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.

• Klíčová slova
• EUS-FNB, KRAS, ctDNA, liquid biopsy, mutation type, pancreatic cancer, prognosis,
• MeSH
• biopsie tenkou jehlou pod endosonografickou kontrolou * MeSH
• cirkulující nádorová DNA genetika   krev   MeSH
• dospělí MeSH
• duktální karcinom slinivky břišní * genetika   patologie   krev   MeSH
• lidé středního věku MeSH
• lidé MeSH
• mutace * MeSH
• nádorové biomarkery genetika   MeSH
• nádory slinivky břišní * genetika   patologie   mortalita   MeSH
• prognóza MeSH
• protoonkogenní proteiny p21(ras) * genetika   MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• tekutá biopsie metody   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cirkulující nádorová DNA MeSH
• KRAS protein, human MeSH Prohlížeč
• nádorové biomarkery MeSH
• protoonkogenní proteiny p21(ras) * 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.

• Klíčová slova
• EUS-FNA, KRAS, Pancreatic cancer, miR-21,
• MeSH
• biopsie tenkou jehlou pod endosonografickou kontrolou MeSH
• cytodiagnostika metody   MeSH
• DNA analýza   MeSH
• duktální karcinom slinivky břišní diagnóza   MeSH
• fixace tkání metody   MeSH
• lidé středního věku MeSH
• lidé MeSH
• mikro RNA analýza   MeSH
• mutace MeSH
• nádorové biomarkery analýza   MeSH
• nádory slinivky břišní diagnóza   MeSH
• odběr biologického vzorku metody   MeSH
• protoonkogenní proteiny p21(ras) genetika   MeSH
• senioři MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH
• Názvy látek
• DNA MeSH
• KRAS protein, human MeSH Prohlížeč
• mikro RNA MeSH
• MIRN21 microRNA, human MeSH Prohlížeč
• nádorové biomarkery MeSH
• protoonkogenní proteiny p21(ras) MeSH

AIM: To compare molecular profiles of proximal colon, distal colon and rectum in large adenomas, early and late carcinomas. To assess feasibility of testing directed at molecular markers from this study in routine clinical practice. METHODS: A prospective 3-year study has resulted in the acquisition of samples from 159 large adenomas and 138 carcinomas along with associated clinical parameters including localization, grade and histological type for adenomas and localization and stage for carcinomas. A complex molecular phenotyping has been performed using multiplex ligation-dependent probe amplification technique for the evaluation of CpG-island methylator phenotype (CIMP), PCR fragment analysis for detection of microsatellite instability and denaturing capillary electrophoresis for sensitive detection of somatic mutations in KRAS, BRAF, TP53 and APC genes. RESULTS: Molecular types according to previously introduced Jass classification have been evaluated for large adenomas and early and late carcinomas. An increase in CIMP+ type, eventually accompanied with KRAS mutations, was notable between large adenomas and early carcinomas. As expected, the longitudinal observations revealed a correlation of the CIMP+/BRAF+ type with proximal location. CONCLUSION: Prospective molecular classification of tissue specimens is feasible in routine endoscopy practice. Increased frequency of some molecular types corresponds to the developmental stages of colorectal tumors. As expected, a clear distinction is notable for tumors located in proximal colon supposedly arising from the serrated (methylation) pathway.

• Klíčová slova
• BRAF, Colorectal cancer, CpG-island methylator phenotype, DNA, Microsatellite instability,
• MeSH
• adenom genetika   patologie   chirurgie   MeSH
• biopsie MeSH
• CpG ostrůvky MeSH
• dospělí MeSH
• karcinom genetika   patologie   chirurgie   MeSH
• kolonoskopie * MeSH
• kolorektální nádory genetika   patologie   chirurgie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• metylace DNA MeSH
• mikrosatelitní nestabilita MeSH
• multiplexová polymerázová řetězová reakce * MeSH
• mutace MeSH
• mutační analýza DNA MeSH
• nádorové biomarkery genetika   MeSH
• nádorový supresorový protein p53 genetika   MeSH
• prediktivní hodnota testů MeSH
• prospektivní studie MeSH
• protein familiární adenomatózní polypózy genetika   MeSH
• protoonkogenní proteiny B-Raf genetika   MeSH
• protoonkogenní proteiny p21(ras) genetika   MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• staging nádorů MeSH
• studie proveditelnosti MeSH
• stupeň nádoru MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• Názvy látek
• APC protein, human MeSH Prohlížeč
• BRAF protein, human MeSH Prohlížeč
• KRAS protein, human MeSH Prohlížeč
• nádorové biomarkery MeSH
• nádorový supresorový protein p53 MeSH
• protein familiární adenomatózní polypózy MeSH
• protoonkogenní proteiny B-Raf MeSH
• protoonkogenní proteiny p21(ras) MeSH
• TP53 protein, human MeSH Prohlížeč

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.

• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

In spite of continuous research efforts directed at early detection and treatment of pancreatic cancer, the outlook for patients affected by the disease remains dismal. With most cases still being diagnosed at advanced stages, no improvement in survival prognosis is achieved with current diagnostic imaging approaches. In the absence of a dominant precancerous condition, several risk factors have been identified including family history, chronic pancreatitis, smoking, diabetes mellitus, as well as certain genetic disorders such as hereditary pancreatitis, cystic fibrosis, familial atypical multiple mole melanoma, and Peutz-Jeghers and Lynch syndromes. Most pancreatic carcinomas, however, remain sporadic. Current progress in experimental molecular techniques has enabled detailed understanding of the molecular processes of pancreatic cancer development. According to the latest information, malignant pancreatic transformation involves multiple oncogenes and tumor-suppressor genes that are involved in a variety of signaling pathways. The most characteristic aberrations (somatic point mutations and allelic losses) affect oncogenes and tumor-suppressor genes within RAS, AKT and Wnt signaling, and have a key role in transcription and proliferation, as well as systems that regulate the cell cycle (SMAD/DPC, CDKN2A/p16) and apoptosis (TP53). Understanding of the underlying molecular mechanisms should promote development of new methodology for early diagnosis and facilitate improvement in current approaches for pancreatic cancer treatment.

• Klíčová slova
• Diabetes, Molecular biology, Pancreatic cancer, Pancreatitis, Risk factors,
• MeSH
• chronická pankreatitida epidemiologie   etiologie   genetika   patofyziologie   MeSH
• diabetes mellitus epidemiologie   genetika   MeSH
• diferenciální diagnóza MeSH
• genetická predispozice k nemoci MeSH
• komorbidita MeSH
• lidé MeSH
• míra přežití MeSH
• molekulární biologie * MeSH
• nádory slinivky břišní epidemiologie   etiologie   genetika   patofyziologie   MeSH
• rizikové faktory MeSH
• signální transdukce fyziologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH