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.

• MeSH
• biopsie tenkou jehlou pod endosonografickou kontrolou * MeSH
• cirkulující nádorová DNA genetika   krev   MeSH
• dospělí MeSH
• duktální karcinom pankreatu * 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

• Publikační typ
• abstrakt z konference MeSH

• Publikační typ
• abstrakt z konference MeSH

• Publikační typ
• abstrakt z konference MeSH

Purpose: To investigate potential association between selected tumor markers and laboratory parameters (lactate dehydrogenase [LDH], neutrophils, hemoglobin, neutrophils, lymphocytes, C-reactive protein, albumin, carcinoembryonic antigen, and cytokeratin 19 fragment 21-1 [CYFRA 21-1]) and circulating tumor DNA (ctDNA) with survival in patients with advanced non-small cell lung cancer (NSCLC). Patients and Methods: The study encompassed 82 patients from a single center. All patients had (localy-) advanced adenocarcinomas. ctDNA was determined before starting therapy and at 6 weeks follow-up. Laboratory parameters were measured before each cycle of therapy and oncomarkers before starting the therapy as standard clinical practice. Mann-Whitney U test, Cox proportional hazards model, Fisher's exact test, and Kaplan-Meier survival estimation with Gehan-Wilcoxon test were used for statistical analysis of the corresponding variables. Results: We have confirmed predictive or prognostic significance for some of the selected laboratory markers and oncomarkers. Above all, we demonstrate a significant relationship between the levels of LDH and the oncomarker CYFRA 21-1 and the presence or absence of ctDNA at the time of diagnosis. We also demonstrate significantly lower CRP levels in patients within whom the ctDNA disappeared during treatment. A similar but statistically insignificant trend was observed for LDH. Conclusions: CYFRA 21-1, LDH and probably CRP correlate with ctDNA levels in NSCLC. Repeated measurement of these markers could thus help in early detection of disease progression in the same way as does ctDNA monitoring.

• Publikační typ
• časopisecké články MeSH

• Publikační typ
• abstrakt z konference MeSH

• Publikační typ
• abstrakt z konference MeSH

In years 2017–2020, there will be 200 patients with removed colorectal adenomas >10 mm included. In adenomas high risk areas identified by endoscopist, the histopathology evaluation and molecular examination of intratumoral heterogenity and clonal profiles of highly frequent mutated genes (KRAS, TP53, PIK3CA and APC) will be done. The level of heterogenity will be determined based on the presence and quantification of mutations (score of heterogenity 1-5). All patients will have follow-up colonoscopy in one-year intervals. In a high-risk patients group (high grade dysplasia; high heterogenity score; adenoma recurrence) and selected control subjects, the extensive somatic mutations profiling will be carried out according to multitarget sequencing by NGS technologies (next generation sequencing). The aims are to identify the candidate genes for the prediction of colorectal neoplasia recurrence after endoscopic therapy and detailed adenomas topographic heterogenity characterization to set the biopsy sampling recommendations.

V letech 2017 – 2020 bude vyšetřeno 200 pacientů s odstraněnými adenomy >10 mm. Za asistence endoskopisty bude v rizikových oblastech adenomu provedeno histopatologické hodnocení a molekulární vyšetření intratumorové heterogenity a klonálních profilů s využitím panelu genů obsahující frekventně mutované geny (KRAS, TP53, PIK3CA, APC). Bude stanovena míra heterogenity na základě výskytu a kvantifikace sledovaných mutací (skóre 1 – 5). Následně budou pacienti kolonoskopicky sledováni v jednoletém intervalu. U rizikových pacientů (vysoký stupeň dysplázie, vysoké skóre heterogenity, rekurence adenomů) a vybraných kontrolních subjektů bude provedeno rozsáhlé profilování somatických mutací cíleným „multitarget“ sekvenováním technologií NGS (sekvenování nové generace). Cílem projektu je provedení statistické analýzy za účelem vytipování kandidátních genů pro predikci rekurence kolorektální neoplázie po endoskopické terapii a detailní charakterizace topografické heterogenity adenomů se stanovením doporučení pro odběr bioptických vzorků.

• Klíčová slova
• adenom, rekurence, adenoma, follow-up, kolonoskopie, colonoscopy, kolorektální neoplázie, dispenzarizace, genová mutace, multiparametrický test, colorectal neoplasia, gene mutation, recurrence, multiparametric test,

• NLK Publikační typ
• závěrečné zprávy o řešení grantu AZV MZ ČR

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.

• MeSH
• cirkulující nádorová DNA * genetika   MeSH
• DNA nádorová genetika   MeSH
• elektroforéza kapilární MeSH
• lidé MeSH
• mutace genetika   MeSH
• nádorové biomarkery genetika   MeSH
• nádory plic * farmakoterapie   MeSH
• nemalobuněčný karcinom plic * genetika   terapie   MeSH
• reziduální nádor MeSH
• vysoce účinné nukleotidové sekvenování metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

(1) Background: this prospective study was focused on detailed analysis of the mutation heterogeneity in colorectal lesions removed during baseline (index) colonoscopy to identify patients at high risk of early occurrence of metachronous adenomas. (2) Methods: a total of 120 patients after endoscopic therapy of advanced colorectal neoplasia size ≥10 mm (index lesion) with subsequent surveillance colonoscopy after 10-18 months were included. In total, 143 index lesions and 84 synchronous lesions in paraffin blocks were divided into up to 30 samples. In each of them, the detection of somatic mutations in 11 hot spot gene loci was performed. Statistical analysis to correlate the mutation profiles and the degree of heterogeneity of the lesions with the risk of metachronous adenoma occurrence was undertaken. (3) Results: mutation in exon 7 of the TP53 gene found in the index lesion significantly correlated with the early occurrence of metachronous adenoma (log-rank test p = 0.003, hazard ratio 2.73, 95% confidence interval 1.14-6.56). We did not find an association between the risk of metachronous adenomas and other markers monitored. (4) Conclusions: the findings of this study could lead to an adjustment of existing recommendations for surveillance colonoscopies in a specific group of patients with mutations in exon 7 of the TP53 gene in an index lesion, where a shortening of surveillance interval may be warranted.

• Publikační typ
• časopisecké články MeSH