Krypuy Michael, Ahmed Ahmed Ashour, Etemadmoghadam Dariush, Hyland Sarah J, DeFazio Anna, Fox Stephen B, Brenton James D, Bowtell David D, Dobrovic Alexander
Molecular Pathology Research and Development Laboratory, Department of Pathology, Peter MacCallum Cancer Centre, Locked Bag 1, A'Beckett St, Melbourne, Victoria 8006, Australia.
BMC Cancer. 2007 Aug 31;7:168. doi: 10.1186/1471-2407-7-168.
p53 is commonly inactivated by mutations in the DNA-binding domain in a wide range of cancers. As mutant p53 often influences response to therapy, effective and rapid methods to scan for mutations in TP53 are likely to be of clinical value. We therefore evaluated the use of high resolution melting (HRM) as a rapid mutation scanning tool for TP53 in tumour samples.
We designed PCR amplicons for HRM mutation scanning of TP53 exons 5 to 8 and tested them with DNA from cell lines hemizygous or homozygous for known mutations. We assessed the sensitivity of each PCR amplicon using dilutions of cell line DNA in normal wild-type DNA. We then performed a blinded assessment on ovarian tumour DNA samples that had been previously sequenced for mutations in TP53 to assess the sensitivity and positive predictive value of the HRM technique. We also performed HRM analysis on breast tumour DNA samples with unknown TP53 mutation status.
One cell line mutation was not readily observed when exon 5 was amplified. As exon 5 contained multiple melting domains, we divided the exon into two amplicons for further screening. Sequence changes were also introduced into some of the primers to improve the melting characteristics of the amplicon. Aberrant HRM curves indicative of TP53 mutations were observed for each of the samples in the ovarian tumour DNA panel. Comparison of the HRM results with the sequencing results revealed that each mutation was detected by HRM in the correct exon. For the breast tumour panel, we detected seven aberrant melt profiles by HRM and subsequent sequencing confirmed the presence of these and no other mutations in the predicted exons.
HRM is an effective technique for simple and rapid scanning of TP53 mutations that can markedly reduce the amount of sequencing required in mutational studies of TP53.
在多种癌症中，p53通常因DNA结合结构域的突变而失活。由于突变型p53常常影响治疗反应，因此，有效且快速的TP53突变检测方法可能具有临床价值。我们因此评估了高分辨率熔解曲线分析（HRM）作为肿瘤样本中TP53突变快速扫描工具的应用。
我们设计了用于TP53第5至8外显子HRM突变扫描的PCR扩增子，并用已知突变的半合子或纯合子细胞系的DNA进行检测。我们使用细胞系DNA与正常野生型DNA的稀释液评估每个PCR扩增子的敏感性。然后，我们对先前已进行TP53突变测序的卵巢肿瘤DNA样本进行盲法评估，以评估HRM技术的敏感性和阳性预测值。我们还对TP53突变状态未知的乳腺肿瘤DNA样本进行了HRM分析。
扩增第5外显子时，一种细胞系突变不易观察到。由于第5外显子包含多个熔解结构域，我们将该外显子分为两个扩增子进行进一步筛查。还对一些引物引入了序列改变以改善扩增子的熔解特性。在卵巢肿瘤DNA样本组中，每个样本均观察到指示TP53突变的异常HRM曲线。将HRM结果与测序结果进行比较，发现每个突变均在正确的外显子中被HRM检测到。对于乳腺肿瘤样本组，我们通过HRM检测到7个异常熔解图谱，随后的测序证实了这些预测外显子中存在这些突变且无其他突变。
HRM是一种用于简单快速扫描TP53突变的有效技术，可显著减少TP53突变研究中所需的测序量。
