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BackgroundCalcineurin inhibitors, including tacrolimus, remain a cornerstone of immunosuppressive therapy after kidney transplantation. However, the therapeutic window is narrow, and nephrotoxic side effects occur with overdose, while the risk of alloimmunization and graft rejection increases with underdose. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) allows quantification of tacrolimus in biological samples from patients. This study investigates the feasibility of quantifying tacrolimus in scalp hair from kidney transplant (KT) recipients and correlates hair tacrolimus concentrations with tacrolimus dosage and blood trough levels. The aim was to provide proof-of-principle for hair tacrolimus drug monitoring in KT recipients.MethodSingle-center prospective study between September 9, 2021 and December 4, 2021, including KT recipients under tacrolimus. Minors, patients with active skin or hair diseases, and patients with scalp hair shorter than 4 cm were excluded from participation. Scalp hair was collected from the posterior vertex of patients, cut into segments, and analyzed for tacrolimus by LC-MS/MS. Patients filled out a questionnaire on hair treatments and washing habits. In parallel, tacrolimus trough levels were measured in whole blood and correlated with hair tacrolimus concentrations.ResultsIn total, 39 consenting KT recipients were included, and hair samples were collected at 53 visits. Tacrolimus was detected in 98% of hair samples from patients exposed to the drug. Tacrolimus hair levels and whole blood trough levels were correlated with a beta coefficient of 0.42 (95% CI: −0.22–1.1, p = n.s.). Age and dark hair affected hair tacrolimus measurements, while different tacrolimus formulations (immediate release vs. extended release), hair washes, and permanent coloring did not. Longitudinal measurements in a subgroup of patients indicate that long-term measurement of hair tacrolimus levels is feasible.ConclusionMeasuring tacrolimus in hair is a potentially reliable method to monitor drug exposure in KT patients. Rapid wash-in effects and consistent concentrations over time indicate that tacrolimus is incorporated into the hair matrix, allowing temporal resolution in the analysis of recent exposure and exposure history. This method provides a simple and low-risk alternative to regular blood sampling, sparing patients from frequent hospital visits through the self-collection of hair samples.

The diagnosis of prostate cancer brain metastasis (PCBM) has increased. Here, the authors investigate the landscape of somatic genetic alterations in brain metastases in a PCBM cohort of 51 patients with non-synchronous matched primary samples available for 20 patients.

In allograft monitoring of solid organ transplant recipients, liquid biopsy has emerged as a novel approach using quantification of donor-derived cell-free DNA (dd-cfDNA) in plasma. Despite early clinical implementation and analytical validation of techniques, direct comparisons of dd-cfDNA quantification methods are lacking. Furthermore, data on dd-cfDNA in urine is scarce and high-throughput sequencing-based methods so far have not leveraged unique molecular identifiers (UMIs) for absolute dd-cfDNA quantification. Different dd-cfDNA quantification approaches were compared in urine and plasma of kidney and liver recipients: A) Droplet digital PCR (ddPCR) using allele-specific detection of seven common HLA-DRB1 alleles and the Y chromosome; B) high-throughput sequencing (HTS) using a custom QIAseq DNA panel targeting 121 common polymorphisms; and C) a commercial dd-cfDNA quantification method (AlloSeq® cfDNA, CareDx). Dd-cfDNA was quantified as %dd-cfDNA, and for ddPCR and HTS using UMIs additionally as donor copies. In addition, relative and absolute dd-cfDNA levels in urine and plasma were compared in clinically stable recipients. The HTS method presented here showed a strong correlation of the %dd-cfDNA with ddPCR (R2 = 0.98) and AlloSeq® cfDNA (R2 = 0.99) displaying only minimal to no proportional bias. Absolute dd-cfDNA copies also correlated strongly (τ = 0.78) between HTS with UMI and ddPCR albeit with substantial proportional bias (slope: 0.25; 95%-CI: 0.19–0.26). Among 30 stable kidney transplant recipients, the median %dd-cfDNA in urine was 39.5% (interquartile range, IQR: 21.8–58.5%) with 36.6 copies/μmol urinary creatinine (IQR: 18.4–109) and 0.19% (IQR: 0.01–0.43%) with 5.0 copies/ml (IQR: 1.8–12.9) in plasma without any correlation between body fluids. The median %dd-cfDNA in plasma from eight stable liver recipients was 2.2% (IQR: 0.72–4.1%) with 120 copies/ml (IQR: 85.0–138) while the median dd-cfDNA copies/ml was below 0.1 in urine. This first head-to-head comparison of methods for absolute and relative quantification of dd-cfDNA in urine and plasma supports a method-independent %dd-cfDNA cutoff and indicates the suitability of the presented HTS method for absolute dd-cfDNA quantification using UMIs. To evaluate the utility of dd-cfDNA in urine for allograft surveillance, absolute levels instead of relative amounts will most likely be required given the extensive variability of %dd-cfDNA in stable kidney recipients.

Methylation sequencing is a promising approach to infer the tissue of origin of cell-free DNA (cfDNA). In this study, a single- and a double-stranded library preparation approach were evaluated with respect to their technical biases when applied on cfDNA from plasma and urine. Additionally, tissue of origin (TOO) proportions were evaluated using two deconvolution methods. Sequencing cfDNA from urine using the double-stranded method resulted in a substantial within-read methylation bias and a lower global methylation (56.0% vs. 75.8%, p ≤ 0.0001) compared to plasma cfDNA, both of which were not observed with the single-stranded approach. Individual CpG site-based TOO deconvolution resulted in a significantly increased proportion of undetermined TOO with the double-stranded method (urine: 32.3% vs. 1.9%; plasma: 5.9% vs. 0.04%; p ≤ 0.0001), but no major differences in proportions of individual cell types. In contrast, fragment-level deconvolution led to multiple cell types, with significantly different TOO proportions between the two methods. This study thus outlines potential limitations of double-stranded library preparation for methylation analysis of cfDNA especially for urinary cfDNA. While the double-stranded method allows jagged end analysis in addition to TOO analysis, it leads to significant methylation bias in urinary cfDNA, which single-stranded methods can overcome.

The implementation of pharmacogenetic testing into clinical practice has been a slow process so far. Here, we review the implementation of pre-treatment testing of dihydropyrimidine dehydrogenase gene (DPYD) risk variants to prevent early-onset fluoropyrimidine (FP)-related toxicity in cancer patients in Switzerland based on data of a large Swiss diagnostic center. In January 2017, the Swiss Federal Office of Public Health introduced the reimbursement of DPYD testing by the compulsory health insurance in Switzerland based on evidence for the clinical relevance of DPYD-risk variants and the cost-effectiveness of pre-treatment testing, and on the availability of international guidelines. However, we did not observe a strong increase in DPYD testing at our diagnostic center from 2017 to 2019. Only a low number of DPYD-testing requests (28–42 per year), concerning mostly retrospective investigations of suspected FP-toxicity, were received. In contrast, we observed a 14-fold increase in DPYD testing together with a strong shift from retrospective to pre-treatment test requests upon the release of recommendations for DPYD testing prior to FP-treatment in April 2020 by the European Medicines Agency. This increase was mainly driven by three geographic regions of Switzerland, where partner institutions of previous research collaborations regarding FP-related toxicity are located and who acted as early-adopting institutions of DPYD testing. Our data suggest the important role of early adopters as accelerators of clinical implementation of pharmacogenetic testing by introducing these policies to their working environment and educating health workers from their own and nearby institutions.

BackgroundApplying a single anti-Xa assay, calibrated to unfractionated heparin to measure rivaroxaban, apixaban, and edoxaban would simplify laboratory procedures and save healthcare costs.AimWe hypothesized that a heparin-calibrated anti-Xa assay would accurately measure rivaroxaban, apixaban, and edoxaban drug concentrations and correctly predict clinically relevant drug levels.MethodsThis analysis is part of the Simple-Xa study, a prospective multicenter cross-sectional study conducted in clinical practice. Patients treated with rivaroxaban, apixaban, or edoxaban were included. Anti-Xa activity was measured using the Siemens INNOVANCE® Heparin assay. Drug concentrations were determined using ultra-high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). Cut-off levels were determined in a derivation dataset (50% of patients) and sensitivities and specificities were calculated in a verification dataset (50% of patients).ResultsOverall, 845 patients were available for analysis. Correlation coefficients (rs) between the heparin-calibrated anti-Xa assay and drug concentrations were 0.97 (95% CI 0.97, 0.98) for rivaroxaban, 0.96 (0.96, 0.97) for apixaban, and 0.96 (0.94, 0.99) for edoxaban. The area under the receiver operating characteristics curve (ROC) was 0.99 for all clinically relevant drug concentrations. In the verification dataset, the sensitivity was 94.2% (95% CI 90.8–96.6) for 30 μg L–1, 95.8% (92.4–98.0) for 50 μg L–1, and 98.7% (95.5–99.9) for 100 μg L–1. Specificities were 86.3% (79.2–91.7), 89.8% (84.5–93.7), and 88.7% (84.2–92.2), respectively.ConclusionIn a large prospective study in clinical practice, a strong correlation of heparin-calibrated anti-Xa measurements with LC-MS/MS results was observed and clinically relevant drug concentrations were predicted correctly.

Fluoropyrimidines (FPs), mainly 5-fluorouracil (5-FU) and its oral prodrug capecitabine (Cap), remain the backbone of the treatment of many different solid tumors. Despite their broad use in clinical routine, 10–40% of patients experience severe, and in rare cases (0.2–0.5%) even lethal, FP-related toxicity in early chemotherapy cycles. Today, there is a plethora of evidence that genetic variants in the gene encoding for the 5-FU catabolising enzyme dihydropyrimidine dehydrogenase (DPD, encoded by DPYD) are predictive of severe FP-related toxicities, and international clinical practice recommendations for DPYD genotype-guided FP dosing and therapeutic drug monitoring (TDM) are available. In spite of this strong evidence and DPYD genotyping becoming standard practice in other countries, it is has not been widely adopted in Switzerland to date. Here, we discuss current guidelines on genotype-guided FP dosing and TDM, and propose recommendations tailored to the situation in Switzerland to facilitate their clinical uptake for the further individualisation of FP chemotherapy. We recommend preemptive testing of four DPYD variants (c.1905+1G>A (rs3918290), c.1679T>G (rs55886062), c.2846A>T (rs67376798) and c.1129-5923C>G (rs75017182, c.1236G>A/HapB3)) in patients with an indication for FP-based chemotherapy, with the costs reimbursed through the compulsory health insurance in Switzerland. Carriers of these variants (6.5% in the Swiss population) have a 40–50% risk of developing severe early-onset toxicity when treated with standard FP doses. In these patients, we therefore recommend the use of a reduced starting dose, based on a dose adjustment scheme provided herein. Furthermore, we recommend the use of infusional 5-FU in patients with a DPYD risk genotype in order to enable TDM-based dose escalation. Only if the use of an infusional 5-FU regimen is not feasible should a slow titration of Cap, starting with the recommended reduced dose and basing further doses on monitoring of toxicity, be considered. Given that several studies have shown that TDM in 5-FU treatment improves not only the therapy’s safety, but potentially also its efficacy, we also include detailed TDM-based dosing guidelines and discuss the pre-analytical aspects of 5-FU TDM.

Background and Objective: Agranulocytosis is a rare and potentially life-threatening complication of metamizole (dipyrone) intake that is characterized by a loss of circulating neutrophil granulocytes. While the mechanism underlying this adverse drug reaction is not well understood, involvement of the immune system has been suggested. In addition, associations between genetic variants in the Human Leukocyte Antigen (HLA) region and agranulocytosis induced by other drugs have been reported. The aim of the present study was to assess whether genetic variants in classical HLA genes are associated with the susceptibility to metamizole-induced agranulocytosis (MIA) in a European population by targeted resequencing of eight HLA genes.Design: A case-control cohort of Swiss patients with a history of neutropenia or agranulocytosis associated with metamizole exposure (n = 53), metamizole-tolerant (n = 39) and unexposed controls (n = 161) was recruited for this study. A high-throughput resequencing (HTS) and high-resolution typing method was used to sequence and analyze eight HLA loci in a discovery subset of this cohort (n = 31 cases, n = 38 controls). Identified candidate alleles were investigated in the full Swiss cohort as well as in two independent cohorts from Germany and Spain using HLA imputation from genome-wide SNP array data. In addition, variant calling based on HTS data was performed in the discovery subset for the class I genes HLA-A, -B, and -C using the HLA-specific mapper hla-mapper.Results: Eight candidate alleles (p < 0.05) were identified in the discovery subset, of which HLA-C∗04:01 was associated with MIA in the full Swiss cohort (p < 0.01) restricted to agranulocytosis (ANC < 0.5 × 109/L) cases. However, no candidate allele showed a consistent association in the Swiss, German and Spanish cohorts. Analysis of individual sequence variants in class I genes produced consistent results with HLA typing but did not reveal additional small nucleotide variants associated with MIA.Conclusion: Our results do not support an HLA-restricted T cell-mediated immune mechanism for MIA. However, we established an efficient high-resolution (three-field) eight-locus HTS HLA resequencing method to interrogate the HLA region and demonstrated the feasibility of its application to pharmacogenetic studies.