Lucie B.,Genomac International Ltd. |
Marek M.,Genomac International Ltd. |
Dana J.,Faculty Hospital Pilsen |
Barbora B.,Genomac International Ltd. |
Milos P.,Faculty Hospital Pilsen
Anticancer Research | Year: 2010
Background: Concurrent presence of EGFR and KRAS mutations in non-small cell lung cancer (NSCLC) patients is relatively rare and their appearance is believed to be mutually exclusive. Tumours harbouring KRAS mutation are perceived as not being capable of response to tyrosine kinase inhibitor (TKI) therapy. Patients and Methods: This paper presents 5 case reports of patients with tumours harbouring multiple EGFR andlor KRAS mutations. There were 3 patients with EGFR mutations (2 x exon 19 deletions, 1 x L858R) combined with KRAS mutations (2 x Gly12Asp, 1 x Gly12Val), 1 patient with two EGFR mutations (exon 19 deletion + L858R) and 1 patient with two KRAS mutations (Ala11Pro + Gly12Val). Results: All EGFR+/KRAS+ patients had initially showed positive response to TK1 treatment. The EGFR+/EGFR + patient has exhibited strong rash and good response with the best survival, while the KRAS+/KRAS+ patient did not respond to TKI therapy. Conclusion: EGFR+/KRAS+ combination does not necessarily pose a negative prediction. This is probably due to the multiclonal character of the tumour displaying partial response in the EGFR+ subpopulation.
Pesek M.,Faculty Hospital Pilsen |
Kopeckova M.,Genomac International Ltd. |
Kopeckova M.,Applied Genomics |
Benesova L.,Genomac International Ltd. |
And 6 more authors.
Anticancer Research | Year: 2011
Background: DNA methylation is one of major factors in cancer progression. We observed multiple genes involved in cancer-related signaling and focused on patients with advanced non-small cell lung cancer (NSCLC) and evaluated methylation in relation to various clinical parameters. Patients and Methods: Thirty genes were examined in 121 NSCLC patients using the methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) method. Correlations to gender, smoking status, tumor subtype, disease stage and EGFR/KRAS mutation status were performed by chi-square test. Results: 90% of tumors exhibited methylation of at least one gene. Most frequently methylated were cadherin-13 (CDH13), Ras associated domain-containing protein (RASSF1A), Wilms' tumor protein (WT1), adenomatous polyposis coli protein (APC), paired box protein Pax-5 (PAX5), estrogen receptor (ESR1), an inhibitor of cyclin-dependent kinase p15 (CDKN2B), paired box protein Pax-6 (PAX6), transcription factor GATA-5 (GATA5) and cell adhesion molecule 4 (IGSF4). Overall methylation (any gene) was increased in adenocarcinomas (p=0.0329), unrelated to gender or disease stage. Several genes exhibited variable methylation with gender (CDH13, p<0.001; GATA5, p=0.02; PAX6, p=0.01 and ESR1, p=0.03), smoking (CDH13, p=0.002), or epidermal growth factor receptor (EGFR) mutation status [Von Hippel-Lindau disease tumor supresor (VHL), p=0.001; CDKN2B, p=0.02; CDH13, p=0.02; APC, p=0.04 and ESR1, p=0.04]. Conclusion: Differences in gene methylation associated with gender, smoking and EGFR mutation suggest potential for prediction in relation to management of tyrosine kinase inhibitor therapy.
Kobr J.,Charles University |
Fremuth J.,Faculty Hospital Pilsen |
Pizingerova K.,Faculty Hospital Pilsen |
Sasek L.,Charles University |
And 3 more authors.
Cardiovascular Ultrasound | Year: 2011
Background: The aim of this study was to verify the benefits and limitations of repeated bedside echocardiographic examinations in children during mechanical ventilation. For the purposes of this study, we selected the data of over a time period from 2006 to 2010. Methods. A total of 235 children, average age 3.21 (SD 1.32) years were included into the study and divided into etiopathogenic groups. High-risk groups comprised: Acute lung injury and acute respiratory distress syndrome (ALI/ARDS), return of spontaneous circulation after cardiopulmonary resuscitation (ROSC), bronchopulmonary dysplasia (BPD), cardiomyopathy (CMP) and cardiopulmonary disease (CPD). Transthoracic echocardiography was carried out during mechanical ventilation. The following data were collated for statistical evaluation: right and left ventricle myocardial performance indices (RV MPI; LV MPI), left ventricle shortening fraction (SF), cardiac output (CO), and the mitral valve ratio of peak velocity of early wave (E) to the peak velocity of active wave (A) as E/A ratio. The data was processed after a period of recovery, i.e. one hour after the introduction of invasive lines (time-1) and after 72 hours of comprehensive treatment (time-2). The overall development of parameters over time was compared within groups and between groups using the distribution-free Wilcoxons and two-way ANOVA tests. Results: A total of 870 echocardiographic examinations were performed. At time-1 higher average values of RV MPI (0.34, SD 0.01 vs. 0.21, SD 0.01; p < 0.001) were found in all groups compared with reference values. Left ventricular load in the high-risk groups was expressed by a higher LV MPI (0.39, SD 0.13 vs. 0.29, SD 0.02; p < 0.01) and lower E/A ratio (0.95, SD 0.36 vs. 1.36, SD 0.64; p < 0.001), SF (0.37, SD 0.11 vs. 0.47, SD 0.02; p < 0.01) and CO (1.95, SD 0.37 vs. 2.94, SD 1.03; p < 0.01). At time-2 RV MPI were lower (0.25, SD 0.02 vs. 0.34, SD 0.01; p < 0.001), but remained higher compared with reference values (0.25, SD 0.02 vs. 0.21, SD 0.01; p < 0.05). Other parameters in high-risk groups were improved, but remained insignificantly different compared with reference values. Conclusion: Echocardiography complements standard monitoring of valuable information regarding cardiac load in real time. Chest excursion during mechanical ventilation does not reduce the quality of the acquired data. © 2011 Kobr et al; licensee BioMed Central Ltd.
Kobr J.,Faculty Hospital in Pilsen |
Kuntscher V.,Faculty Hospital Pilsen |
Molacek J.,Faculty Hospital Pilsen |
Hes O.,Faculty Hospital Pilsen |
And 3 more authors.
In Vivo | Year: 2010
Background: The aim of the study was to determine how tidal volumes may affect the lung and haemodynamics during mechanical ventilation. Materials and Methods: With the approval of the Ethics Committee, the study included a total of 24 healthy piglets, average weight 30 kg (range 28-33 kg). The animals were ventilated for 90 minutes under general anaesthesia with two different tidal volume strategies and allocated into three groups. Group A, animals were healthy controls, Group B, animals comprised 8 piglets with an abdominal aortic aneurysm and ventilated with a low tidal volume strategy (VTexp 7ml/kg). Group C comprised 8 animals seven days after kidney transplantation, ventilated with a high tidal volume strategy (VTexp 12 ml/kg). Changes in lung mechanics and hemodynamics were assessed at 30th and 90th minutes. Lung tissue samples were examined histologically. Results: Protective ventilation in Group A and B did not confer any haemodynamic and lung mechanic differences. Significant differences were only found in Group C at 90 minutes for increased preload of both heart ventricles (CVP; t-test 4.07, p<0.01 and PAoP; t-test 8.43, p<0.01), pulmonary vascular resistance (t-test 3.11, p<0.05), and decreased expiratory tidal volume (t-test 6.07, p<0.01), dynamic lung compliance (t-test 3.83, p<0.01) and cardiac output (t-test 2.07, p<0.01). Diffuse alveolar damage was detected histologically. Conclusion: Mechanical ventilation at high tidal volumes reaching 12 ml/kg caused functional changes in the lungs, diffuse alveolar damage and reduction of cardiac output within 90 minutes.
Kucera R.,Faculty Hospital Pilsen |
Kucera R.,Charles University |
Topolcan O.,Faculty Hospital Pilsen |
Topolcan O.,Charles University |
And 10 more authors.
Clinica Chimica Acta | Year: 2015
Background: IGF1 is responsible for regulation of growth, metabolism and differentiation of human cells. IGFBP3 is the most abundant of the carrier proteins for IGF1 in the blood. IGF1/IGFBP3 molar ratio is an indicator of IGF1 bioavailability. We decided to create a file of reference ranges of IGF1, IGFBP3 and IGF1/IGFBPP3 ratio for the adult Czech population across the age spectrum. Methods: We selected a group of 1022 subjects, 467 males and 555 females (ages 20-98. years), from several regions in the Czech Republic. The group consisted of blood donors and patients undergoing regular preventive examinations. Serum levels of IGF1 and IGFBP3 were measured using the following radioimmunoassay kits: IRMA IGF1 (Immunotech, Marseille, France) and IRMA IGFBP3 (Immunotech, Prague, Czech Republic). The IGF1/IGFBP3 ratio was also calculated. The following groups of patients were excluded: patients with diabetes, high blood glucose, high insulin levels, post-surgery patients, polymorbid patients, and subjects with oncological diseases. Subjects were divided into seven age-groups. Changes in the levels of observed analytes in each decade across the age spectrum were evaluated. All statistical analyses were performed by SAS 9.3 (Statistical Analysis Software release 9.3; SAS Institute Inc., Cary, NC, USA). Results: All three parameters IGF1, IGFBP3 and IGF1/IGFBP3 decreased in parallel with decrease in age: p<. 0.0001, r=. -. 0.64, -. 0.35 and -. 0.54, respectively. The dynamics of the decline was different between males and females.Linear regression models with age as independent variable fitted by gender are displayed in Fig. 1. Non-parametric reference interval curves (medians and 2.5th-97.5th percentiles) for IGF1, IGFBP3 and IGF1/IGFBP3 ratio as function of age by gender are displayed in Fig. 2(a,b,c). All medians and 2.5th-97.5th percentiles were plotted by cubic spline.For males, linear regression models were as follows: IGF1. =. 291.34619. -. 2.41211. ×. age, IGFBP3. =. 2931.62778. -. 6.11659. ×. age, IGF1/IGFBP3. =. 0.02897. -. 0.00021213. ×. age. For females, we plotted the following: IGF1. =. 241.67406. -. 1.98466. ×. age, IGFBP3. =. 3688.60561. -. 16.39560. ×. age, IGF1/IGFBP3. =. 0.02029. -. 0.00013233. ×. age.IGF1 was statistically significantly higher in males with p<. 0.0001 (Wilcoxon test) but decreased faster (. p=. 0.0121). IGFBP3 was statistically significantly higher in females with p=. 0.0004 (Wilcoxon test) but decreased faster (. p<. 0.0001). IGF1/IGFBP3 was statistically significantly higher in males with p<. 0.0001 (Wilcoxon test) but decreased faster (. p<. 0.0001). Conclusion: Authors recommend using of a linear regression model based reference ranges for IGF1, IGFBP3 and IGF1/IGFBP3 ratio and using different reference ranges for genders. © 2015 Elsevier B.V.
Minarik M.,Laboratory for Molecular Genetics and Oncology |
Minarik M.,Applied Genomics |
Gassman M.,Agilent Technologies |
Belsanova B.,Laboratory for Molecular Genetics and Oncology |
And 7 more authors.
Electrophoresis | Year: 2010
There is a growing interest in evaluating molecular markers as predictors of response to new generation of targeted cancer therapies. One of such areas is biological therapy targeting epidermal growth factor receptor gene (EGFR) in lung cancer. The testing of tumor tissue is focused on specific EGFR mutations and EGFR gene amplification, since tumors exhibiting positivity of either of the two marker types are highly sensitive towards the treatment. Although traditional methods of DNA sequencing and fluorescence in situ hybridization are still in use for the detection of EGFR mutations and gene amplification, respectively, there is a need for new dedicated techniques with the primary emphasis on simplicity, sensitivity, speed and cost effectiveness. The main purpose of this work was to integrate diverse assays for both EGFR tests onto a single platform to eliminate the need for different instruments and separate processing. We demonstrate a chip capillary electrophoresis (chipCE) application for EGFR mutation detection by a combination of fragment analysis and denaturing CE along with multiplex ligation-dependent probe amplification (MLPA) for evaluation of EGFR amplification. All separations are carried out in denaturing sieving polymer on a modified Bioanalyzer 2100 chipCE instrument running at temperatures of up to 65°C. The main strength of the resulting high-resolution chipCE application is in its simplicity, speed of analysis and minimal amount of sample required for complete testing of EGFR status. Such an approach could potentially fit medium throughput laboratories providing molecular pathology services for clinical oncologists with fast turnaround times and limited consumption of tissue material. © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
PubMed | Institute of Endocrinology, Faculty Hospital Pilsen, Institute for Clinical and Experimental Medicine and Charles University
Type: Journal Article | Journal: PloS one | Year: 2014
Abnormal postprandial elevation of plasma glucose and lipids plays an important role in the pathogenesis of diabetes and strongly predicts cardiovascular mortality. In patients suffering from type 2 diabetes (T2D) postprandial state is associated with oxidative stress, cardiovascular risk and, probably, with impairment of both secretion and the effect of gastrointestinal peptides. Evaluating postprandial changes of gastrointestinal hormones together with changes in oxidative stress markers may help to understand the mechanisms behind the postprandial state in diabetes as well as suggest new preventive and therapeutical strategies.A standard meal test has been used for monitoring the postprandial concentrations of gastrointestinal hormones and oxidative stress markers in patients with T2D (n=50) compared to healthy controls (n=50). Blood samples were drawn 0, 30, 60, 120 and 180 minutes after the standard meal.Both basal and postprandial plasma concentrations of glucose and insulin proved to be significantly higher in patients with T2D, whereas plasma concentrations of ghrelin showed significantly lower values during the whole meal test. In comparison with healthy controls, both basal and postprandial concentrations of almost all other gastrointestinal hormones and lipoperoxidation were significantly increased while ascorbic acid, reduced glutathione and superoxide dismutase activity were decreased in patients with T2D. A positive relationship was found between changes in GIP and those of glucose and immunoreactive insulin in diabetic patients (p<0.001 and p<0.001, respectively) and between changes in PYY and those of glucose (p<0.01). There was a positive correlation between changes in GIP and PYY and changes in ascorbic acid in patients with T2D (p<0.05 and p<0.001, respectively).Apart from a positive relationship of postprandial changes in GIP and PYY with changes in ascorbic acid, there was no direct link observed between gastrointestinal hormones and oxidative stress markers in diabetic patients.ClinicalTrials.gov NCT01572402.
PubMed | Faculty Hospital Pilsen
Type: Journal Article | Journal: Anticancer research | Year: 2011
DNA methylation is one of major factors in cancer progression. We observed multiple genes involved in cancer-related signaling and focused on patients with advanced non-small cell lung cancer (NSCLC) and evaluated methylation in relation to various clinical parameters.Thirty genes were examined in 121 NSCLC patients using the methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) method. Correlations to gender, smoking status, tumor subtype, disease stage and EGFR/KRAS mutation status were performed by chi-square test.90% of tumors exhibited methylation of at least one gene. Most frequently methylated were cadherin-13 (CDH13), Ras associated domain-containing protein (RASSF1A), Wilms tumor protein (WT1), adenomatous polyposis coli protein (APC), paired box protein Pax-5 (PAX5), estrogen receptor (ESR1), an inhibitor of cyclin-dependent kinase p15 (CDKN2B), paired box protein Pax-6 (PAX6), transcription factor GATA-5 (GATA5) and cell adhesion molecule 4 (IGSF4). Overall methylation (any gene) was increased in adenocarcinomas (p=0.0329), unrelated to gender or disease stage. Several genes exhibited variable methylation with gender (CDH13, p<0.001; GATA5, p=0.02; PAX6, p=0.01 and ESR1, p=0.03), smoking (CDH13, p=0.002), or epidermal growth factor receptor (EGFR) mutation status [Von Hippel-Lindau disease tumor supresor (VHL), p=0.001; CDKN2B, p=0.02; CDH13, p=0.02; APC, p=0.04 and ESR1, p=0.04].Differences in gene methylation associated with gender, smoking and EGFR mutation suggest potential for prediction in relation to management of tyrosine kinase inhibitor therapy.
PubMed | Faculty Hospital Pilsen
Type: Journal Article | Journal: EJIFCC | Year: 2016
Multiplex analysis is intended to simultaneously look for multiple targets in one sample. This approach has been largely adopted in genomics and progressively expands to various domains of laboratory investigation. In protein analysis, immunoassays are the fundamental methods and their multiplexing and miniaturization is of great applicability to both basic and applied research. Furthermore, the potential of these high-throughput methodologies can be foreseen in the field of clinical diagnostics. The following text describes planar and bead-based arrays, two main strategies of immunoassay multiplexing. Principles, detection methods and strengths of each are shortly discussed. Finally, we mention several challenges linked with the integration of these methods to diagnostics.