Prospective validation of microRNA signatures for detecting pancreatic malignant transformation in endoscopic-ultrasound guided fine-needle aspiration biopsies

Abstract

BackgroundPancreatic ductal adenocarcinoma (PDAC) is a lethal disease. Novel biomarkers are required to aid treatment decisions and improve patient outcomes. MicroRNAs (miRNAs) are potentially ideal diagnostic biomarkers, as they are stable molecules, and tumour and tissue specific.ResultsLogistic regression analysis revealed an endoscopic-ultrasound fine-needle aspiration (EUS-FNA) 2-miRNA classifier (miR-21 + miR-155) capable of distinguishing benign from malignant pancreatic lesions with a sensitivity of 81.5% and a specificity of 85.7% (AUC 0.930). Validation FNA cohorts confirmed both miRNAs were overexpressed in malignant disease, while circulating miRNAs performed poorly.MethodsFifty-five patients with a suspicious pancreatic lesion on cross-sectional imaging were evaluated by EUS-FNA. At echo-endoscopy, the first part of the FNA was sent for cytological assessment and the second part was used for total RNA extraction. Candidate miRNAs were selected after careful review of the literature and expression was quantified by qRT-PCR. Validation was performed on an independent cohort of EUS-FNAs, as well as formalin-fixed paraffin embedded (FFPE) and plasma samples.ConclusionsWe provide further evidence for using miRNAs as diagnostic biomarkers for pancreatic malignancy. We demonstrate the feasibility of using fresh EUS-FNAs to establish miRNA-based signatures unique to pancreatic malignant transformation and the potential to enhance risk stratification and selection for surgery.

Factors that drive the increasing use of FFPE tissue in basic and translational cancer research

“…Furthermore, How et al (2015) reported that a prognostic 9-mi-RNA signature set for cervical carcinoma identified in frozen samples could not be validated in an independent cohort of FFPE samples and that concordance between frozen and FFPE samples was lacking. A 2-miRNA classifier (miR-21 + miR-155), validated in small cohorts of EUS-FNA and FFPE needle biopsies, exhibited 81.5% sensitivity and 85.7% specificity in distinguishing pancreatic cancer from benign pancreatic lesions (Frampton et al 2016). Kakimoto et al (2016) reported that mi-RNA stability in FFPE tissue correlated with guanine and cytosine (GC) content: GC-rich mi-RNAs were less degraded than those with < 40% GC content.…”

Utility of endoscopic ultrasound and endoscopic ultrasound‐guided fine‐needle aspiration for the diagnosis and management of pancreatic cystic lesions: Differences between the guidelines

“…More recently, next‐generation sequencing (NGS) of DNA, miRNA, and proteomic and metabolomic approaches were evaluated to identify possible biomarkers in cyst fluid in the hopes of improving the diagnostic capability of PCL . A study by Singhi et al .…”

“…Aspirated cyst fluid occasionally does not contain enough PCL cellular material. A meta-analysis of EUS-FNA for diagnosing PCL, including 18 studies and a total of 1438 patients, showed a pooled sensitivity and specificity of cytological diagnostic capability for differentiating mucinous from non-mucinous cysts as 54% (95% CI, [49][50][51][52][53][54][55][56][57][58][59] and 93% (95% CI, 90-95), respectively. 40 A recent systematic review for the detection of malignant IPMN, including 193 studies with 12,297 patients, showed cyst fluid cytology as area under the curve of 0.84, sensitivity of 54% and specificity of 91% to identify malignant IPMN.…”

“…More recently, next-generation sequencing (NGS) of DNA, miRNA, and proteomic and metabolomic approaches were evaluated to identify possible biomarkers in cyst fluid in the hopes of improving the diagnostic capability of PCL. [53][54][55][56][57][58] A study by Singhi et al 57 prospectively and preoperatively evaluated NGS of cyst fluid obtained by EUS-FNA from PCL and also determined the accuracy of molecular analysis using both NGS and Sanger sequencing. Based on 102 patients with surgical follow up, KRAS/GNAS mutations on NGS were detected in 56 (100%) IPMN and in three (30%) MCN, and associated with 89% sensitivity and 100% specificity for mucinous cystic lesions compared with 65% sensitivity and 100% specificity with KRAS/GNAS mutations by Sanger sequencing.…”

Noncoding Rnas as Novel Biomarkers in Pancreatic Cancer: what do we Know?

“…Of note, a recent analysis of miRNA expression in 55 endoscopic ultrasound-guided fine needle aspiration biopsies found that miR-21 and miR-155 levels could be used to differentiate malignant from benign lesions with a greater accuracy than standard pathology. However, comparison with other biomarkers (such as CEA) was not possible [80]. Another clinical challenge is making a differential diagnosis between PDAC and bile duct cancers.…”