Trisolini Elena, Armellini Elia, Paganotti Alessia, Veggiani Claudia, Bozzola Cristina, Frattini Milo, Pizio Corinna, Mancuso Giuseppe, Andorno Silvano, Boldorini Renzo
Department of Health Science, School of Medicine, University of Eastern Piedmont 'Amedeo Avogadro', Novara, Italy.
Unit of Gastroenterology, 'Maggiore della Carità' Hospital, Novara, Italy.
Pathology. 2017 Jun;49(4):379-386. doi: 10.1016/j.pathol.2016.12.348. Epub 2017 Apr 24.
Endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) is the procedure of choice for the cytologic diagnosis of pancreatic masses. The specificity of EUS-FNA approaches 100%, but the sensitivity is still low, and the high rate of indeterminate (atypical and suspicious) and false-negative results needs improvement. KRAS gene is frequently mutated in pancreatic ductal adenocarcinoma (PDAC) (up to 90%), and mutation analysis of KRAS has been proposed as diagnostic biomarker of PDAC. In most laboratories, KRAS mutation testing is performed by Sanger sequencing or real time-quantitative polymerase chain reaction (RT-qPCR), but these methods may give false-negative results in routine samples, mainly due to low cellularity. In order to increase the sensitivity of EUS-FNA, we propose a sequential approach for detecting KRAS mutations using mutant enriched-PCR (ME-PCR, sensitivity up to 0.1%) in cytologically indeterminate and negative samples tested wild-type by RT-qPCR. EUS-FNA specimens from 107 patients with pancreatic masses (51 males, 56 females, mean age 67 years) were cytologically examined. According to the Papanicolaou Society of Cytopathology guidelines, 50 cases (47%) were classified malignant, 15 (14%) suspicious, 13 (12%) atypical and 10 (9%) negative for malignancy; 18 cases (17%) were non-diagnostic. The overall specificity and sensitivity of cytological examination were 100% and 61%, respectively, when only negative and positive cases were considered; when atypical and suspicious were added to positive cases, the sensitivity increased to 95.1% and the specificity decreased to 85.7%. In all the cases, DNA was extracted from the cell-block and KRAS mutations were investigated by RT-qPCR, followed by ME-PCR in non-amplifiable and negative cases. The overall sensitivity and specificity of KRAS mutation testing alone were 79.3% and 100%; when KRAS mutation testing was performed in indeterminate and negative cytology, the sensitivity increased to 90% with specificity to 100%. Our data indicate that conventional cytology from EUS-FNA samples is highly specific for the diagnosis of pancreatic cancer. Indeterminate and negative cases need to be screened for KRAS mutations; this two-step approach may greatly improve the diagnostic accuracy of this method.
内镜超声引导下细针穿刺抽吸术（EUS-FNA）是胰腺肿块细胞学诊断的首选方法。EUS-FNA的特异性接近100%，但敏感性仍然较低，不确定（非典型和可疑）及假阴性结果的高发生率有待改善。KRAS基因在胰腺导管腺癌（PDAC）中经常发生突变（高达90%），KRAS突变分析已被提议作为PDAC的诊断生物标志物。在大多数实验室中，KRAS突变检测通过桑格测序或实时定量聚合酶链反应（RT-qPCR）进行，但这些方法在常规样本中可能会得出假阴性结果，主要是由于细胞数量少。为了提高EUS-FNA的敏感性，我们提出一种序贯方法，即在通过RT-qPCR检测为野生型的细胞学不确定和阴性样本中，使用突变富集PCR（ME-PCR，敏感性高达0.1%）检测KRAS突变。对107例胰腺肿块患者（51例男性，56例女性，平均年龄67岁）的EUS-FNA标本进行了细胞学检查。根据帕潘尼古拉乌细胞病理学协会的指南，50例（47%）被分类为恶性肿瘤，15例（14%）可疑，13例（12%）非典型，10例（9%）恶性肿瘤阴性；18例（17%）无法诊断。仅考虑阴性和阳性病例时，细胞学检查的总体特异性和敏感性分别为100%和61%；当将非典型和可疑病例加入阳性病例时，敏感性提高到95.1%，特异性降至85.7%。在所有病例中，从细胞块中提取DNA，通过RT-qPCR研究KRAS突变，然后对不可扩增和阴性病例进行ME-PCR。单独进行KRAS突变检测的总体敏感性和特异性分别为79.3%和100%；当在细胞学不确定和阴性的情况下进行KRAS突变检测时，敏感性提高到90%，特异性为100%。我们的数据表明，EUS-FNA样本的传统细胞学检查对胰腺癌的诊断具有高度特异性。不确定和阴性病例需要筛查KRAS突变；这种两步法可能会大大提高该方法的诊断准确性。
