Gontareva

Cell-free DNA (cfDNA) analysis-based liquid biopsy is a rapidly emerging diagnostic and prognostic tool in pancreatic ductal adenocarcinoma (PDAC). KRAS point mutations are the main biomarkers used for the detection of tumor cfDNA. However, there is another less studied yet frequent genetic alteration in this gene, namely copy number gain (CNG). The aim of this study was to evaluate the diagnostic and prognostic potential of KRAS CNG analysis in plasma and bile of patients with PDAC using ddPCR. This study included healthy volunteers (n = 69), patients with PDAC (n = 94), and other pancreatic neoplasms (OPN) (n = 17). The sensitivity and specificity of KRAS CNG compared to the control group were 16% and 100% (AUC-ROC—0.580), and compared to the OPN group, 16% and 94% (AUC-ROC—0.554), respectively. Addition of KRAS point mutations to the analysis increased the sensitivity to 65% (AUC-ROC—0.824 and 0.801, respectively). Bile exhibited an equal KRAS CNG detection rate compared to plasma (20% vs. 16%). KRAS CNG was not associated with clinical parameters, except prognosis. The probability of survival was worse in patients with KRAS CNG (HR—3.54; 95% CI: 1.55–8.12; p = 0.001). KRAS CNG in cfDNA might be a promising biomarker for both diagnostic and prognostic purposes in PDAC.

The key challenge of cell-free tumor DNA (cftDNA) analysis in pancreatic ductal adenocarcinoma (PDAC) is overcoming its low detection rate, which is mainly explained by the overall scarcity of this biomarker in plasma. Obstructive jaundice is a frequent event in PDAC, which enables bile collection as a part of routine treatment. The aim of this study was to evaluate the performance of KRAS-mutated cftDNA detection-based liquid biopsy of plasma and bile in patients with pancreatic neoplasms using digital droplet PCR. The study included healthy volunteers (n = 38), patients with PDAC (n = 95, of which 20 had obstructive jaundice) and other pancreatic neoplasms (OPN) (n = 18). The sensitivity and specificity compared to the control group were 61% and 100% (AUC-ROC—0.805), and compared to the OPN group, they were 61% and 94% (AUC-ROC—0.794), respectively. Bile exhibited higher cftDNA levels than plasma (248.6 [6.743; 1068] vs. 3.26 [0; 19.225] copies/mL) and a two-fold higher detection rate (p < 0.01). Plasma cftDNA levels were associated with distant metastases, tumor size, and CA 19-9 (p < 0.05). The probability of survival was worse in patients with higher levels of cftDNA in plasma (hazard ratio—2.4; 95% CI: 1.3–4.6; p = 0.005) but not in bile (p > 0.05). Bile is a promising alternative to plasma in patients with obstructive jaundice, at least for the diagnostic purposes of liquid biopsy.

INTRODUCTION: Plasma liquid biopsy with tumor cell-free DNA detection is one of the most promising technologies in pancreatic ductal adenocarcinoma diagnosis. Due to low levels of this biomarker in plasma its detectability turns out to be lower than expected. However, some patients with this disease present with biliary obstruction, which enables collection of bile for tumor cell-free DNA analysis. THE AIM: To comparison of diagnostic potential of cell-free tumor DNA detection-based liquid biopsy of plasma and bile in pancreatic ductal adenocarcinoma patients. MATERIAL AND METHODS: The pilot study included 15 primary untreated pancreatic ductal adenocarcinoma patients with biliary obstruction. Cell-free DNA was isolated from 5 mL of plasma and 5 mL of bile. Tumor cell-free DNA detection was performed using digital droplet PCR with KRAS gene mutations analysis: G12A, G12C, G12D, G12R, G12S, G12V, G13D и Q61H (183AC), Q61H (183AT), Q61K, Q61L, Q61R. False-positive droplets threshold was established during the analysis of plasma cell-free DNA samples from 15 healthy volunteers. RESULTS: Tumor cell-free DNA was detected in 13 out of 15 bile samples, whereas among paired plasma samples only 9 were positive. All plasma positive samples had a paired positive bile sample. Tumor cell-free DNA levels were statistically significantly higher in bile than in plasma: 538.0 (4.11960.0) vs. 4.4 (027.6) copies/mL (p=0.005). CONCLUSION: Bile liquid biopsy in pancreatic ductal adenocarcinoma patients with biliary obstruction is a promising alternative to plasma analysis, due to higher concentrations of tumor cell-free DNA.