Two-round coamplification at lower denaturation temperature-PCR (COLD-PCR)-based sanger sequencing identifies a novel spectrum of low-level mutations in lung adenocarcinoma.
Two-round coamplification at lower denaturation temperature-PCR (COLD-PCR)-based sanger sequencing identifies a novel spectrum of low-level mutations in lung adenocarcinoma.
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DOI：
10.1002/humu.21112
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发表时间：
2009-11
期刊：
影响因子：
3.9
通讯作者：
Makrigiorgos, G. Mike
中科院分区：
文献类型：
--
作者：
Li, Jin;Milbury, Coren A.;Li, Cheng;Makrigiorgos, G. Mike
TP53 中与癌症相关的突变的可靠鉴定通常存在问题，因为这些突变可以随机分布在众多密码子中，并且它们在临床样本中的相对丰度可能会低于传统测序的灵敏度极限。为了确保突变检测的最高灵敏度，我们采用了最近描述的 COLD-PCR 方法，采用连续两轮 COLD-PCR，然后进行桑格测序。使用这种高度敏感的方法，我们筛查了 48 个非显微解剖肺腺癌样本的 TP53 突变。在检查的 48 个肺腺癌样本中的 23 个 (48%) 中，发现了 24 个遍及外显子 5-8 的错义/移码 TP53 突变，其中包括 8 个丰度约为 1-17% 的低水平突变，其中大多数突变使用传统方法会被遗漏。已发现的改变包括两种罕见的肺腺癌突变，其中一种是目前肺癌突变数据库中尚未记录的“破坏性”突变。含有低水平突变（约 2% 丰度）和克隆突变（80% 丰度）的样本揭示了肿瘤内 TP53 突变异质性。通过基于 COLD-PCR 的桑格测序，能够在没有精细显微解剖的情况下识别和测序低水平突变，为临床样本中准确的突变分析以及使用 TP53 作为预后/预测生物标志物、评估癌症风险、复发和进一步了解癌症生物学提供了一个平台。
Reliable identification of cancer-related mutations in TP53 is often problematic as these mutations can be randomly distributed throughout numerous codons and their relative abundance in clinical samples can fall below the sensitivity limits of conventional sequencing. To ensure the highest sensitivity in mutation detection, we adapted the recently described COLD-PCR method to employ two consecutive rounds of COLD-PCR followed by Sanger sequencing. Using this highly sensitive approach we screened 48 non-microdissected lung-adenocarcinoma samples for TP53 mutations. Twenty-four missense/frameshift TP53 mutations throughout exons 5–8 were identified in 23 of 48 (48%) lung-adenocarcinoma samples examined, including 8 low-level mutations at an abundance of ~1–17%, most of which would have been missed using conventional methodologies. The identified alterations include two rare lung-adenocarcinoma mutations, one of which is a ‘disruptive’ mutation currently undocumented in the lung cancer mutation-databases. A sample harboring a low-level mutation (~2% abundance) concurrently with a clonal mutation (80%-abundance) revealed intra-tumoral TP53 mutation heterogeneity. The ability to identify and sequence low-level mutations in the absence of elaborate micro-dissection, via COLD-PCR-based Sanger-sequencing, provides a platform for accurate mutation profiling in clinical specimens and the use of TP53 as a prognostic/predictive biomarker, evaluation of cancer risk, recurrence, and further understanding of cancer biology.
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影响因子：
9.3
作者：
Milbury CA;Li J;Makrigiorgos GM
通讯作者：
Makrigiorgos GM
影响因子：
3.7
作者：
Chen Z;Feng J;Buzin CH;Sommer SS
通讯作者：
Sommer SS
影响因子：
3.9
作者：
Olivier, M;Eeles, R;Hainaut, P
通讯作者：
Hainaut, P
DOI：
10.1073/pnas.0607057103
发表时间：
2006-11-28
影响因子：
11.1
作者：
Bielas, Jason H.;Loeb, Keith R.;Loeb, Lawrence A.
通讯作者：
Loeb, Lawrence A.
影响因子：
7.5
作者：
Delaney, David;Diss, Tim C.;Flanagan, Adrienne M.
通讯作者：
Flanagan, Adrienne M.