Next Generation Sequencing Improves the Accuracy of KRAS Mutation Analysis in Endoscopic Ultrasound Fine Needle Aspiration Pancreatic Lesions
Next Generation Sequencing Improves the Accuracy of KRAS Mutation Analysis in Endoscopic Ultrasound Fine Needle Aspiration Pancreatic Lesions
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DOI：
10.1371/journal.pone.0087651
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发表时间：
2014-02-04
期刊：
影响因子：
3.7
通讯作者：
Tallini, Giovanni
中科院分区：
文献类型：
--
作者：
de Biase, Dario;Visani, Michela;Tallini, Giovanni
超声内镜的使用使得细针抽吸物的检测和病理分析能够更好地用于胰腺病变的诊断。KRAS的分子分析进一步提高了术前分析的临床敏感性。因此，在对来自细针抽吸标本的材料进行分析时，使用高度分析灵敏度和特异性的分子测试变得非常重要。在本研究中，使用三种不同的技术：桑格测序、等位基因特异性锁核酸PCR和下一代测序（454 GS-Junior，Roche），分析了60份来自内镜超声细针抽吸物的标本的KRAS外显子2和外显子3突变。此外，还在野生型样品中测试了KRAS，从病理学评价后从细胞学涂片获得的DNA开始。桑格测序显示KRAS突变检测的临床灵敏度为42.1%，等位基因特异性锁核酸检测的临床灵敏度为52.8%，下一代检测的临床灵敏度为73.7%。在两个野生型病例中，从选定材料开始的重新测序允许检测KRAS突变，将下一代测序的临床灵敏度提高到78.95%。本研究表明，使用高分析灵敏度的技术可以提高分子分析的性能。新一代测序技术可以提高检测的临床灵敏度，而不会降低分析的特异性。此外，我们观察到，从可选择的材料开始重复分析可能是有用的，例如细胞学涂片，以避免假阴性结果。
The use of endoscopic ultrasonography has allowed for improved detection and pathologic analysis of fine needle aspirate material for pancreatic lesion diagnosis. The molecular analysis of KRAS has further improved the clinical sensitivity of preoperative analysis. For this reason, the use of highly analytical sensitive and specific molecular tests in the analysis of material from fine needle aspirate specimens has become of great importance. In the present study, 60 specimens from endoscopic ultrasonography fine needle aspirate were analyzed for KRAS exon 2 and exon 3 mutations, using three different techniques: Sanger sequencing, allele specific locked nucleic acid PCR and Next Generation sequencing (454 GS-Junior, Roche). Moreover, KRAS was also tested in wild-type samples, starting from DNA obtained from cytological smears after pathological evaluation. Sanger sequencing showed a clinical sensitivity for the detection of the KRAS mutation of 42.1%, allele specific locked nucleic acid of 52.8% and Next Generation of 73.7%. In two wild-type cases the re-sequencing starting from selected material allowed to detect a KRAS mutation, increasing the clinical sensitivity of next generation sequencing to 78.95%. The present study demonstrated that the performance of molecular analysis could be improved by using highly analytical sensitive techniques. The Next Generation Sequencing allowed to increase the clinical sensitivity of the test without decreasing the specificity of the analysis. Moreover we observed that it could be useful to repeat the analysis starting from selectable material, such as cytological smears to avoid false negative results.