Radka Fuchsova

Reference values of IGF1, IGFBP3 and IGF1/IGFBP3 ratio in adult population in the Czech Republic

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.

Serum Vitamin D Status, Vitamin D Receptor Polymorphism, and Glucose Homeostasis in Healthy Subjects

Low vitamin D status has been frequently associated with impaired glucose metabolism. We examined associations between 25-hydroxyvitamin D (25-OH-D) and several parameters of glucose homeostasis in virtually healthy subjects, and explored possible interaction with vitamin D receptor (VDR) polymorphism. Nondiabetic subjects without chronic medication or any known significant manifest disease were selected from large general-population based population survey. Insulin sensitivity and β cell secretion were calculated by homeostasis model assessment (HOMA) and soluble isoform of receptor for advanced glycation end-products (sRAGE) using commercial ELISA. Subjects were also genotyped for rs2228570 polymorphism of VDR. After adjustment for potential confounders, we observed a significant relationship between 25-OH-D and fasting glycemia (β coefficient=-5.904; p=0.002) or insulin sensitivity (β=0.042; p=0.001), but not with β cell secretion or sRAGE. We found also an interaction with VDR polymorphism. Subjects with low 25-OH-D and AA genotype had significantly lower insulin sensitivity than those with GG genotype plus highest 25-OH-D concentrations (107.3% vs. 183.9%, p=0.021). In conclusion, low vitamin D status was in virtually healthy subjects associated with decreased insulin sensitivity, namely in those with GG genotype of rs2228570 VDR polymorphism.

Synergistic effect of low K and D vitamin status on arterial stiffness in a general population

Both vitamins K and D are nutrients with pleiotropic functions in human tissues. The metabolic role of these vitamins overlaps considerably in calcium homeostasis. We analyzed their potential synergetic effect on arterial stiffness. In a cross-sectional study, we analyzed aortic pulse wave velocity (aPWV) in 1023 subjects from the Czech post-MONICA study. Desphospho-uncarboxylated matrix γ-carboxyglutamate protein (dp-ucMGP), a biomarker of vitamin K status, was measured by sandwich ELISA and 25-hydroxyvitamin D3 (25-OH-D3) by a commercial immunochemical assay. In a subsample of 431 subjects without chronic disease or pharmacotherapy, we detected rs2228570 polymorphism for the vitamin D receptor. After adjustment for confounders, aPWV was independently associated with both factors: dp-ucMGP [β-coefficient(S.E.M.)=13.91(4.87); P=.004] and 25-OH-D3 [0.624(0.28); P=.027]. In a further analysis, we divided subjects according to dp-ucMGP and 25-OH-D3 quartiles, resulting in 16 subgroups. The highest aPWV had subjects in the top quartile of dp-ucMGP plus bottom quartile of 25-OH-D3 (i.e., in those with insufficient status of both vitamin K and vitamin D), while the lowest aPVW had subjects in the bottom quartile of dp-ucMGP plus top quartile of 25-OH-D3 [9.8 (SD2.6) versus 6.6 (SD1.6) m/s; P<.0001]. When we compared these extreme groups of vitamin K and D status, the adjusted odds ratio for aPWV≥9.3 m/s was 6.83 (95% CI:1.95-20.9). The aPWV was also significantly higher among subjects bearing the GG genotype of rs2228570, but only in those with a concomitantly poor vitamin K status. In conclusion, we confirmed substantial interaction of insufficient K and D vitamin status in terms of increased aortic stiffness.

Effects of Icodextrin and Glucose Bicarbonate/Lactate-Buffered Peritoneal Dialysis Fluids on Effluent Cell Population and Biocompatibility Markers IL-6 and CA125 in Incident Peritoneal Dialysis Patients

Icodextrin peritoneal dialysis (PD) solution has been shown to increase interleukin‐6 (IL‐6) levels in PD effluent as well as leukocyte and mesothelial cell count. Mesothelial cells release cancer antigen 125 (CA125), which is used as a marker of mesothelial cell mass. This 1‐year prospective study was designed to compare peritoneal effluent cell population, its inflammatory phenotype and biocompatibility biomarkers IL‐6 and CA125 between icodextrin (E) and glucose bicarbonate/lactate (P) based PD solutions. Using baseline peritoneal ultrafiltration capacity, 19 stable incident PD patients were allocated either to P only (N = 8) or to P plus E for the overnight dwell (N = 11). Flow cytometry was used to measure white blood cell count and differential and the expression of inflammatory molecules on peritoneal cells isolated from timed overnight peritoneal effluents. Compared to P, E effluent showed higher leukocyte (10.9 vs. 7.9), macrophages (6.1 vs. 2.5) and mesothelial cells (0.3 vs. 0.1)×106/L count, as well as expression of HLA DR on mesothelial cells and IL‐6 (320.5 vs. 141.2 pg/min) on mesothelial cells and CA125 appearance rate (159.6 vs. 84.3 IU/min), all P < 0.05. In the E group, correlation between IL‐6 and CA125 effluent levels (r = 0.503, P < 0.05) as well as appearance rates (r = 0.774, P < 0.001) was demonstrated. No effect on systemic inflammatory markers or peritoneal permeability was found. Icodextrin PD solution activates local inflammation without systemic consequences so the clinical relevance of this observation remains obscure. Correlation between effluent IL‐6 and CA125 suggests that CA125 might be upregulated due to inflammation and thus is not a reliable marker of mesothelial cell mass and/or biocompatibility.

The Ability of Prostate Health Index (PHI) to Predict Gleason Score in Patients With Prostate Cancer and Discriminate Patients Between Gleason Score 6 and Gleason Score Higher Than 6—A Study on 320 Patients After Radical Prostatectomy

Aim: The purpose of this study was to investigate the Prostate Health Index as a marker for tumor aggressiveness in prostate biopsy and the optimization of indication for treatment options. Methods: Our cohort consisted of 320 patients indicated for radical prostatectomy with preoperative measurements of total prostate-specific antigen, free prostate-specific antigen, [-2]proPSA, calculated %freePSA, and Prostate Health Index. The Gleason score was determined during biopsy and after radical prostatectomy. Using the Gleason score, we divided the group of patients into the 2 subgroups: Gleason score ≤6 and Gleason score >6. This division was performed according to the biopsy Gleason score and according to the postoperative Gleason score. We compared total prostate-specific antigen, [-2]proPSA, %freePSA, and Prostate Health Index in the subgroups Gleason score ≤6 and Gleason score >6 after biopsy and the definitive score. Results: On evaluation of the subgroups created by Gleason score ≤6 and Gleason score >6, we observed agreement between biopsy Gleason score and definitive Gleason score in only 45.3% of cases. Of the calculated biopsy, Gleason score ≤6 and Gleason score >6 subgroups, [-2]proPSA, and Prostate Health Index (P = .0003 and P = .0005) were statistically significant. Of the definitive Gleason score ≤6 and Gleason score >6 subgroups, Prostate Health Index, [-2]proPSA, %freePSA, and PSA (P < .0001, P < .0001, P = .0003, and P = .0043) were statistically significant. The best area under the curve value (0.7496) was achieved by Prostate Health Index when the subgroups were established according to the postoperative Gleason score. Conclusion: Prostate Health Index is the best of the tested markers for the categorization of Gleason score 6 tumors and for facilitating the management of patients with prostate cancer. Prostate Health Index can be a helpful marker for indication of active surveillance or radical prostatectomy. Prostate health index can also simplify the decision of whether to perform nerve-sparing radical prostatectomy.

An Assessment of Novel Biomarkers in Bone Metastatic Disease Using Multiplex Measurement and Multivariate Analysis

Aim: Current diagnostics of bone metastatic disease is not satisfactory for early detection or regular process monitoring. The combination of biomarkers and the multiparametric approach was described as effective in other oncology diagnoses. The aim of the study was to improve the difference diagnostics between bone-metastatic disease and solid tumors using mutivariate logistic regression model. Methods: We assessed the group of 131 patients with the following diagnoses: prostate cancer, breast cancer, lung cancer, and colorectal cancer. According to the results of scintigraphy, the cohort was divided into 2 groups based on the occurrence of bone metastases. Group 0 was a control group of 75 patients with no signs of bone metastases and group 1 included 56 patients with bone metastases. Results: We used stepwise selection multivariate logistic regression for choosing the multimarker formula for calculation of risk score for bone metastases diagnostics. For detection of bone metastasis, it was shown to be most effective measurement of 3 biomarkers: procollagen type 1 N-terminal propeptide, growth differentiation factor-15, and osteonectin and combining with calculation of risk score by designating measured concentrations in mathematical formula: bone risk score = procollagen type 1 N-terminal propeptide × 0.0500 + growth differentiation factor-15 × 1.4179 + osteonectin × 0.00555. Conclusion: We identified growth differentiation factor-15 as the best individual marker for bone metastasis diagnostics. The best formula for risk score includes levels of 3 biomarkers—procollagen type 1 N-terminal propeptide, growth differentiation factor-15, and osteonectin. The new score has better performance described by higher area under the curve than individual biomarkers. A further study is necessary to confirm these findings incorporating a larger number of patients.

Stability of total prostate-specific antigen and free prostate-specific antigen after 10 years’ storage

Introduction: PSA is a serine protease composed of 240 amino acids in a single polypeptide chain and is a routine parameter in prostate cancer diagnostics. The aim of our study was to test the long-term stability of tPSA and fPSA after 10 years’ storage at −80°C. Materials and methods: We analyzed two aliquots from 55 serum samples. The first was assayed in routine testing at the time of establishing the diagnosis. The second was thawed for further testing after approximately 10 years’ storage at −80°C. The mean of storage time was 10.41 years (min–max: 9.35–11.40 years). We compared the results of tPSA and fPSA. We calculated the fPSA/tPSA ratio and compared the results of clinical evaluation. Serum tPSA and fPSA levels were assayed using chemiluminescent kits Access Hybritech PSA and free PSA. All measurements were performed using the instrument UniCel® DxI 800. Results: tPSA decreased 3.59% on average with a correlation r=0.9213, and fPSA increased at an average of 2.41% with a correlation r=0.9338. The fPSA/tPSA ratio increased 0.80% on average with a correlation r=0.9174. On clinical evaluation, five samples had fallen to a less malignant category and three samples had risen to a higher malignant category compared with the original results. Conclusion: The stability of tPSA and fPSA levels in serum is sufficient after 10 years’ storage at −80°C. Calculation of the fPSA/tPSA ratio is not recommended due to the change in the category of malignancy of 15% of the samples.

Serum levels of markers in early detection of prostate cancer (pilote study)

Technological approaches The Immunoanalytical Laboratory of University Hospital in Pilsen examined sera of 76 patients from the Urology department of the University Hospital with suspected prostate cancer who have undergone TRUS biopsy. We assessed the levels of PSA and, if the interval of PSA was between 0-30 ng/mL, we also assessed the levels of freePSA, [-2]proPSA and we calculated %freePSA and Prostate Health Index (PHI). The monitored biomarkers were measured using the chemiluminescent DxI 800 instrument (Beckman Coulter, USA). The peripheral blood was drawn by VACUETTE ® (Greiner Bio-One, Austria). All specimens were immediately aliquoted, frozen and stored at -80°C. Samples were thaw only ones just before the processing. SAS 9.2 software was used for all statistical analysis.

Education in personalized medicine in Czech Republic

Personalized medicine is a new philosophy in the health care. It consists in the application of innovative diagnostic methods and biotechnologies to the prediction of human pathologies and in the development of prevention and individual therapy-planning. The principal tool of personalized medicine is the application of new methods, mainly those of molecular biology (abbreviated as – omics methods) into all areas of medicine in order to comply with two principal goals: 1/ optimization of early diagnostics, prediction and prognosis, and 2/ rigorous individualization of therapy-planning as well as an individual approach to the patient. Final effect should be a completely new way to the prevention assurance. Actual knowledge of the principles of personalized medicine among clinicians and therefore their application are still low. Technological approaches Our first approach was to find out the level of knowledge among different population groups by means of a surway. The results confirmed the above mentioned statement. We have initiated the education of personalized medicine which is targeted at students of bachelor and master programs as well as at post graduate students, clinicians, academicians and researchers. The education is provided by the means of in-room teaching as well as e-learning and internet resources. Our goal is to provide the teaching in cooperation with national and international medical associations.