandible | Cystic neoplasm | Curettage | Non recurrent, 4 months |
| 12 | 22 | F | Left mandible | Swelling, firm | Curettage | Recurrent, 40 months |
| 13 | 67 | M | Right mandible | Swelling, firm, adhering to the bone |
Right mandibular segmental osteotomy and left fibula flap repair |
Non recurrent, 3 years |
| 14 | 41 | M | Left mandible | Swelling, repeated decline and growth |
Left mandibular segmental osteotomy and left fibula flap repair |
Non recurrent,16 months |
| 15 | 16 | M | Right mandible | Cystic neoplasm | Curettage | Non recurrent, 21 months |
| 16 | 6 | F | Left mandible | Swelling | Curettage | Non recurrent, 6 years |
1.
成釉细胞纤维瘤中检测到BRAF突变
Assessment of BRAF mutations in ameloblastic fibroma
A, PCR amplification with AB 3730xl identifies a BRAF exon 15 mutation encoding p.Val600Glu; B, wide-type BRAF of the normal tissue.
3. 讨论
AF由类似于牙乳头的间充质和类似于成釉器和牙板的上皮组织组成,不含釉质和牙本质等牙体硬组织[8]。现有临床资料显示,该病非常罕见,仅占所有牙源性肿瘤的1.5%~6.5%, 约80%的患者发病年龄在22岁以下,男、女比例为1.4 :1.0,上颌与下颌的比例为1.0 :3.3,特别好发于下颌骨后部区域[9,10]。AF生长缓慢,最常见的症状是下颌无痛性膨隆,约12%的患者是在拍摄X线片时偶然发现,其中56%的患者表现为边界清楚的单房透射影[11]。AF典型的组织学表现是同时包含外胚间叶和上皮成分。间质成分呈黏液状,可见幼稚纤维结缔组织聚集并形成少量的胶原纤维;间叶组织内富含类似于牙乳头的细胞,偶见局部玻璃样变性和钙化成骨。上皮细胞的形态类似于成釉细胞,可排列成条索状、小梁状或(和)岛状,从立方体到柱状过度,中央可见少量星形网状细胞[8]。除了组织病理表现之外,AF的诊断主要依靠病史、临床表现和影像学表现,鉴别诊断有牙源性角化囊肿和成釉细胞瘤等[8, 12]。在选择治疗方法时,患者的年龄应作为重要考虑因素。AF常被认为是非侵袭性肿瘤,复发与肿瘤的残留有关[13]。儿童、年轻人不宜采用根治性手术治疗,可采取剜除术、刮除术、单纯切除术等保守治疗方法,但对于成年人,由于其较高复发率和向成釉细胞纤维肉瘤恶性转化的特点[14,15,16],临床上往往按照“临界瘤”处理原则对其进行治疗:颌骨部分节段或方块切除,对突破骨皮质者行颌骨和肿瘤扩大切除,以降低复发恶性转化的风险[17,18]。
BRAF是鸟类原癌基因c-Rmil的人类同源物,该基因位于人染色体7q34,由18个外显子组成,长约190 kb,功能编码区由2 150 对碱基组成。细胞表面酪氨酸激酶受体激活了RAS蛋白家族的三磷酸鸟苷蛋白,激活的RAS结合并激活RAF蛋白激酶,RAF蛋白磷酸化后激活MEK1/2,进而激活ERK1/2从而促进细胞的分化、增殖和生长[19]。在人类癌症中发现了40多种不同的BRAF基因突变,但90%的突变位点集中于第15外显子的1 799位点,T 被A 替代,编码的氨基酸由缬氨酸变为谷氨酸(V600E)。该突变发生在激酶结构域,使得BRAF可不需要RAS蛋白磷酸化激活而处于持续活化状态,进而模拟T599 和S602 两个位点的磷酸化过程持续激活BRAF 蛋白激酶,造成RAF/MEK/ERK /MAPK信号通路持续活化,细胞无限制分裂增殖进而形成肿瘤[20]。
随着高通量测序技术的飞速发展,全基因组测序揭示了众多肿瘤的发生发展机制,且寻找到了新的驱动基因作为治疗靶点。多所研究机构发现在成釉细胞瘤中存在BRAF高突变率,综合文献报道BRAF在成釉细胞瘤中的突变率高达64.6%[21,22,23,24,25,26,27]。针对BRAF突变的靶向治疗也已取得令人满意的效果。Kaye等[28]报道了1例先后接受3次手术治疗的成釉细胞瘤男性患者,不幸的是经过这些手术治疗后该患者被诊断为Ⅳ期转移性成釉细胞瘤。确定携带BRAF突变后该患者选择了靶向抑制剂进行治疗,在使用达拉菲尼和曲美替尼4 d后,该患者的症状有了明显的改善,肺中的氟脱氧葡萄糖活性基本消失,8周后颈部、下颌和面部中的肿瘤的体积缩小明显。Faden等[29]报道了1例83岁的患者,该患者早在16年前被诊断为成釉细胞瘤,经历过两次手术后已不能耐受手术治疗。基因检测显示该患者伴有BRAF V600E突变,在服用达拉菲尼8个月后,MRI显示该患者的肿瘤体积减少了75%,并且在12个月时肿瘤体积仍在持续缩小。Tan等[30]报道了一名85岁携带BRAF突变的成釉细胞瘤男性患者,该患者在使用达拉菲尼进行治疗的前10周内肿瘤体积只有轻微的改变,但16周后肿瘤体积减少了90%以上。AF和成釉细胞瘤在组织学发生、临床和病理表现上有诸多相似之处,但AF中是否也存在BRAF的高突变率,文献回顾并没有得到确切答案[25]。本实验证实了AF同样存在BRAF的高突变率,尽管BRAF突变抑制剂在AF中的研究很少,但BRAF抑制剂在突变型成釉细胞瘤的治疗中所取得的临床效果同样为AF的靶向治疗带来了启发。
AF中存在BRAF V600E的高突变率,一方面,该突变可作为AF潜在的诊断标志物;另一方面,可考虑应用BRAF抑制剂作为AF的治疗措施,以提高治愈率,避免手术创伤及降低复发和恶变的概率。目前对AF中BRAF突变基因的研究较少,仍需进行大量的细胞实验、动物实验及临床试验来寻求AF切实可行的靶向治疗新方法。
Funding Statement
国家自然科学基金（30901680）; 北京大学医学科技创新平台发展基金（BMU2018PY023）
Supported by the National Natural Science Foundation of China(30901680); and the Fund for Fostering Young Scholars of Peking University Health Science Center(BMU2018PY023)
Footnotes
The authors have declared that no competing interests exist.
作者已声明无竞争性利益关系。
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