Radek Kucera

Changes in the expression of N- and O-glycopeptides in patients with colorectal cancer and hepatocellular carcinoma quantified by full-MS scan FT-ICR and multiple reaction monitoring

Alternations in the glycosylation of proteins have been described in connection with several cancers, including hepatocellular carcinoma (HCC) and colorectal cancer. Analytical tools, which use combination of liquid chromatography and mass spectrometry, allow precise and sensitive description of these changes. In this study, we use MRM and FT-ICR operating in full-MS scan, to determine ratios of intensities of specific glycopeptides in HCC, colorectal cancer, and liver metastasis of colorectal cancer. Haptoglobin, hemopexin and complement factor H were detected after albumin depletion and the N-linked glycopeptides with fucosylated glycans were compared with their non-fucosylated forms. In addition, sialylated forms of an O-linked glycopeptide of hemopexin were quantified in the same samples. We observe significant increase in fucosylation of all three proteins and increase in bi-sialylated O-glycopeptide of hemopexin in HCC of hepatitis C viral (HCV) etiology by both LC-MS methods. The results of the MRM and full-MS scan FT-ICR analyses provide comparable quantitative readouts in spite of chromatographic, mass spectrometric and data analysis differences. Our results suggest that both workflows allow adequate relative quantification of glycopeptides and suggest that HCC of HCV etiology differs in glycosylation from colorectal cancer and liver metastasis of colorectal cancer. SIGNIFICANCE The article compares N- and O-glycosylation of several serum proteins in different diseases by a fast and easy sample preparation procedure in combination with high resolution Fourier transform ion cyclotron resonance mass spectrometry. The results show successful glycopeptides relative quantification in a complex peptide mixture by the high resolution instrument and the detection of glycan differences between the different types of cancer diseases. The presented method is comparable to conventional targeted MRM approach but allows additional curation of the data.

Anti-Müllerian hormone in serum and seminal plasma in comparison with other male fertility parameters.

ABSTRACT Anti-Müllerian hormone (AMH) is a factor most associated with female fertility and especially with the ovarian reserve. AMH is also used as a parameter of fertility in men as it arises from the epithelium of the seminiferous tubules that contain Sertoli cells which produce the AMH. To investigate the relationship between AMH production and sperm related parameters we compared the AMH levels in serum and seminal plasma between a group of healthy males (n=65) and male patients (n=68) of infertile couples with semen pathology. We assessed the following fertility parameters: sperm count (SC), presence of intra-acrosomal enzymes (IAE), and antispermatozoal antibodies (ASA). Infertile men were divided into four subgroups according to: SC less than 15 million, SC less than 15 million and lack of IAE, SC less than 15 million and presence of ASA, presence of all three pathological parameters. The mean AMH serum level in the healthy group was 6.95 ng/ml and no significant difference was observed in serum AMH levels. The mean AMH seminal plasma level in the healthy group was 14.21 ng/ml. We observed a statistically significant decrease in the group with a SC with less than 15 million (3.29 ng/ml, p=0.0001) sperm, in the group with SC less than 15 million sperm and lack of IAE (3.95 ng/ml, p=0.0046), and in the group with all three pathological parameters (2.65 ng/ml, p=<0.0001). No significant difference was observed in the group with SC less than 15 million sperm and ASA positivity (11.41 ng/ml, p=0.3171). In conclusion AMH serum levels do not correlate with any of the observed parameters. AMH levels in seminal plasma positively correlate with the pathological SC and with SC pathology and IAE together.

Effect of long-term using of hormonal contraception on anti-Müllerian hormone secretion

Abstract Anti-Müllerian hormone (AMH) is an important factor associated with female fertility and the ovarian reserve. There are several past studies available concerning the influence of hormonal contraception (HC) on serum AMH levels. Recent studies have reported that AMH levels in women using HC can be about 30% lower compared to those not using HC. However, earlier studies showed no reduction in AMH levels in HC users. We decided to evaluate the effects of long-term HC use (mean duration of HC use: 11.4 years) on AMH levels in women. To exclude potential shorter and reversible decreasing effects of HC on fertility function, we decided to include women in the study who had stopped using HC 1 year before the AMH sample collection. We examined 105 women who used HC and 44 women who had never used HC. The median concentration of AMH in the group of long-term users of HC was 2.89 and 3.37 ng/ml in the group of women who had never used HC. We found no statistically significant difference (p = 0.3261). In conclusion, we observed no negative impact of HC on the AMH serum levels. AMH can be used as an ovarian reserve marker for these women.

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.

Detection and Quantification of Carbohydrate-Deficient Transferrin by MALDI-Compatible Protein Chips Prepared by Ambient Ion Soft Landing

BACKGROUND Transferrin is synthetized in the liver and is the most important iron-transport carrier in the human body. Severe alcohol consumption leads to alterations in glycosylation of transferrin. Mass spectrometry can provide fast detection and quantification of transferrin isoforms because they have different molecular masses. In this study, we used antibody chips in combination with MALDI-TOF MS for the detection and quantification of transferrin isoforms. METHODS Protein chips were prepared by functionalization of indium tin oxide glass using ambient ion soft landing of electrosprayed antitransferrin antibody. Two microliters of patient serum was applied on the antibody-modified spots, and after incubation, washing, and matrix deposition, transferrin isoforms were detected by MALDI-TOF MS. Peak intensities of each transferrin form were used to calculate total carbohydrate-deficient transferrin (CDT). The CDT values obtained by the MALDI chip method were compared with the results obtained by a standard capillary electrophoresis (CE). RESULTS The chip-based MALDI-TOF MS method was used for enrichment and detection of CDT from human serum. A sample cohort from 186 patients was analyzed. Of these samples, 44 were positively identified as belonging to alcoholic patients, whereas 142 were negative by the MALDI chip approach. The correlation of the data obtained by the CE and the chip-based MALDI was r = 0.986, 95% CI. CONCLUSIONS Functionalized MALDI chips modified by antitransferrin antibody prepared by ambient ion soft landing were successfully used for detection and quantification of CDT from human sera.

PIVKA-II as a Potential New Biomarker for Hepatocellular Carcinoma – A Pilot Study

Aim: The aim of this study was to evaluate the clinical contribution of protein induced by vitamin K absence (PIVKA-II) for hepatocellular carcinoma (HCC) diagnostics and compare it with alpha-foetoprotein (AFP), a routinely used tumour marker. Materials and Methods: A total of 332 participants were enrolled in this study: 64 with HCC, 48 with liver metastases of colorectal cancer origin, 42 with liver cirrhosis and 178 healthy individuals. Serum levels of PIVKA-II were measured using the chemiluminescent assay of the Architect 1000i System (Abbott, USA) and AFP levels using the chemiluminescent assay by DxI 800 (Beckman Coulter, USA). Results: PIVKA-II achieved better clinical sensitivity than AFP and the difference in this sensitivity was statistically significant. PIVKA-II achieved the best sensitivity (96.9%) in distinguishing between the HCC and control groups with the proposed cut-off value of 60 mAU/ml. Conclusion: Our recommendation is for addition of PIVKA-II to the routine panel of HCC tumour markers.

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.

Follicular fluid levels of anti-Müllerian hormone, insulin-like growth factor 1 and leptin in women with fertility disorders

ABSTRACT Anti-Müllerian hormone (AMH), insulin-like growth factor 1 (IGF1) and leptin are produced in the granulosa cells of follicles and play an important role in the growth and maturation of follicles. The aim of our study was to monitor AMH, IGF1 and leptin levels in a group of healthy women and compare them to a group of women with fertility disorders. The second aim was the evaluation of biomarker levels in relation to the identified cause of infertility. Totally, 146 females were enrolled into our study. Seventy-two healthy controls and seventy-four females with fertility disorders were divided into four subgroups: anovulation, endometriosis, fallopian tube damage, unknown reason. IGF1 was the only biomarker with significantly lower levels throughout the entire group with fertility disorders. We did not identify any statistically significant differences for AMH and leptin. Regarding subgroups, significant differences were only observed in the group of anovulatory women. AMH and leptin showed higher levels while IGF1 showed lower levels. In conclusion, levels of AMH, IGF1 and leptin found in follicular fluid are sensitive markers for anovulatory fertility disorders. AMH, IGF1 and leptin levels in follicular fluid have no relation to the fertility disorders caused by endometriosis, fallopian tube damage or disorders with unknown etiology. Abbreviations: AMH: anti-Müllerian hormone; IGF1: insulin-like growth factor 1; PCOS: polycystic ovary syndrome

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.