Cheng Shih-Ping, Hsu Yi-Chiung, Liu Chien-Liang, Liu Tsang-Pai, Chien Ming-Nan, Wang Tao-Yeuan, Lee Jie-Jen
Mackay Junior College of Medicine, Nursing, and Management, Taipei, Taiwan.
Ann Surg Oncol. 2014 Dec;21 Suppl 4:S619-26. doi: 10.1245/s10434-014-3723-5. Epub 2014 Apr 19.
Somatic BRAF mutation is frequently observed in papillary thyroid carcinoma (PTC). Recent evidence suggests that PTCs are heterogeneous tumors containing a subclonal or oligoclonal occurrence of BRAF mutation. Conflicting results have been reported concerning the prognostic significance of the mutant allele frequency. Our present aim was to investigate the association between the percentage of BRAF c.1799T > A (p.Val600Glu) alleles and clinicopathological parameters in PTC.
Genomic DNA was extracted from fresh-frozen specimens obtained from 50 PTC patients undergoing total thyroidectomy. The BRAF mutation status was determined by Sanger sequencing. The percentage of mutant BRAF alleles was quantified by mass spectrometric genotyping, pyrosequencing, and competitive allele-specific TaqMan PCR (castPCR).
Positive rate of BRAF mutation was 72 % by Sanger sequencing, 82 % by mass spectrometric genotying, and 84 % by pyrosequencing or castPCR. The average percentage of mutant BRAF alleles was 22.5, 31, and 30.7 %, respectively. There was a good correlation among three quantification methods (Spearman's rho = 0.87-0.97; p < 0.0001). The mutant allele frequency was significantly correlated with tumor size (rho = 0.47-0.52; p < 0.01) and extrathyroidal invasion. The frequency showed no difference in pathological lymph node metastasis.
The percentage of mutant BRAF alleles is positively associated with tumor burden and extrathyroidal invasion in PTC. Relatively good correlations exist among mass spectrometric genotyping, pyrosequencing, and castPCR in quantification of mutant BRAF allele frequency.
在甲状腺乳头状癌（PTC）中经常观察到体细胞BRAF突变。最近的证据表明，PTC是异质性肿瘤，包含BRAF突变的亚克隆或寡克隆发生。关于突变等位基因频率的预后意义，已有相互矛盾的报道。我们目前的目的是研究PTC中BRAF c.1799T＞A（p.Val600Glu）等位基因百分比与临床病理参数之间的关联。
从50例接受甲状腺全切除术的PTC患者的新鲜冷冻标本中提取基因组DNA。通过Sanger测序确定BRAF突变状态。通过质谱基因分型、焦磷酸测序和竞争性等位基因特异性TaqMan PCR（castPCR）对突变BRAF等位基因的百分比进行定量。
Sanger测序的BRAF突变阳性率为72%，质谱基因分型为82%，焦磷酸测序或castPCR为84%。突变BRAF等位基因的平均百分比分别为22.5%、31%和30.7%。三种定量方法之间具有良好的相关性（Spearman相关系数ρ=0.87 - 0.97；p＜0.0001）。突变等位基因频率与肿瘤大小（ρ=0.47 - 0.52；p＜0.01）和甲状腺外侵犯显著相关。该频率在病理淋巴结转移方面无差异。
PTC中突变BRAF等位基因的百分比与肿瘤负荷和甲状腺外侵犯呈正相关。在突变BRAF等位基因频率的定量方面，质谱基因分型、焦磷酸测序和castPCR之间存在相对良好的相关性。
