Optimization of apolipoprotein(a) genotyping with pulsed field gel electrophoresis

BACKGROUND: Increased lipoprotein(a) is a risk factor for atherosclerosis, and its concentration in serum is inversely correlated with the size of the apoliprotein(a) [apo(a)] component. The size of the apo(a) gene is determined mainly by the Kringle IV size polymorphism. We have optimized and characterized pulsed field gel electrophoresis (PFGE) for apo(a) genotyping. METHODS: Established PFGE protocols were adjusted. The changes included the following: (a) increased DNA yields by the use of all leukocytes for isolation from either 3 mL of fresh EDTA whole blood or 250 microL of frozen buffy coats; (b) increased efficiency of Kpn1 digestion by the inclusion of a digestion buffer wash; (c) reduction of assay time by the use of capillary blotting; (d) increased sensitivity by the use of four digoxigenin-labeled apo(a) probes; and (e) identification using a single film by the inclusion of a digoxigenin-labeled lambda marker probe in addition to apo(a) probes in the hybridization mix. RESULTS: In older Caucasians, 93% (buffy coats, n=468) were heterozygous for apo(a) gene size. An inverse correlation between serum lipoprotein(a) and the sum of Kringle IV alleles was found (y = -23x + 1553; r = -0.442; n = 468). Gel-to-gel variation was minimal (3%). Imprecision (SD) was one Kringle IV repeat (control sample containing eight fragments of 72-233 kb; n=34 electrophoretic runs). CONCLUSIONS: The practicality and sensitivity of the apo(a) genotyping technique by PFGE were improved, and accuracy and reproducibility were preserved. The optimized procedure is promising for apo(a) genotyping on frozen buffy coats from large epidemiological studies

Nonlinear Mixed-Effects Model of Z-Endoxifen Concentrations in Tamoxifen-Treated Patients from the CEPAM Cohort

Tamoxifen is widely used in patients with hormone receptor-positive breast cancer. The polymorphic enzyme CYP2D6 is primarily responsible for metabolic activation of tamoxifen, resulting in substantial interindividual variability of plasma concentrations of its most important metabolite, Z-endoxifen. The Z-endoxifen concentration thresholds below which tamoxifen treatment is less efficacious have been proposed but not validated, and prospective trials of individualized tamoxifen treatment to achieve Z-endoxifen concentration thresholds are considered infeasible. Therefore, we aim to validate the association between Z-endoxifen concentration and tamoxifen treatment outcomes, and identify a Z-endoxifen concentration threshold of tamoxifen efficacy, using pharmacometric modeling and simulation. As a first step, the CYP2D6 Endoxifen Percentage Activity Model (CEPAM) cohort was created by pooling data from 28 clinical studies (> 7,000 patients) with measured endoxifen plasma concentrations. After cleaning, data from 6,083 patients were used to develop a nonlinear mixed-effect (NLME) model for tamoxifen and Z-endoxifen pharmacokinetics that includes a conversion factor to allow inclusion of studies that measured total endoxifen but not Z-endoxifen. The final parent-metabolite NLME model confirmed the primary role of CYP2D6, and contributions from body weight, CYP2C9 phenotype, and co-medication with CYP2D6 inhibitors, on Z-endoxifen pharmacokinetics. Future work will use the model to simulate Z-endoxifen concentrations in patients receiving single agent tamoxifen treatment within large prospective clinical trials with long-term survival to identify the Z-endoxifen concentration threshold below which tamoxifen is less efficacious. Identification of this concentration threshold would allow personalized tamoxifen treatment to improve outcomes in patients with hormone receptor-positive breast cancer

Human activin-A is expressed in the atherosclerotic lesion and promotes the contractile phenotype of smooth muscle cells

Activin is a member of the transforming growth factor-beta superfamily, and it modulates the proliferation and differentiation of various target cells. In this study, we investigated the role of activin in the initiation and progression of human atherosclerosis. The expression of activin, its physiological inhibitor follistatin, and activin receptors were assayed in human vascular tissue specimens that repr

Modification of the association between paroxetine serum concentration and SERT-occupancy by ABCB1 (P-glycoprotein) polymorphisms in major depressive disorder

BACKGROUND: Selective serotonin reuptake inhibitors (SSRIs) exert substantial variability in effectiveness in patients with major depressive disorder (MDD), with up to 50-60% not achieving adequate response. Elucidating pharmacokinetic factors that explain this variability is important to increase treatment effectiveness. OBJECTIVES: To examine potential modification of the relationship between paroxetine serum concentration (PSC) and serotonin transporter (SERT)-occupancy by single nucleotide polymorphisms (SNPs) of the ABCB1 gene, coding for the P-glycoprotein (P-gp) pump, in MDD patients. To investigate the relationship between ABCB1 SNPs and clinical response. METHODS: Patients had MDD and received paroxetine 20 mg/day. We measured PSC after 6 weeks. We quantified SERT-occupancy with SPECT imaging (n = 38) and measured 17-item Hamilton Depression Rating Scale (HDRS17)-scores at baseline and after 6 wee

Clinical Utility of Circulating Tumor DNA in Patients With Advanced KRAS^G12C-Mutated NSCLC Treated With Sotorasib

Introduction: For patients with KRASG12C-mutated NSCLC who are treated with sotorasib, there is a lack of biomarkers to guide treatment decisions. We therefore investigated the clinical utility of pretreatment and on-treatment circulating tumor DNA (ctDNA) and treatment-emergent alterations on disease progression. Methods: Patients with KRASG12C-mutated NSCLC treated with sotorasib were prospectively enrolled in our biomarker study (NCT05221372). Plasma samples were collected before sotorasib treatment, at first-response evaluation and at disease progression. The TruSight Oncology 500 panel was used for ctDNA and variant allele frequency analysis. Tumor response and progression-free survival were assessed per Response Evaluation Criteria in Solid Tumors version 1.1. Results: Pretreatment KRASG12C ctDNA was detected in 50 of 66 patients (76%). Patients with detectable KRASG12C had inferior progression-free survival (hazard ratio [HR] 2.13 [95% confidence interval [CI]: 1.06–4.30], p = 0.031) and overall survival (HR 2.61 [95% CI: 1.16–5.91], p = 0.017). At first-response evaluation (n = 40), 29 patients (73%) had a molecular response. Molecular nonresponders had inferior overall survival (HR 3.58 [95% CI: 1.65–7.74], p = 0.00059). The disease control rate was significantly higher in those with a molecular response (97% versus 64%, p = 0.015). KRAS amplifications were identified as recurrent treatment-emergent alterations. Conclusions: Our data suggest detectable pretreatment KRASG12C ctDNA as a marker for poor prognosis and on-treatment ctDNA clearance as a marker for treatment response. We identified KRAS amplifications as a potential recurring resistance mechanism to sotorasib. Identifying patients with superior prognosis could aid in optimizing time of treatment initiation, and identifying patients at risk of early progression could allow for earlier treatment decisions.</p

Critical appraisal of the views of healthcare professionals with respect to pharmacogenomics and personalized medicine in Greece

Aim: In the postgenomic era, in many European countries, very little is known regarding the level of awareness of healthcare professionals with respect to pharmacogenomics and personalized medicine. Methods: Here, we report the findings of an in-depth study, involving 86 pharmacists and 208 physicians, to assess their level of awareness of pharmacogenomics and personalized medicine. Results: Our findings indicate that approximately 60% of pharmacists consider their level of knowledge of personalized medicine to be very low, while over half of the pharmacists and physicians intimate that they would be unable to explain the results of pharmacogenomic tests to their customers or patients, respectively. This situation may be directly related to the low level of their undergraduate education in genetics and pharmacogenomics. Conclusion: These findings provide the basis for assessing the views of healthcare professionals in relation to personalized medicine in Greece, and should help to facilitate the integration of genomics into the medical decision-making process

High-sensitivity Troponin T in relation to coronary plaque characteristics in patients with stable coronary artery disease; results of the ATHEROREMO-IVUS study

Background and aims: To assess the relationship between the extent and phenotype of coronary atherosclerosis, as assessed by in-vivo grayscale and radiofrequency intravascular ultrasound (IVUS), and circulating Troponin levels in patients with established stable coronary artery disease (CAD). Methods: In this single-center, cross-sectional analysis, high-sensitivity Troponin T (hsTnT) was measured and IVUS was performed in a predefined non-stenotic segment of a non-culprit coronary artery in 231 patients with stable CAD undergoing elective angiography. Results: HsTnT was detectable (>3 pg/mL) in 212 patients (92%) and a concentration above 14 pg/mL was observed in 19.5%. Normalised segmental plaque volumes were positively associated with hsTnT levels (25.0 mm3 increase in segmental plaque volume per SD increase in ln-transformed hsTnT, 95% CI: 6.0-44.0, p = 0.010). Higher hsTnT levels were measured in patients with a virtual histology derived thin-cap fibroatheroma (VH-TCFA, adj. odds ratio for presence of VH-TCFA = 1.52 per SD increase in ln-transformed hsTnT, 95% CI: 1.10-2.11, p = 0.011). Patients with a VH-TCFA had a 2-fold increased prevalence of hsTnT concentration ≥14 pg/mL (adj. OR 2.35, 95% CI: 1

Donor-Derived Cell-Free DNA for the Detection of Heart Allograft Injury:The Impact of the Timing of the Liquid Biopsy

Background: In heart transplant recipients, donor-derived cell-free DNA (ddcfDNA) is a potential biomarker for acute rejection (AR), in that increased values may indicate rejection. For the assessment of ddcfDNA as new biomarker for rejection, blood plasma sampling around the endomyocardial biopsy (EMB) seems a practical approach. To evaluate the effect of the EMB procedure on ddcfDNA values, ddcfDNA values before the EMB were pairwise compared to ddcfDNA values after the EMB. We aimed at evaluating whether it matters whether the ddcfDNA sampling is done before or after the EMB-procedure. Methods: Plasma samples from heart transplant recipients were obtained pre-EMB and post-EMB. A droplet digital PCR method was used for measuring ddcfDNA, making use of single-nucleotide polymorphisms that allowed both relative quantification, as well as absolute quantification of ddcfDNA. Results: Pairwise comparison of ddcfDNA values pre-EMB with post-EMB samples (n = 113) showed significantly increased ddcfDNA concentrations and ddcfDNA% in post-EMB samples: an average 1.28-fold increase in ddcfDNA concentrations and a 1.31-fold increase in ddcfDNA% was observed (p = 0.007 and p = 0.03, respectively). Conclusion: The EMB procedure causes iatrogenic injury to the allograft that results in an increase in ddcfDNA% and ddcfDNA concentrations. For the assessment of ddcfDNA as marker for AR, collection of plasma samples before the EMB procedure is therefore essential

The impact of maternal vulnerability on stress biomarkers and first-trimester growth:the Rotterdam Periconceptional Cohort (Predict Study)

STUDY QUESTION:Is the degree of maternal vulnerability positively associated with stress biomarkers (stress hormones, C-reactive protein, tryptophan metabolites, and one-carbon metabolites), and does long-term exposure to stress hormones reduce first-trimester growth?SUMMARY ANSWER:The maternal vulnerability risk score is positively associated with concentrations of hair cortisol and cortisone and negatively with tryptophan, while higher hair cortisol concentrations are associated with reduced first-trimester growth without mediation of tryptophan.WHAT IS KNOWN ALREADY: A high degree of maternal vulnerability during the periconception period is associated with impaired first-trimester growth and pregnancy complications, with consequences for long-term health of the child and future life course. However, due to the challenges of early identification of vulnerable women, the uptake of periconception care is low in this target group. STUDY DESIGN, SIZE, DURATION:Between June 2022 and June 2023, this study was conducted in a sub-cohort of 160 pregnant women participating in the Rotterdam Periconceptional Cohort (Predict Study), an ongoing prospective tertiary hospital-based cohort.PARTICIPANTS/MATERIALS, SETTING, METHODS:One hundred and thirty-two women with ongoing pregnancies and available stress biomarker data were included in the analysis. Data on periconceptional social, lifestyle, and medical risk factors were collected via self-administered questionnaires, and these factors were used for the development of a composite maternal vulnerability risk score. Stress biomarkers, including stress hormones (hair cortisol and cortisone) and inflammatory and oxidative stress biomarkers (C-reactive protein, total homocysteine, and tryptophan metabolites) were determined in the first trimester of pregnancy. First-trimester growth was assessed by crown-rump length (CRL) and embryonic volume (EV) measurements at 7, 9, and 11 weeks gestation by making use of an artificial intelligence algorithm and virtual reality techniques using 3D ultrasound data sets. The associations between the maternal vulnerability risk score and stress biomarkers were identified using linear regression models, and between stress hormones and CRL- and EV-trajectories using mixed models. A mediation analysis was performed to assess the contribution of tryptophan. All associations were adjusted for potential confounders, which were identified using a data-driven approach. Several sensitivity analyses were performed to check the robustness of the findings.MAIN RESULTS AND THE ROLE OF CHANCE:The maternal vulnerability risk score was positively associated with concentrations of hair cortisol and cortisone (pg/mg) (beta = 0.366, 95% CI = 0.010-0.722; beta = 0.897, 95% CI = 0.102-1.691, respectively), and negatively with tryptophan concentrations (mu mol/L) (beta = -1.637, 95% CI = -2.693 to -0.582). No associations revealed for C-reactive protein and total homocysteine. Higher hair cortisol concentrations were associated with reduced EV-trajectories (3 root EV: beta = -0.010, 95% CI = -0.017 to -0.002), while no associations were found with CRL-trajectories. Mediation by tryptophan was not shown.LIMITATIONS, REASONS FOR CAUTION:Residual confounding cannot be ruled out, and the external validity may be limited due to the study's single-center observational design in a tertiary hospital.WIDER IMPLICATIONS OF THE FINDINGS:There is mounting evidence that a high degree of maternal vulnerability negatively affects maternal and perinatal health, and that of the future life course. The results of our study emphasize the need to identify highly vulnerable women as early as possible, at least before conception. Our findings suggest that the chronic stress response and alterations of the maternal tryptophan metabolism are involved in maternal vulnerability, affecting first-trimester growth, with potential impact on the long-term health of the offspring.STUDY FUNDING/COMPETING INTEREST(S): This study was funded by the Departments of Obstetrics and Gynecology and Clinical Chemistry of the Erasmus MC, University Medical Center, Rotterdam, the Netherlands, and the Junior Award granted by the De Snoo-van 't Hoogerhuijs Foundation in March 2022. There are no conflicts of interest