Droplet digital polymerase chain reaction detection of KRAS mutations in pancreatic FNA samples: Technical and practical aspects for routine clinical implementation.

Background: Pancreatic adenocarcinoma (PDAC) is associated with a 5‐year survival rate of less than 6%, and current treatments have limited efficacy. The diagnosis of PDAC is mainly based on a cytologic analysis of endoscopic ultrasound–guided fine‐needle aspiration (EUS‐FNA) samples. However, the collected specimens may prove noncontributory in a significant number of cases, delaying patient management and treatment. The combination of EUS‐FNA sample examination and KRAS mutation detection can improve the sensitivity for diagnosis. In this context, the material used for molecular analysis may condition performance. Methods: The authors prospectively compared the performance of cytologic analysis combined with a KRAS droplet digital polymerase chain reaction (ddPCR) assay for PDAC diagnosis using either conventional formalin‐fixed, paraffin‐embedded cytologic samples or needle‐rinsing fluids. Results: Molecular testing of formalin‐fixed, paraffin‐embedded cytologic samples was easier to set up, but the authors observed that the treatment of preanalytic samples, in particular the fixation process, drastically reduced ddPCR sensitivity, increasing the risk of false‐negative results. Conversely, the analysis of dedicated, fresh needle‐rinsing fluid samples appeared to be ideal for ddPCR analysis; it had greater sensitivity and was easily to implement in clinical use. In particular, fluid collection by the endoscopist, transportation to the laboratory, and subsequent freezing did not affect DNA quantity or quality. Moreover, the addition of KRAS mutation detection to cytologic examination improved diagnosis performance, regardless of the source of the sample. Conclusions: Considering all of these aspects, the authors propose the use of an integrated flowchart for the KRAS molecular testing of EUS‐FNA samples in clinical routine. The use of fresh needle‐rinsing fluid offers better sensitivity than formalin‐fixed, paraffin‐embedded cytology samples for the detection of KRAS G12/G13 mutations using droplet digital polymerase chain reaction. The addition of KRAS G12/G13 mutation detection by droplet digital polymerase chain reaction analysis to cytologic examination improves pancreatic ductal adenocarcinoma diagnostic performance compared with cytology alone and can be proposed in cases of negative or noncontributory cytologic results. [ABSTRACT FROM AUTHOR]