Recent whole-genome sequencing studies identified two novel recurrent mutations in the enhancer region of GPR126 in urothelial bladder cancer (UBC) tumor samples. This mutational hotspot is the second most common after the TERT promoter in UBC. The aim of the study was to develop a digital droplet PCR screening assay for the simultaneous detection of GPR126 mutations in a single tube. Its performance combined with TERT promoter mutation analysis was evaluated in urine of healthy volunteers (n = 50) and patients with cystitis (n = 22) and UBC (n = 70). The developed assay was validated using DNA constructs carrying the studied variants. None of the mutations were detected in control and cystitis group samples. GPR126 mutations were observed in the urine of 25/70 UBC patients (area under the ROC curve (AUC) of 0.679; mutant allele fraction (MAF) of 21.61 [8.30–44.52] %); TERT mutations–in 40/70 (AUC of 0.786; MAF = 28.29 [19.03–38.08] %); ≥1 mutation–in 47/70 (AUC of 0.836)). The simultaneous presence of GPR126 and TERT mutations was observed in 18/70 cases, with no difference in MAFs for the paired samples (31.96 [14.78–47.49] % vs. 27.13 [17.00–37.62] %, p = 0.349, respectively). The combined analysis of these common non-coding mutations in urine allows the sensitive and non-invasive detection of UBC.

Background: Urothelial bladder carcinoma (UBC) is a disease that lacks robust non-invasive laboratory biomarkers. Recently, urine liquid biopsy has emerged as a promising tool for diagnosis and surveillance of patients with these tumors. The aim of this study was to evaluate the diagnostic potential of a urinary tumor DNA detection panel, which included eight common point mutations in TERT, GPR126, FGFR3, and PIK3CA genes, in UBC. Methods: The study included patients with histologically confirmed UBC (n = 88) and patients with cystitis, bladder leiomyomas, or other non-malignant conditions (control group; n = 72). DNA was extracted from whole urine specimens. ddPCR analysis was performed using the Bio-Rad QX200 AutoDG ddPCR system. Results: Urinary tumor DNA detection panel demonstrated a sensitivity of 78.4% and a specificity of 100% (AUC−ROC = 0.892). Detection rates for the analyzed mutations were the following: 54.5%, 37.5%, 28.4%, and 38.6% for TERT, GPR126, FGFR3, and PIK3CA, respectively. Pairwise comparisons of mutant allele fractions (MAFs) for samples simultaneously positive for ≥2 mutations revealed an absence of significant differences (p > 0.05), except for the pair of FGFR3 vs. PIK3CA (p = 0.03). MAFs were not associated with any clinical and demographic features (p > 0.05), with the only exception being the tumor size: patients with tumors larger than 2.16 cm³ had higher MAFs than the rest (23.4 [1.8; 46.3] vs. 1.6 [0; 24.6] %, respectively, p = 0.02). Conclusions: Upon further validation, the presented tumor DNA detection panel for ddPCR might become a useful tool for diagnostic purposes in UBC.

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.

Gliomas are the reason of fatal outcomes in an overwhelming number of patients with oncology diseases located in the central nervous system. The diagnostics of such neoplasms requires using stereotaxic biopsy, which cannot be performed in a certain percentage of the patients. Besides, this disease is characterized by high recurrence rates, despite the advances in developing resection and chemotherapy — based technologies. The early detection of oncological diseases located in the central nervous system and the differential diagnostics of tumor pseudo progression, not affecting the survival of the patient, represents a challenge for modern Medicine. Liquid biopsy is a minimally invasive diagnostic method based on the analysis of tumor derivatives (such as extracellular tumor DNA and RNA), contained within the biological fluids of the organism. For the purpose of defining the presence of the tumor component, the tests are used to detect the so-called hot-spot mutations and the patterns of epigenetic regulation, found in specific types of tumors. The technology can be used for detecting tumor recurrences and for the differential diagnostics of space-occupying mass lesions in patients, in which stereotaxic biopsy is contraindicated. The review contains a discussion on modern advances of fluid biopsy based on the analysis of the extracellular tumor DNA and RNA levels in blood plasma and in the cerebrospinal fluid of glioma patients.

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.

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.