Background/Objectives: Although the overall survival prognosis of patients in advanced stages of pancreatic ductal adenocarcinoma (PDAC) is poor, typically ranging from days to months from diagnosis, there are rare cases of patients remaining in therapy for longer periods of time. Early estimations of survival prognosis would allow rational decisions on complex therapy interventions, including radical surgery and robust systemic therapy regimens. Understandably, there is great interest in finding prognostic markers that can be used for patient stratification. We determined the role of various KRAS mutations in the prognosis of PDAC patients using biopsy samples and circulating tumor DNA. Methods: A total of 118 patients with PDAC, clinically confirmed by endoscopic ultrasound-guided fine-needle aspiration biopsy (EUS-FNB), were included in the study. DNA was extracted from cytological slides following a standard cytology evaluation to ensure adequacy (viability and quantity) and to mark the tumor cell fraction. Circulating tumor DNA (ctDNA) was extracted from plasma samples of 45 patients in stage IV of the disease. KRAS mutations in exons 12 and 13 were detected by denaturing capillary electrophoresis (DCE), revealing a minute presence of mutation-specific heteroduplexes. Kaplan-Meier survival curves were calculated for individual KRAS mutation types. Results:KRAS mutations were detected in 90% of tissue (106/118) and 44% of plasma (20/45) samples. All mutations were localized at exon 2, codon 12, with G12D (GGT > GAT) being the most frequent at 44% (47/106) and 65% (13/20), followed by other types including G12V (GGT > GTT) at 31% (33/106) and 10% (2/20), G12R (GGT > CGT) at 17% (18/106) and 10% (2/20), G12C (GGT/TGT) at 5% (5/106) and 0% (0/20) and G12S (GGT/AGT) at 1% (1/106) and 5% (1/20) in tissue and plasma samples, respectively. Two patients had two mutations simultaneously (G12V + G12S and G12D + G12S) in both types of samples (2%, 2/106 and 10%, 2/20 in tissue and plasma samples, respectively). The median survival of patients with the G12D mutation in tissues was less than half that of other patients (median survival 101 days, 95% CI: 80-600 vs. 228 days, 95% CI: 184-602), with a statistically significant overall difference in survival (p = 0.0080, log-rank test), and furthermore it was less than that of all combined patients with other mutation types (101 days, 95% CI: 80-600 vs. 210 days, 95% CI: 161-602, p = 0.0166). For plasma samples, the survival of patients with this mutation was six times shorter than that of patients without the G12D mutation (27 days, 95% CI: 8-334 vs. 161 days, 95% CI: 107-536, p = 0.0200). In contrast, patients with detected KRAS G12R in the tissue survived nearly twice as long as other patients in the aggregate (286 days, 95% CI: 70-602 vs. 162 days, 95% CI: 122-600, p = 0.0374) or patients with other KRAS mutations (286 days, 95% CI: 70-602 vs. 137 days, 95% CI: 107-600, p = 0.0257). Conclusions: Differentiation of specific KRAS mutations in EUS-FNB and ctDNA (above all, the crucial G12D and G12R) is feasible in routine management of PDAC patients and imperative for assessment of prognosis.

• Keywords
• EUS-FNB, KRAS, ctDNA, liquid biopsy, mutation type, pancreatic cancer, prognosis,
• MeSH
• Endoscopic Ultrasound-Guided Fine Needle Aspiration * MeSH
• Circulating Tumor DNA genetics   blood   MeSH
• Adult MeSH
• Carcinoma, Pancreatic Ductal * genetics   pathology   blood   MeSH
• Middle Aged MeSH
• Humans MeSH
• Mutation * MeSH
• Biomarkers, Tumor genetics   MeSH
• Pancreatic Neoplasms * genetics   pathology   mortality   MeSH
• Prognosis MeSH
• Proto-Oncogene Proteins p21(ras) * genetics   MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Liquid Biopsy methods   MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Circulating Tumor DNA MeSH
• KRAS protein, human MeSH Browser
• Biomarkers, Tumor MeSH
• Proto-Oncogene Proteins p21(ras) * MeSH

Background: Observation of anticancer therapy effect by monitoring of minimal residual disease (MRD) is becoming an important tool in management of non-small cell lung cancer (NSCLC). The approach is based on periodic detection and quantification of tumor-specific somatic DNA mutation in circulating tumor DNA (ctDNA) extracted from patient plasma. For such repetitive testing, complex liquid-biopsy techniques relying on ultra-deep NGS sequencing are impractical. There are other, cost-effective, methods for ctDNA analysis, typically based on quantitative PCR or digital PCR, which are applicable for detecting specific individual mutations in hotspots. While such methods are routinely used in NSCLC therapy prediction, however, extension to cover broader spectrum of mutations (e.g., in tumor suppressor genes) is required for universal longitudinal MRD monitoring. Methods: For a set of tissue samples from 81 NSCLC patients we have applied a denaturing capillary electrophoresis (DCE) for initial detection of somatic mutations within 8 predesigned PCR amplicons covering oncogenes and tumor suppressor genes. Mutation-negative samples were then subjected to a large panel NGS sequencing. For each patient mutation found in tissue was then traced over time in ctDNA by DCE. Results: In total we have detected a somatic mutation in tissue of 63 patients. For those we have then prospectively analyzed ctDNA from collected plasma samples over a period of up to 2 years. The dynamics of ctDNA during the initial chemotherapy therapy cycles as well as in the long-term follow-up matched the clinically observed response. Conclusion: Detection and quantification of tumor-specific mutations in ctDNA represents a viable complement to MRD monitoring during therapy of NSCLC patients. The presented approach relying on initial tissue mutation detection by DCE combined with NGS and a subsequent ctDNA mutation testing by DCE only represents a cost-effective approach for its routine implementation.

• Keywords
• KRAS mutations, NSCLC, TP53 mutations, capillary electrophoresis, ctDNA, liquid biopsy, minimal residual disease,
• MeSH
• Circulating Tumor DNA * genetics   MeSH
• DNA, Neoplasm genetics   MeSH
• Electrophoresis, Capillary MeSH
• Humans MeSH
• Mutation genetics   MeSH
• Biomarkers, Tumor genetics   MeSH
• Lung Neoplasms * drug therapy   MeSH
• Carcinoma, Non-Small-Cell Lung * genetics   therapy   MeSH
• Neoplasm, Residual MeSH
• High-Throughput Nucleotide Sequencing methods   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Circulating Tumor DNA * MeSH
• DNA, Neoplasm MeSH
• Biomarkers, Tumor MeSH

We compare two types of pancreatic carcinoma samples obtained by EUS-guided fine needle biopsy (EUS-FNB) in terms of the success rates and clinical validity of analysis of two most commonly investigated DNA/RNA pancreatic cancer markers, KRAS mutations and miR-21 expression. 118 patients with pancreatic ductal adenocarcinoma underwent EUS-FNB. The collected sample was divided, one part was stored in a stabilizing solution as native aspirate (EUS-FNA) and second part was processed into the cytological smear (EUS-FNC). DNA/RNA extraction was followed by analysis of KRAS mutations and miR-21 expression. For both sample types, the yields of DNA/RNA extraction and success rates of KRAS mutation and miRNA expression were evaluated. Finally, the resulting KRAS mutation frequency and miR-21 prognostic role were compared to literature data from tissue resections. The overall amount of isolated DNA/RNA from EUS-FNC was lower compared to the EUS-FNA, average yield 10 ng vs 147 ng for DNA and average yield 164 vs. 642 ng for RNA, but the success rates for KRAS and miR-21 analysis was 100% for both sample types. The KRAS-mutant detection frequency in EUS-FNC was 12% higher than in EUS-FNA (90 vs 78%). The prognostic role of miR-21 was confirmed in EUS-FNC (p = 0.02), but did not reach statistical significance in EUS-FNA (p = 0.06). Although both types of EUS-FNB samples are suitable for DNA/RNA extraction and subsequent DNA mutation and miRNA expression analysis, reliable results with clinical validity were only obtained for EUS-FNC.

• Keywords
• EUS-FNA, KRAS, Pancreatic cancer, miR-21,
• MeSH
• Endoscopic Ultrasound-Guided Fine Needle Aspiration MeSH
• Cytodiagnosis methods   MeSH
• DNA analysis   MeSH
• Carcinoma, Pancreatic Ductal diagnosis   MeSH
• Tissue Fixation methods   MeSH
• Middle Aged MeSH
• Humans MeSH
• MicroRNAs analysis   MeSH
• Mutation MeSH
• Biomarkers, Tumor analysis   MeSH
• Pancreatic Neoplasms diagnosis   MeSH
• Specimen Handling methods   MeSH
• Proto-Oncogene Proteins p21(ras) genetics   MeSH
• Aged MeSH
• Check Tag
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Comparative Study MeSH
• Names of Substances
• DNA MeSH
• KRAS protein, human MeSH Browser
• MicroRNAs MeSH
• MIRN21 microRNA, human MeSH Browser
• Biomarkers, Tumor MeSH
• Proto-Oncogene Proteins p21(ras) MeSH

Pancreatic cancer is one of the most fatal malignancies with increasing incidence and high mortality. Possibilities for early diagnosis are limited and there is currently no efficient therapy. Molecular markers that have been introduced into diagnosis and treatment of other solid tumors remain unreciprocated in this disease. Recent discoveries have shown that certain microRNAs (miRNAs) take part in fundamental molecular processes associated with pancreatic cancer initiation and progression including cell cycle, DNA repair, apoptosis, invasivity, and metastasis. The mechanism involves both positive and negative regulation of expression of protooncogenes and tumor suppressor genes. Various miRNAs are expressed at different levels among normal pancreatic tissue, chronic pancreatitis, and pancreatic cancer and may therefore serve as a tool to differentiate chronic pancreatitis from early stages of cancer. Other miRNAs can indicate the probable course of the disease or determine the survival prognosis. In addition, there is a growing interest directed at the understanding of miRNA-induced molecular mechanisms. The possibility of intervention in the molecular mechanisms of miRNAs regulation could begin a new generation of pancreatic cancer therapies. This review summarizes the recent reports describing functions of miRNAs in cellular processes underlying pancreatic cancerogenesis and their utility in diagnosis, survival prognosis, and therapy.

• Publication type
• Journal Article MeSH
• Review MeSH