Highly sensitive detection of the PIK3CA (H1047R) mutation in colorectal cancer using a novel PCR-RFLP method.
Highly sensitive detection of the PIK3CA (H1047R) mutation in colorectal cancer using a novel PCR-RFLP method.
复制标题
使用新型 PCR-RFLP 方法高灵敏度检测结直肠癌中的 PIK3CA(H1047R) 突变
DOI：
10.1186/s12885-016-2493-9
复制
发表时间：
2016-07-12
期刊：
影响因子：
3.8
通讯作者：
Fang J
中科院分区：
文献类型：
--
作者：
Li WM;Hu TT;Zhou LL;Feng YM;Wang YY;Fang J
PIK 3CAH 1047 R突变被认为是EGFR靶向治疗的潜在预测生物标志物。在这项研究中，我们开发了一种新的PCR-PFLP方法来检测PIK 3CAH 1047 R突变的高效率。PCR扩增PIK 3CA基因外显子20的126 bp片段，经FspI酶切、3%琼脂糖凝胶电泳后进行PCR-RFLP分析。将PIK 3CAH 1047 R的突变体序列以降低的比率掺入相应的野生型序列中用于灵敏度分析。对86例福尔马林固定石蜡包埋的结直肠癌标本进行PCR-RFLP分析，以评价该方法的适用性。PCR-RFLP方法检测率为0.4%，86例大肠癌组织中PIK 3CAH 1047 R突变检出率为16.3%，显著高于DNA测序法的检出率（9.3%）。首次发现PIK 3CAH 1047 R突变与患者年龄呈正相关，但与肿瘤分化程度呈负相关。此外，使用单独的PCR-RFLP方法实现了PIK 3CA、KRAS和BRAF组合突变的高灵敏度检测。我们建立了一种灵敏、简便、快速的方法检测真实的CRC标本中低丰度PIK 3 CAH 1047 R突变，为指导癌症靶向治疗提供了有效工具。
The PIK3CAH1047R mutation is considered to be a potential predictive biomarker for EGFR-targeted therapies. In this study, we developed a novel PCR-PFLP approach to detect the PIK3CAH1047R mutation in high effectiveness. A 126-bp fragment of PIK3CA exon-20 was amplified by PCR, digested with FspI restriction endonuclease and separated by 3 % agarose gel electrophoresis for the PCR-RFLP analysis. The mutant sequence of the PIK3CAH1047R was spiked into the corresponding wild-type sequence in decreasing ratios for sensitivity analysis. Eight-six cases of formalin-fixed paraffin-embedded colorectal cancer (CRC) specimens were subjected to PCR-RFLP to evaluate the applicability of the method. The PCR-RFLP method had a capability to detect as litter as 0.4 % of mutation, and revealed 16.3 % of the PIK3CAH1047R mutation in 86 CRC tissues, which was significantly higher than that discovered by DNA sequencing (9.3 %). A positive association between the PIK3CAH1047R mutation and the patients’ age was first found, except for the negative relationship with the degree of tumor differentiation. In addition, the highly sensitive detection of a combinatorial mutation of PIK3CA, KRAS and BRAF was achieved using individual PCR-RFLP methods. We developed a sensitive, simple and rapid approach to detect the low-abundance PIK3CAH1047R mutation in real CRC specimens, providing an effective tool for guiding cancer targeted therapy.