Introduction: Patients with locally advanced rectal cancer (LARC) are undergoing neoadjuvant chemoradiotherapy (NCRT) prior to surgery. Although in some patients the NCRT is known to prevent local recurrence, it is also accompanied by side effects. Accordingly, there is an unmet need to identify predictive markers allowing to identify non-responders to avoid its adverse effects. We monitored circulating tumor DNA (ctDNA) as a potential liquid biopsy-based biomarker. We have investigated ctDNA changes plasma during the early days of NCRT and its relationship to the overall therapy outcome. Methods and Patients: The studied cohort included 36 LARC patients (stage II or III) undergoing NCRT with subsequent surgical treatment. We have detected somatic mutations in tissue biopsies taken during endoscopic examination prior to the therapy. CtDNA was extracted from patient plasma samples prior to therapy and at the end of the first week. In order to optimize the analytical costs of liquid-biopsy testing, we have utilized a two-level approach in which first a low-cost detection method of denaturing capillary electrophoresis was used followed by examination of initially negative samples by a high-sensitivity BEAMING assay. The ctDNA was related to clinical parameters including tumor regression grade (TRG) and TNM tumor staging. Results: We have detected a somatic mutation in 33 out of 36 patients (91.7%). Seven patients (7/33, 21.2%) had ctDNA present prior to therapy. The ctDNA positivity before treatment reduced post-operative disease-free survival and overall survival by an average of 1.47 and 1.41 years, respectively (p = 0.015, and p = 0.010). In all patients, ctDNA was strongly reduced or completely eliminated from plasma by the end of the first week of NCRT, with no correlation to any of the parameters analyzed. Conclusions: The baseline ctDNA presence represented a statistically significant negative prognostic biomarker for the overall patient survival. As ctDNA was reduced indiscriminately from circulation of all patients, dynamics during the first week of NCRT is not suited for predicting the outcome of LARC. However, the general effect of rapid ctDNA disappearance apparently occurring during the initial days of NCRT is noteworthy and should further be studied.

• Klíčová slova
• biomarker, circulating tumor ctDNA, neoadjuvant chemoradiotherapy, prediction, prognosis, rectal cancer, response,
• Publikační typ
• časopisecké články 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

Malignant transformation in gliomas is frequently supplemented by somatic mutations in isocitrate dehydrogenase 1 and isocitrate dehydrogenase 2 genes. It has recently emerged that mutations in these genes are associated with prolonged survival and should be used as prognostic factor in management of brain cancer patients. There are several approaches in use for the detection of isocitrate dehydrogenase 1 and 2 mutations; however, these often exhibit shortcomings such as convoluted protocols with long processing time, complex (and costly) dedicated fluorescent probes, and/or demand on amounts of input DNA. Therefore, a simple and rapid method would be highly desired. Here, we present development and validation of simple and reliable isocitrate dehydrogenase 1 and 2 mutation detection assay using denaturing capillary electrophoresis. The detection sensitivity in terms of the limiting mutated allele fraction detectable estimated from a series of dilution runs was 2.9%. The method was validated by comparing to results obtained by a widely accepted detection technique, the multiplex ligation-dependent probe amplification, on a set of 85 brain tumors. The concordance of both methods was 100%, but denaturing capillary electrophoresis assay required fivefold lower input of DNA (1 versus 5 μL of DNA at concentrations typically between 10 and 30 ng/μL).

• Klíčová slova
• DNA mutation, brain cancer, capillary electrophoresis, glioma, isocitrate dehydrogenase,
• MeSH
• alely MeSH
• elektroforéza kapilární metody   MeSH
• isocitrátdehydrogenasa genetika   MeSH
• lidé MeSH
• mutace * MeSH
• nádory mozku diagnóza   enzymologie   genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• Názvy látek
• IDH2 protein, human MeSH Prohlížeč
• isocitrátdehydrogenasa MeSH

The synthesis and determination of the structure of a Förster resonance energy transfer probe intended for the detection of specific nucleic acid sequences are described here. The probe is based on the hybridization of oligonucleotide modified quantum dots with a fluorescently labeled nucleic acid sample resulting in changes of the fluorescence emission due to the energy transfer effect. The stoichiometry distribution of oligonucleotides conjugated to quantum dots was determined by capillary electrophoresis separation. The results indicate that one to four molecules of oligonucleotide are conjugated to the surface of a single nanoparticle. This conclusion is confirmed by the course of the dependence of Förster resonance energy transfer efficiency on the concentration of fluorescently labeled complementary single-stranded nucleic acid, showing saturation. While the energy transfer efficiency of the probe hybridized with complementary nucleic acid strands was 30%, negligible efficiency was observed with a noncomplementary strand.

• Klíčová slova
• Förster resonance energy transfer, capillary electrophoresis, nucleic acids, quantum dots, sensors,
• Publikační typ
• časopisecké články MeSH

Differential diagnosis of solid pancreatic masses using EUS FNA is in 1015 % of cases still challenging. Promising method, which helps to distinguish between chronic pancreatitis and cancer, is point mutations of the proto-oncogene KRAS test. This method is not established in routine clinical practice yet.Objectives were the determination of the sensitivity of the KRAS assay using various kinds of samples of patients with pancreatic mass and testing the effect of the presence of KRAS mutations on the prognosis of survival. 147 patients underwent EUS-FNA examination of pancreatic mass, accompanied by blood sampling with subsequent separation of plasma for the detection of circulating tumor DNA. Part of biopsy sample was left native in a stabilizing solution and part as cytological smear. Samples (native aspirates, cytological smears, plasma) were examined for the presence of KRAS mutation by heteroduplex analysis, denaturing capillary electrophoresis.Among 147 patients with pancreatic masses, 118 were diagnosed as a cancer, 26 chronic pancreatitis, 3 neuroendocrine tumor. In total 147 native aspirates, 118 cytological smears and 94 plasma samples were examined. The highest sensitivity of KRAS mutation was reached in the group of pancreatic cancer patients using cytology, in which 90 % of KRAS mutation was detected (106/118 of the samples). When using the native cellular aspirates, mutation was detected in 78 % (92/118 samples), and examination of plasma was positive in 27 % (24/90 samples). In four patients with chronic pancreatitis KRAS mutations was detected, although none has been cytologically confirmed as a cancer. Two of these four patients were confirmed in the course of the disease as a cancer, one patient died because of alcoholic delirium and the last one was indicated for surgery recently.Examination of KRAS mutations can be performed in all patients undergoing EUS-FNA, with the cytology being the most reliable type of sample for genetic tests. KRAS examination would be reasonable to introduce into routine clinical practice in a group of patients with unclear differential diagnosis of chronic pancreatitis, especially in those with suspicion of cancer in inflammatory terrain.Kexwords: pancreatic cancer, chronic pancreatitis, KRAS mutation , EUS-FNA.

• MeSH
• biopsie tenkou jehlou pod endosonografickou kontrolou * MeSH
• chronická pankreatitida genetika   patologie   MeSH
• diferenciální diagnóza MeSH
• duktální karcinom slinivky břišní genetika   patologie   MeSH
• endosonografie MeSH
• lidé středního věku MeSH
• lidé MeSH
• mutace MeSH
• mutační analýza DNA metody   MeSH
• nádory slinivky břišní genetika   patologie   MeSH
• prognóza MeSH
• protoonkogen Mas 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
• Názvy látek
• KRAS protein, human MeSH Prohlížeč
• MAS1 protein, human MeSH Prohlížeč
• protoonkogen Mas MeSH
• protoonkogenní proteiny p21(ras) MeSH

BACKGROUND: The presence of circulating tumor cells (CTC) has been reported in patients with advanced colorectal cancer. Monitoring CTC (also known as a liquid-biopsy) has recently become the center of interest for low-invasive monitoring of cancer progression and predictive biomarkers testing. Along with high-cost technology and a complex methodology, a straightforward method based on magnetic beads enrichment followed by RT-PCR is set to allow for routine CTC analysis in colorectal cancer patients. OBJECTIVES: The main purpose of this study was to evaluate the possibility of CTC detection in routine monitoring of patients starting before and continuing after surgery. MATERIAL AND METHODS: The investigated group consisted of 30 patients mainly in advanced stages of colorectal cancer. In all patients, CTC detection was performed prior to surgery, in a subset of 14 patients additional sampling was done during and after surgery. In all cases, peripheral blood was processed using AdnaTest ColonCancer kit, which relies on enriching CTCs using EpCAM-functionalized magnetic beads and subsequently identifying tumorspecific CEA, EGFR and GA733-2 mRNA transcripts. RESULTS: Out of all the tested samples, CTC were found in one patient suffering from advanced disease with lung and liver metastases. There, however, the positive finding was confirmed in 3 consecutive samples acquired before, during and shortly after palliative R2 resection. CONCLUSIONS: The presence of CTC may be used to observe post-operative disease development. Due to the overall low CTC detection, further technology development may be necessary before its universal applicability to manage colorectal cancer patients.

• Klíčová slova
• CEA, CTC, EGFR, GA733-2, colorectal cancer,
• MeSH
• imunomagnetická separace metody   MeSH
• kolorektální chirurgie * MeSH
• kolorektální nádory patologie   chirurgie   MeSH
• lidé MeSH
• nádorové cirkulující buňky patologie   MeSH
• polymerázová řetězová reakce s reverzní transkripcí metody   MeSH
• senioři MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

Background. Gastric cancer is known for a notable variety in the course of the disease. Clinical factors, such as tumor stage, grade, and localization, are key in patient survival. It is expected that molecular factors such as somatic mutations and gene amplifications are also underlying tumor biological behavior and may serve as factors for prognosis estimation. Aim. The purpose of this study was to examine gene amplifications from a panel of genes to uncover potential prognostic marker candidates. Methods. A panel of gene amplifications including 71 genes was tested by multiplex ligation-dependent probe amplification (MLPA) technique in 76 gastric cancer samples from a Caucasian population. The correlation of gene amplification status with patient survival was determined by the Kaplan-Meier method. Results. The amplification of two cell cycle regulators, CCND1 and CDKN1B, was identified to have a negative prognostic role. The medial survival of patients with gastric cancer displaying amplification compared to patients without amplification was 192 versus 725 days for CCND1 (P = 0.0012) and 165 versus 611 days for CDKN1B (P = 0.0098). Conclusion. Gene amplifications of CCND1 and CDKN1B are potential candidates to serve as prognostic markers for the stratification of patients based on the estimate of survival in the management of gastric cancer patients.

• Publikační typ
• časopisecké články 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

OBJECTIVES: A substantial proportion of patients with hypertrophic cardiomyopathy (HCM) do not have causative mutations in the genes for heart sarcomere. The purpose of this study was to evaluate the association between microRNA (miRNA) sequence variants and HCM. METHODS: We performed genetic testing on 56 HCM patients who had previously been found to be negative for mutations in the 4 major genes for sarcomeric proteins. The coding and adjacent regions (120-220 nt) of selected miRNAs were analyzed for the presence of sequence variants. The testing was based on PCR amplification of DNA-encoding miRNAs and subsequent denaturing capillary electrophoresis. RESULTS: A total of 3 different variants were detected in the 11 selected miRNAs. These included polymorphisms rs45489294 in miRNA 208b, rs13136737 in miRNA 367 and rs9989532 in miRNA 1-2. In the patient group, the most frequent polymorphism was in miRNA 208b (10 times) followed by miRNA 367 (7 times). Both polymorphisms were found to occur with similar frequencies in the group of healthy controls. The remaining detected variant was not present in the control group, but was not connected with the HCM phenotype in the children of the probands. CONCLUSION: Sequence variants in miRNAs of patients with HCM are not frequent and the contribution of these variants to the development of this disease was not demonstrated.

• MeSH
• DNA MeSH
• dospělí MeSH
• fenotyp MeSH
• genetická variace * MeSH
• genetické testování MeSH
• hypertrofická kardiomyopatie genetika   MeSH
• lidé středního věku MeSH
• lidé MeSH
• mikro RNA genetika   MeSH
• mutace MeSH
• polymorfismus genetický MeSH
• rodokmen MeSH
• senioři nad 80 let MeSH
• senioři 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
• práce podpořená grantem MeSH
• Názvy látek
• DNA MeSH
• mikro RNA MeSH
• MIRN1-2 microRNA, human MeSH Prohlížeč
• MIRN208 microRNA, human MeSH Prohlížeč
• MIRN367 microRNA, human MeSH Prohlížeč

The role of KRAS mutations in molecular targeted therapy by epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) in non-small cell lung cancer (NSCLC) has not been fully understood. The present investigation is aimed at an elucidation of the role of specific KRAS mutation types in predicting outcomes of patients with advanced NSCLC receiving EGFR-TKI therapy. Initially, 448 NSCLC patients were tested for the presence of KRAS mutations, to obtain frequencies of specific KRAS mutation types. Subsequently, the clinical outcome of treatment was evaluated in a subgroup of 38 KRAS-positive patients receiving EGFR-TKI therapy. KRAS mutations were detected in 69 of 448 patients (15.4%), mostly in smokers (17.86% vs. 5.8%, P = 0.0048), and appeared more frequently in adenocarcinomas than in squamous cell NSCLC or NSCLC that is not otherwise specified (21% vs. 6.99% vs. 4.4%, P = 0.0004). The most frequent type of KRAS mutation was G12C. The progression-free survival (PFS) was doubled in a group of non-G12C patients compared with that of the G12C group (9.0 wk vs. 4.3 wk, P = 0.009). The overall survival (OS) was not significantly different between non-G12C and G12C groups (12.1 wk vs. 9.3 wk, P = 0.068). The G12C KRAS mutation is a strong negative predictor for EGFR-TKI treatment, whereas other KRAS mutation types have not negatively predicted treatment efficacy compared with that for the wild-type KRAS genotype.

• MeSH
• biomarkery farmakologické metabolismus   MeSH
• chemorezistence genetika   MeSH
• cystein genetika   MeSH
• dospělí MeSH
• erbB receptory antagonisté a inhibitory   MeSH
• glycin genetika   MeSH
• inhibitory proteinkinas terapeutické užití   MeSH
• kohortové studie MeSH
• lidé středního věku MeSH
• lidé MeSH
• missense mutace fyziologie   MeSH
• nádorové biomarkery genetika   fyziologie   MeSH
• nádory plic diagnóza   farmakoterapie   genetika   MeSH
• nemalobuněčný karcinom plic diagnóza   farmakoterapie   genetika   MeSH
• prognóza MeSH
• protinádorové látky terapeutické užití   MeSH
• protoonkogenní proteiny p21(ras) MeSH
• protoonkogenní proteiny genetika   metabolismus   fyziologie   MeSH
• Ras proteiny genetika   metabolismus   fyziologie   MeSH
• senioři MeSH
• studie případů a kontrol MeSH
• substituce aminokyselin fyziologie   MeSH
• tyrosinkinasy antagonisté a inhibitory   MeSH
• výsledek terapie MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• Názvy látek
• biomarkery farmakologické MeSH
• cystein MeSH
• erbB receptory MeSH
• glycin MeSH
• inhibitory proteinkinas MeSH
• KRAS protein, human MeSH Prohlížeč
• nádorové biomarkery MeSH
• protinádorové látky MeSH
• protoonkogenní proteiny p21(ras) MeSH
• protoonkogenní proteiny MeSH
• Ras proteiny MeSH
• tyrosinkinasy MeSH