Tamoxifen's pharmacokinetics are strongly influenced by the highly polymorphic CYP2D6, while the influence of other genetic variants has been inconclusive. To further delineate this genotypic-phenotypic impact, we conducted a multi-ancestry genome-wide association study in 636 hormone-receptor-positive (HR+) breast cancer (BC) patients treated with 20 mg tamoxifen daily for ≥8 weeks and validated these genetic determinants in another 869 patients. Association with clinical outcomes was examined in 1326 non-metastatic HR+ patients receiving adjuvant tamoxifen. A genome-wide significant association with Z-endoxifen levels was observed at the CYP2D6 locus on chromosome 22 and its downstream region of TCF20 rs932376 A > G. Both CYP2D6 metabolizer status and TCF20 rs932376 A > G were independent predictors of endoxifen levels in multivariable analysis. CYP2D6 metabolizer status accounted for greater variability of mean endoxifen levels compared to TCF20 rs932376 A > G (91.2% vs 48.8%). These findings were replicated in validation cohorts. Neither TCF20 rs932376 nor CYP2D6 metabolizer status was significantly associated with BC outcomes after adjustment for known prognostic factors. Our study confirmed that CYP2D6 metabolizer status remains as the prime predictor of steady-state Z-endoxifen levels, while TCF20 rs932376 A > G has a smaller, independent effect. Both genetic factors were not associated with BC clinical outcomes.

Targeting ACKR3/CXCR7 regulates enzymatic generation of pro-thrombotic, while favoring anti-thrombotic lipids that inhibit platelets through AC-cAMP-PKA pathway in coordination with prostacyclin-IP receptor. This investigation validated the impact of CXCR7 in modulating non-enzymatic lipid (per)oxidation, platelet response to lipoproteins-(LDL, oxLDL), mitochondrial metabolism and procoagulatory functions. Pharmacological CXCR7-agonist-(VUF11207) preserved mitochondrial membrane integrity-(Δψm), counteracted activation-induced mitochondrial superoxide generation-(MitoSOXRed), and nonenzymatic lipid (per)oxidation. Additionally, CXCR7-agonist regulated lipoprotein-induced platelet adhesion on thrombogenic matrices, degranulation, αIIbβIII-integrin activation, aggregation and thrombotic response, by reducing lipoprotein uptake through scavenger receptors-(CD36, ApoER2). CXCR7-ligation triggered activation of metabolic energy sensor Adenosine MonoPhosphate-dependent Kinase-(AMPKSer-172), prompted AMPK-mediated inhibitory phosphorylation of Acetyl-CoA-Carboxylase-(ACC)Ser-79, to foster lipolysis over lipogenesis. Consequently AMPKSer-172-ACCSer-79 pathway increased anticoagulatory FXa-inhibitory long-chain acylcarnitine-(LC-CARs)-(16:0, 18:1, 18:2) generation in platelets from healthy subjects and CAD patients. Increased intraplatelet LC-CARs was not due to dysregulated mitochondrial respiration; since CXCR7-agonist improved maximal respiration, spare respiratory capacity, and ATP-linked respiration in thrombin-activated platelets, suggesting sustained mitochondrial metabolism. Exerting a two-pronged effect on procoagulant function, CXCR7-agonist downregulated phosphatidylserine exposure on activated platelets, reducing FX/FXa binding, while platelet-derived anticoagulatory-LC-CARs regulated thrombin generation. CXCR7-agonist administration reduced thrombus formation, platelet degranulation, αIIbβIII-integrin activation, procoagulant activity, circulatory platelet-leukocyte aggregates in murine venous thrombosis model; besides, decreased plasma procoagulant lipids-(platelet COX-1, 12-LOX, and leukocyte 5/15-LOX-derived) and thrombo-inflammatory mediators-(IL-1β, IL-6, IFN-γ, TNF-α, MCP-1), and increased plasma LC-CAR levels. Therefore, pharmacological targeting of CXCR7 could regulate (non)enzymatic lipid processing, and promote anticoagulatory LC-CAR generation to check platelet-directed thrombotic propensity, and hypercoagulation, moreover, replenish reduced levels of circulatory anticoagulant-LC-CARs in STEMI and venous thromboembolism-(VTE) patients.

Polychlorinated biphenyls (PCBs) are widespread environmental contaminants that interfere with xenobiotic metabolism, primarily by modulating cytochrome P450 (CYP) enzymes. However, their pharmacokinetic consequences in exposed individuals remain poorly defined. Here, we investigated the impact of PCB exposure on CYP enzyme activity using a combined clinical pharmacokinetic and in vitro mechanistic approach. Ten occupationally PCB-exposed individuals from the German HELPcB cohort and ten controls received a CYP phenotyping cocktail to assess enzyme function (Clinical Trial Registry: DRKS00028922). Plasma drug and metabolite concentrations were quantified to evaluate CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, and CYP3A activity, considering genetically defined metabolizer status. PCB118 exposure was significantly associated with reduced CYP1A2 activity (R2 = 0.155, t = − 2.115, p = 0.049, β = − 0.446), indicating a decrease in CYP1A2 activity with higher PCB118 levels. This was further supported by in vitro assays demonstrating dose-dependent inhibition. In addition, PCB74 exposure showed a trend toward increased CYP2C9 activity, suggesting a potential inductive effect. Mechanistic studies revealed that PCB118 acts as both an inhibitor and a substrate of CYP1A2, generating reactive arene oxide intermediates that may contribute to mechanism-based inhibition. These findings challenge the traditional view of PCBs as merely chronic toxicants, showing that they can acutely alter drug metabolism and potentially impact drug efficacy and safety. Given the widespread presence of PCBs and other persistent organic pollutants, these results highlight the need to integrate environmental toxicant exposure into pharmacokinetic models to optimize drug therapy and minimize adverse effects.

Background and purposeThiopurines are used in paediatric inflammatory bowel disease (IBD), but some patients do not respond. Because the gut microbiota influences drug efficacy and IBD-patient microbiota presents increased bacterial abundance, we investigated the impact of candidate Enterobacteriaceae on drug cytotoxicity, metabolism and efficacy.Experimental approachThiopurines were exposed in vitro to bacteria for 4 h at 37 degrees C and drug concentrations measured by UV spectrophotometry. Cytotoxic effects and drug metabolite concentrations on NALM6 and JURKAT cells were determined after treatment with thiopurines exposed or not to bacteria. Drugs were measured in Klebsiella pneumoniae lysates and bacterial conditioned media were used for metabolomic analyses. Shotgun metagenomic sequencing was performed on eight IBD-patient faecal stools.Key resultsIncubation of thiopurines with K. pneumoniae, but not Escherichia coli and Salmonella enterica, reduced thiopurine concentrations and cytotoxicity on NALM6 and JURKAT cells. Thiopurine metabolites were lower in cells treated with drugs previously exposed to K. pneumoniae. Internalisation of drugs was demonstrated by their detection in lysates after bacterial incubation. Untargeted metabolomics revealed biotransformation of thiopurines by K. pneumoniae, as reactions of deconjugation, reduction, glycosylation, acetylation or conjugation with propionic acid. Incubation with thiopurines led to changes in the secretion of endogenous bacterial metabolites. K. pneumoniae faecal abundance was associated with lower thiopurine metabolite concentrations in erythrocytes of paediatric IBD-patients.Conclusions and ImplicationsK. pneumoniae decreases the cytotoxicity of thiopurines through internalisation of MP and TG. We revealed potential bacterial drug biotransformation, as well as negative correlations between bacterial abundance and drug metabolites.

PURPOSE::Tamoxifen undergoes bioactivation to its active metabolite (Z)-endoxifen, which blocks estrogen-dependent breast tumor growth at high potency. We tested the feasibility and safety of supplementing standard tamoxifen therapy with low-dose (Z)-endoxifen in patients with breast cancer with compromised tamoxifen bioactivation. PATIENTS AND METHODS::We conducted a prospective, interventional, three group randomized trial including 235 patients with hormone receptor-positive breast cancer who received standard tamoxifen therapy (20 mg/day). Patients were stratified by CYP2D6 genotype (n = 78), defining poor, intermediate, and normal metabolizers, or by baseline (Z)-endoxifen plasma concentration (n = 78), defining ≤15, 15 to 25, and ≥25 nmol/L. Co-treatment with (Z)-endoxifen 3 and 1.5 mg/day or placebo was performed, respectively. A control group (n = 79) received placebo regardless of metabolizer phenotype. The primary endpoint was the number of patients with (Z)-endoxifen levels >32 nmol/L after 6 weeks of treatment. Adverse events were continuously monitored. RESULTS:A higher proportion of patients in both intervention groups achieved target concentrations >32 nmol/L compared with control (P < 0.0001). At 3 mg (Z)-endoxifen supplementation, 92.3% of CYP2D6 poor metabolizer patients and all patients with baseline (Z)-endoxifen ≤15 nmol/L achieved the target concentration. At 1.5 mg (Z)-endoxifen supplementation, 88% of CYP2D6 intermediate metabolizer patients and 95% of patients with 15 to 25 nmol/L baseline (Z)-endoxifen levels achieved the target concentration. Similar proportions of patients receiving (Z)-endoxifen (6/80, 7.5%) or placebo (8/155, 5.2%) experienced grade 3 adverse events. CONCLUSIONS:Adding low-dose (Z)-endoxifen to standard tamoxifen is safe and provides a new approach to personalized antiestrogen treatment for patients with low endoxifen plasma levels.

Glucuronidation is a crucial pathway for the metabolism and detoxification of drugs and endobiotics, and primarily occurs in the liver. UGT2B17 is one of the 22 glycosyltransferases (UGT) that catalyze this reaction. In a large proportion of the population, UGT2B17 is absent due to complete gene deletion. We hypothesized that a UGT2B17 human deficiency affects the composition and function of the liver proteome, potentially provoking compensatory responses, and altering interconnected pathways and regulatory networks. The objective was to elucidate the liver proteome of UGT2B17-deficient individuals. Liver specimens from UGT2B17-deficient and proficient individuals were compared by mass spectrometry-based proteomics using data-independent acquisition. In UGT2B17-deficient livers, 80% of altered proteins showed increased abundance with a notable enrichment in various metabolic and chemical defense pathways, cellular stress and immune-related responses. Enzymes involved in the homeostasis of steroids, nicotinamide, carbohydrate and energy metabolism, and sugar pathways were also more abundant. Some of these changes support compensatory mechanisms, but do not involve other UGTs. An increased abundance of non-metabolic proteins suggests an adaptation to endoplasmic reticulum stress, and activation of immune responses. Data implies a disrupted hepatocellular homeostasis in UGT2B17-deficient individuals and offers new perspectives on functions and phenotypes associated with a complete UGT2B17 deficiency.

Clear cell renal cell carcinoma (ccRCC) is characterized by a metabolic shift towards enhanced aerobic glycolysis and increased lactate production. The survival rate for metastatic RCC is still poor. We evaluated the lactate monocarboxylate transporter 4 (MCT4), encoded by SLC16A3, as drug target for metastatic disease. MCT4 protein expression in 209 distant ccRCC metastases, including 40 recurrent metastases, was generally as high as compared to primary tumor tissue and significantly increased compared to non-tumor tissue (P<1E-15). MCT4 expression was irrespective of affected organs and mutations in RCC driver genes. DNA methylation in the SLC16A3 promoter, assessed by MALDI TOF mass spectrometry and correlated with clinicopathological data, were not significantly different in metastases of all investigated organ sites, and between paired tumor and metastases samples. Visualization of expression in single-cell and spatial RNA sequencing datasets reveals main expression of SLC16A3 in cells derived from tumor, tumor-normal interface, metastatic and lymph node tissue. Alone or combined with inhibition of mitochondrial respiration by metformin and phenformin, the MCT4 inhibitor syrosingopine significantly inhibits lactate efflux, induces cell viability reduction in four different RCC cell lines and patient-derived 2D/3D models, and alterations in cellular metabolism and mitochondrial respiration. Six patient-derived RCC air-liquid interface models, mimicking the complex RCC architecture, corroborate these data. Beyond potential prediction of patient outcome using MCT4 expression and DNA methylation at specific CpG sites, drug targeting of MCT4 and inhibiting mitochondrial respiration synergistically is a novel treatment strategy for metastatic ccRCC.

The classical approach of using adjacent pieces of fresh-frozen tissue for various omics analysis from the same sample possesses a risk of biological mismatch between arising from intrinsic tissue heterogeneity. We propose an alternative approach of tissue cryogenic pulverization and lyophilization before distribution for omics studies for a more reliable analysis. Here, we compare individual omics layer readouts from fresh-frozen adjacent tissue pieces and homogenized powder in mouse brain, kidney, and liver. Genomics, transcriptomics, proteomics, and metabolomics analyses showed comparable RNA integrity, DNA methylation, and coverage of transcripts, proteins, and metabolites across both methods. Moreover, the homogenized-lyophilized powder usage led to reduced heterogeneity between biological replicates. We conclude that the cryogenically pulverized-lyophilized tissue approach not only maintains a critical molecular feature coverage and quality but also provides a homogenous basis for various omics analysis enhancing reproducibility, sample transport, storage and enabling multi omics base on one and the same tissue aliquot.

BACKGROUND:Membrane transport proteins are critical determinants of systemic and intracellular drug levels, thereby contributing substantially to drug response and/or adverse drug reactions. Therefore, the U.S. Food and Drug Administration and the European Medicines Agency, the regulatory authorities for drug approval in the U.S. and Europe, respectively, recommend pre-clinical testing of selected drug transporters during the drug development process to elucidate clinically relevant drug-drug interactions (DDIs). In the current proof-of-principle study, we describe the generation of fruit flies expressing human membrane drug transporters in their salivary glands to enable DDI studies in a time-saving manner and at low costs. METHODS:Using the Gal4/upstream activation sequence (UAS) expression system, we established fruit flies expressing human organic cation transporters (hOCTs) 1 and 2 and genetic variants thereof. Both transporters are key drug uptake transporters in humans and are recommended for pre-clinical DDI studies. After injecting fluorescent hOCT substrates, their accumulation in salivary gland cells was observed by confocal laser scanning microscopy. RESULTS:We demonstrate the feasibility of expressing hOCT1 and hOCT2 in the salivary glands of fruit fly embryos and subsequent alteration by clinically relevant genetic variants, corroborating results from mammalian cell experiments. Moreover, we show an OCT-dependent accumulation of the prototypic fluorescent OCT substrates ethidium (Et+) and 4-Di-1-ASP (4-(4-(dimethylamino)styryl)-N-methylpyridinium, ASP+) in the salivary gland cells and subsequent inhibition by clinically-used OCT drug inhibitors. CONCLUSIONS:Based on the handling procedure and the lack of need for Animal Protection Act approval, we propose that the humanized Drosophila melanogaster fruit fly model opens a new avenue for pre-clinical functional transporter studies.

Immunotherapy with immune checkpoint inhibitors (ICI) has substantially improved the treatment of advanced renal cell carcinoma (aRCC). Since treatment effects vary among patients, identifying biomarkers to predict response is crucial for optimizing clinical management. Expression of programmed death ligand 1 (PD-L1) is associated with better response to ICI treatment, but is not a valid biomarker. To advance patient stratification for aRCC, we analyzed molecular and clinical data from the JAVELIN Renal 101 (n=726) and IMmotion151 (n=823) trials, both of which compared a combination of PD-L1 inhibitors and anti-angiogenic treatments with sunitinib monotherapy. In JAVELIN Renal 101, we found correlations between genes and progression-free survival (PFS) in PD-L1-positive tumors treated with sunitinib. These associations were independent of variations in the proportion of PD-L1-positive immune cells present in the tumors. Clustering of PD-L1-positive tumors based on PFS-related genes revealed an IMmune CHeckpoint Inhibitor-responsive Phenotype (IMCHIP). In PD-L1-positive tumors with IMCHIP, combined therapy with ICI led to significantly longer PFS relative to sunitinib (13.3 months vs. 4.4 months median PFS), while in PD-L1-positive tumors without IMCHIP, PFS was comparable for both treatments (11.1 months). By de-correlating gene expression measurements from PD-L1 expression, the clustering could be extended to PD-L1-negative tumors resulting in two groups (aRCC1/2) that were independent of PD-L1 status. Stratification based on aRCC1/2 and PD-L1 status allowed treatment effects to be categorized into four groups. A significant difference in PFS between treatments arms was only observed in patients with tumors of type aRCC2/PD-L1-positive, which corresponded to the IMCHIP profile and covered 32.4% of JAVELIN Renal 101. These findings could be validated in the IMmotion151 trial, where IMCHIP represented 20% of the cohort. Compared to other biomarkers currently being investigated, the combination of aRCC1/2 with PD-L1 status exhibited the greatest potential as a predictive biomarker for patient selection, which was confirmed in both cohorts. The prognostic potential of the aRCC1/2 subdivision was evaluated in 476 clear cell RCC from TCGA. Here, aRCC2 was associated with significantly worse overall survival and cancer-specific survival in both univariate and multivariable analyses, accounting for age, sex, and clinicopathological parameters (TNM). In summary, categorization into aRCC1/2 proved to be a new risk stratification of aRCC which, in combination with PD-L1 status, enabled the retrospective identification of responders who benefited from combined therapy with ICI relative to sunitinib monotherapy. The analysis of IMmotion151 trial data utilized data generated by Genentech/Genentech Research and Early Development. Florian A. Büttner, Viktor Grünwald, Elfriede Noessner, Igor Tsaur, Jens Bedke, Matthias Schwab, Elke Schaeffeler. A PD-L1-independent phenotype uncovers responders to combined therapy with immune checkpoint inhibitors in kidney cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7155.

AbstractPurpose :Tamoxifen undergoes Patients and Methods :We conducted a prospective, interventional, three group randomized trial including 235 patients with hormone receptor–positive breast cancer who received standard tamoxifen therapy (20 mg/day). Patients were stratified by CYP2D6 genotype (n = 78), defining poor, intermediate, and normal metabolizers, or by baseline (Z)-endoxifen plasma concentration (n = 78), defining ≤n = 79) received placebo regardless of metabolizer phenotype. The primary endpoint was the number of patients with (Z)-endoxifen levels >32 nmol/L after 6 weeks of treatment. Adverse events were continuously monitored. Results: A higher proportion of patients in both intervention groups achieved target concentrations >32 nmol/L compared with control (P < 0.0001). At 3 mg (Z)-endoxifen supplementation, 92.3% of CYP2D6 poor metabolizer patients and all patients with baseline (Z)-endoxifen ≤15 nmol/L achieved the target concentration. At 1.5 mg (Z)-endoxifen supplementation, 88% of CYP2D6 intermediate metabolizer patients and 95% of patients with 15 to 25 nmol/L baseline (Z)-endoxifen levels achieved the target concentration. Similar proportions of patients receiving (Z)-endoxifen (6/80, 7.5%) or placebo (8/155, 5.2%) experienced grade 3 adverse events. Conclusions: Adding low-dose (Z)-endoxifen to standard tamoxifen is safe and provides a new approach to personalized antiestrogen treatment for patients with low endoxifen plasma levels.

Suppl Fig. S1: Concentration time course of tamoxifen, N-desmentyl tamoxifen, (Z)-4-hydroxy tamoxifen, and (Z)-endoxifen

A recent phase Ⅰ trial published in Nature by Braun et al.1 showed data on personalized cancer vaccination(PCV)in patients with high-risk,resected clear cell renal cell carcinoma(ccRCC)targeting neoantigens including to key RCC driver somatic mutations and resulting in antitumour immunity.Notably,recurrence was not observed in any of the nine patients at median follow-up of 40.2 months,as well as dose-limiting toxicities,which renders neoantigen-targeting PCV as promising RCC therapy in the adjuvant setting.

Inter- and intra-assay variability

Background:Pharmacogenetics (PGx) aims to revolutionize healthcare by individualizing drug doses and medication choices. However, clinical uptake will require positive evaluation evidence of both clinical utility and cost-effectiveness. We have recently demonstrated the clinical utility of this approach, using a panel-based PGx-guided treatment of patients from various indications recruited in seven countries (PREPARE study). Methods:Here, we provide economic evidence from a multinational cost-utility analysis of PGx-guided treatment in 6930 patients participating in the PREPARE study. The study was conducted from March 2017 to June 2020. We used the national healthcare system's perspective in each participating country, including only direct medical costs that budget holders cover. A Visual Analog Scale was used to measure utility and the quality of life was estimated by averaging the Visual Analog Scale scores of participants over four specific time points in the study, namely baseline visit (day 1), week 4, week 12, and 18 months from the baseline visit. Findings:Our analysis showed that the PGx-guided treatment is marginally cost-effective at the threshold of €11,000 QALYs. Cost drivers were hospitalization and ADRs costs, accounting for most of the resources used in both groups (46% and 37.5% in the PGx-guided group versus 49% and 48% in the control group, respectively), as a result of the average duration of hospitalization [1.51 days (95% CI: 1.23-1.82) for the PGx-guided group and 2.37 days (95% CI: 1.95-2.89) for the control group, resulting in a mean difference of 0.86 days (95% CI: 0.37-1.44). The difference in QALYs gained was 0.00178 (95% CI: 0.00176-0.00180). The ICER was €12,020 (95% CI: €10,957-€13,356) per QALY on average (SD: €116). When comparing cost and effectiveness of actionable PGx-guided versus actionable control patients, the total cost for the PGx-guided group was €491 (95% CI: €384-€613), versus €767 (95% CI: €583-€982) in the control group, with an incremental cost difference of €276 (95% CI: €62-€511), favoring the PGx-guided group. Also, the difference in effectiveness was 0.007 QALYs (95% CI: -0.021 to 0.033). Lastly, the difference in the mean total cost was estimated to be €21.4 (95% CI: €19.5-€23.8), while without considering the PGx test cost, indicative of a pre-emptive genetic testing approach, the PGx-guided treatment becomes a cost-saving option, with an estimated savings of approximately €103.6 (€124-€21.4) per patient. Interpretation:These data suggest that panel-based PGx testing is cost-effective, which, together with the clinically beneficial outcomes already demonstrated in the PREPARE study, provides additional evidence of the need to implement PGx into clinical practice. Funding:European Union Horizon 2020.

Representativeness of study participants

The Ubiquitous Pharmacogenomics consortium (www.upgx.eu) has recently completed and published the Preemptive Pharmacogenomic Testing for Preventing Adverse Drug Reactions (PREPARE) study on the implementation of panel-based pharmacogenetic testing. PREPARE has provided interesting lessons for the design, execution, and interpretation of future clinical implementation studies. In this paper, we share our experience and lessons learned from the PREPARE study for future pharmacogenetic implementation studies. Issues addressed are the study population, intervention, endpoint, randomization, blinding, crossover, ethics, real-world changes during the study, and data analysis and reporting.