Zeng Qian, Xie Li, Zhou Na, Liu Min, Song Xianrang
Clinical Laboratory, Shandong Cancer Hospital & Institute, 440 Ji-Yan Road, Jinan, 250117, Shandong Province, People's Republic of China.
Clinical Laboratory, Qilu Children's Hospital of Shandong University, 23976 Jing-Shi Road, Jinan, 250022, Shandong, People's Republic of China.
Mol Diagn Ther. 2017 Aug;21(4):443-451. doi: 10.1007/s40291-017-0269-9.
Mutant Phosphatidylinositol-4, 5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) has been shown to be associated with the occurrence, development and prognosis in colorectal cancer (CRC). However, its detection has been limited because of complicated procedures and the low sensitivity of the present approaches.
We established an ultra-sensitive peptide nucleic acid-mediated polymerase chain reaction (PNA-PCR) assay to detect PIK3CA gene mutation in exon 9 and exon 20 with cell-free DNA (cfDNA). Using this technology, we detected the mutation status of PIK3CA in 128 colorectal cancer patients. 6 CRC patients receiving targeted therapy were chosen at random to undergo continuous PIK3CA mutation detection.
The results showed that the sensitivity of PNA-PCR clamping method was 0.1% for the exon 9 and 0.2% for the exon 20 variant alleles. When the PIK3CA mutation status was determined by PNA-PCR plus sequencing, 38.3% (49/128) of CRC carried at least one mutation, either E545Kor H1047R. The clinic-pathological parameters of age (p = 0.358), gender (p = 0.622), disease stage (p = 0.353) and disease location (p = 0.307) were not associated with the PIK3CA mutation. In the continuous monitoring study, we found that the gene status was associated with the effect of treatment. Furthermore, when the PIK3CA variant was determined by only the PNA-PCR method, there was a good linear relationship between ΔCp values and the proportion of variant DNA. The accuracy of PNA-PCR was 93.75 and 92.27% respectively when the cut-off values of ΔCp at 9.0 and 8.0 were set for determining the E545K and H1047R mutations.
A simple, noninvasive, ultra-sensitive PNA-PCR technology was developed and was especially suitable for the dynamic detection of PIK3CA variants using cfDNA.
突变型磷脂酰肌醇-4,5-二磷酸3-激酶催化亚基α（PIK3CA）已被证明与结直肠癌（CRC）的发生、发展及预后相关。然而，由于检测程序复杂且现有方法灵敏度较低，其检测受到限制。
我们建立了一种超灵敏的肽核酸介导的聚合酶链反应（PNA-PCR）检测方法，用于检测游离DNA（cfDNA）中PIK3CA基因第9外显子和第20外显子的突变。利用该技术，我们检测了128例结直肠癌患者中PIK3CA的突变状态。随机选择6例接受靶向治疗的CRC患者进行PIK3CA突变的连续检测。
结果显示，PNA-PCR钳夹法对外显子9变异等位基因的灵敏度为0.1%，对外显子20变异等位基因的灵敏度为0.2%。当通过PNA-PCR加测序确定PIK3CA突变状态时，38.3%（49/128）的CRC患者携带至少一种突变，即E545K或H1047R。年龄（p = 0.358）、性别（p = 0.622）、疾病分期（p = 0.353）和疾病部位（p = 0.307）的临床病理参数与PIK3CA突变无关。在连续监测研究中，我们发现基因状态与治疗效果相关。此外，当仅通过PNA-PCR方法确定PIK3CA变异时，ΔCp值与变异DNA比例之间存在良好的线性关系。当将ΔCp的截断值分别设为9.0和8.0来确定E545K和H1047R突变时，PNA-PCR的准确率分别为93.75%和92.27%。
开发了一种简单、无创、超灵敏的PNA-PCR技术，特别适用于使用cfDNA对PIK3CA变异进行动态检测。
