de Biase Dario, Visani Michela, Baccarini Paola, Polifemo Anna Maria, Maimone Antonella, Fornelli Adele, Giuliani Adriana, Zanini Nicola, Fabbri Carlo, Pession Annalisa, Tallini Giovanni
Department of Medicine (DIMES) - Anatomic Pathology Unit, Bellaria Hospital, University of Bologna, Bologna, Italy ; Department of Pharmacology and Biotechnology (FaBiT), University of Bologna, Bologna, Italy.
Department of Pharmacology and Biotechnology (FaBiT), University of Bologna, Bologna, Italy.
PLoS One. 2014 Feb 4;9(2):e87651. doi: 10.1371/journal.pone.0087651. eCollection 2014.
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.
内镜超声检查的应用有助于提高胰腺病变诊断中细针穿刺抽吸物的检测及病理分析水平。KRAS的分子分析进一步提高了术前分析的临床敏感性。因此，在细针穿刺抽吸标本材料分析中使用高分析灵敏度和特异性的分子检测方法变得至关重要。在本研究中，采用三种不同技术对60例内镜超声引导下细针穿刺抽吸标本进行KRAS第2外显子和第3外显子突变分析：桑格测序、等位基因特异性锁核酸PCR和下一代测序（454 GS-Junior，罗氏公司）。此外，从病理评估后的细胞学涂片获得的DNA开始，对野生型样本也进行了KRAS检测。桑格测序检测KRAS突变的临床敏感性为42.1%，等位基因特异性锁核酸为52.8%，下一代测序为73.7%。在两例野生型病例中，从选定材料重新测序可检测到KRAS突变，将下一代测序的临床敏感性提高到78.95%。本研究表明，使用高分析灵敏度技术可提高分子分析的性能。下一代测序在不降低分析特异性的情况下提高了检测的临床敏感性。此外，我们观察到从可选择的材料（如细胞学涂片）重新开始分析以避免假阴性结果可能是有用的。
