The Clinical Pharmacogenetics Implementation Consortium (CPIC) TPMT/NUDT15 Pharmacogene Curation Expert Panel (PCEP) conducted a comprehensive review of clinical, laboratory, and computational evidence to determine the clinical function assignments for TPMT and NUDT15 star alleles. These genes are critical for the metabolism of thiopurines, which are widely used in the treatment of cancer and autoimmune disorders. Standardized allele function assignment is essential for predicting metabolizer phenotypes and pharmacogenetics-guided thiopurine dosing. The work presented here includes the first designation of decreased function alleles for both TPMT and NUDT15, reflecting new clinical data that demonstrate partial loss of enzymatic activity and reduced dose tolerance. The panel also reclassified several alleles previously assigned uncertain or unknown function. The functional assignments were informed by a standardized framework incorporating clinical data, such as thiopurine tolerance and toxicity, as well as in vitro protein activity, ex vivo enzymatic measurements, and in silico variant effect prediction tools. These updates enhance the precision of genotype-to-phenotype mapping and support more personalized thiopurine therapy across diverse patient populations.

BACKGROUND AND PURPOSE:Metabolic dysfunction-associated steatohepatitis (MASH) is linked to activation of hepatic stellate cells (HSCs) to α-smooth muscle actin-positive myofibroblasts that produce collagen and proinflammatory cytokines. Quiescent HSCs express the NO-cGMP signalling axis. Modulating this pathway could alter HSC activation and fibrosis during MASH progression. EXPERIMENTAL APPROACH:Using transgenic cGMP sensor mice, we monitored NO-induced cGMP in living HSCs. The relevance of this pathway was analyzed using HSC-specific mouse models, ApoE-deficient mice on high-fat diet as a MASH model, human liver sections, and published scRNA-seq datasets. For pharmacological activation of NO-cGMP signalling, BAY-543, an activator of NO-sensitive guanylyl cyclase (NO-GC) was used. KEY RESULTS:HSCs in primary culture and liver tissue generated NO-induced cGMP and expressed NO-GC and cGMP-dependent protein kinase type I (cGKI). Compared to controls, HSC-specific cGKI knockout livers showed enhanced myofibroblast marker expression, indicating increased HSC activation and MASH susceptibility. MASH mice developed steatosis, fibrosis, and inflammation, and showed a high number of HSCs expressing NO-GC and cGKI. cGKI expression was also increased in human fibrotic livers as compared to healthy tissue. In MASH livers, oxidative stress could lead to reduced sensitivity of NO-GC to NO. Treatment of MASH mice with BAY-543, which targets oxidized/NO-insensitive NO-GC, significantly attenuated HSC activation, inflammation, collagen deposition, macro-steatosis, fibrosis, and serum liver enzymes. CONCLUSION AND IMPLICATIONS:The NO-cGMP-cGKI axis serves as both a functional pathway marker and regulator of HSCs. Pharmacological elevation of cGMP with an NO-GC activator represents a promising therapeutic strategy for MASH.

5-hydroxytryptamine type 3 (5-HT3) receptor antagonists are used to treat nausea and vomiting and in the prevention of chemotherapy-induced, radiation-induced, and postoperative nausea and vomiting. Most of the 5-HT3 receptor antagonists (i.e., ondansetron, tropisetron, dolasetron, palonosetron, and ramosetron) are metabolized by CYP2D6, but the extent of CYP2D6 involvement varies. CYP2D6 genetic variation can influence the metabolism of these medications, particularly ondansetron and tropisetron, thereby affecting drug efficacy. This guideline is an update to the 2016 Clinical Pharmacogenetics Implementation Consortium (CPIC) guideline for CYP2D6 genotype and use of ondansetron and tropisetron and includes updated information on CYP2D6 genetic testing and evidence tables. We summarize evidence from the published literature supporting these associations and provide therapeutic recommendations for 5-HT3 receptor antagonists based on CYP2D6 genotype, particularly where genetic variation is associated with reduced drug efficacy (updates at https://www.clinpgx.org/guideline/PA166251457).

This study investigated genetic determinants of the pharmacokinetics of the CYP2C8 index drugs repaglinide and gemfibrozil, and their interaction in healthy participants. Sequencing data from a study with montelukast revealed a novel functional CYP2C8 allele (rs2071426, CYP2C8*19), predicted to create an intronic splice donor site. In human liver samples, CYP2C8*19 associated with transcript-specific changes in CYP2C8 mRNA expression, reduced CYP2C8 protein expression, and decreased enzyme activity. Consistently, participants with the CYP2C8*19/*19 genotype had 45% greater area under the plasma repaglinide concentration-time curve from time zero to infinity (AUC0-∞) than participants with CYP2C8*1/*1 (P = 1.6 × 10-4). Participants with CYP2C8*1/*3 had 26% smaller AUC0-∞ (P = 0.0033) and those with CYP2C8*1/*4 had 51% greater AUC0-∞ (P = 8.2 × 10-4). The fold increase in repaglinide AUC0-∞ caused by gemfibrozil was 36% (P = 1.3 × 10-4) smaller in CYP2C8*19/*19 participants than in CYP2C8*1/*1 participants. In a genome-wide association study (GWAS), SLCO1B1 c.521 T>C (rs4149056) associated with increased repaglinide AUC0-∞ (P = 4.5 × 10-15; n = 172) and SLCO1A2 variants associated with decreased AUC0-∞ (P < 10-8). In a GWAS of repaglinide after gemfibrozil pretreatment, SLCO1C1 variants associated with decreased AUC0-∞ (P < 1.6 × 10-8; n = 66). Participants with the poor function SLCO1B1 genotype showed a 32% smaller fold increase in repaglinide AUC0-∞ following gemfibrozil than participants with the normal function SLCO1B1 genotype (P = 0.0045). This study characterizes CYP2C8*19 as a novel decreased function allele and shows that CYP2C8 and SLCO1B1 genotypes affect the gemfibrozil-repaglinide interaction.

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.

Brain volume changes and infratentorial involvement are key predictors of disability in multiple sclerosis (MS) and can be assessed using magnetic resonance imaging (MRI) planimetry. Although MRI planimetry is less susceptible to methodological and patient-related confounders than volumetry, it currently depends on manual measurements by unblinded experts, an approach that is time-consuming and vulnerable to bias. In this study, we present a fully automated deep learning framework for deriving brainstem planimetric measurements from MRI. The pipeline integrates an automated midsagittal plane (MSP) detection algorithm with a convolutional neural network trained to perform the segmentations required for planimetry. The automated method shows strong agreement with manual measurements and remains robust across scanners and acquisition protocols. These findings suggest that the proposed framework enables reliable, reproducible, and scalable MRI planimetry, supporting objective assessment of disease progression and treatment response in patients with MS.

Background: Medication errors (MEs) are a frequent cause of preventable harm but remain insufficiently quantified in emergency care. This study assessed the frequency, characteristics, and clinical impact of MEs among adverse drug reaction (ADR)–related emergency department (ED) admissions in Germany. Methods: We conducted a prospective multicenter study across six EDs over six years (n=7,967). ADRs and MEs were classified using standard causality (World Health Organization-Uppsala Monitoring Centre (WHO-UMC)) and preventability criteria (Schumock). Patient, drug, symptom, and outcome characteristics were compared between ADRs with and without MEs. Regression models assessed predictors of MEs and length of stay in hospital. Results: 20.1% of ADR-related cases, involved a preventable ME. Clinical presentation between groups; symptom burden, triage severity, and discharge outcomes, were similar. MEs clustered around chronic medications (pantoprazole, torasemide, metoprolol, ramipril, phenprocoumon, ibuprofen). Schumock analysis showed preventability as primarily linked to dosing errors (30%), non-adherence (28%), contraindications (26%), and monitoring (20%). Drug-specific symptom clusters mirrored expected pharmacology but were not error-specific. Multimorbidity was modestly protective (OR 0.84, 95%CI 0.71–1.00), while age, sex, polypharmacy, and number of diagnoses were not. Length of hospital stay was slightly longer in ME cases (+0.37 days; p = 0.037). Conclusion: MEs were identified in a substantial proportion of ADR-related ED admissions. Most MEs arose from routine prescribing and monitoring processes involving commonly used drugs suggesting that preventive efforts should focus on upstream safeguards, including medication reviews, electronic prescribing support, pharmacist involvement, and adherence monitoring, rather than detection at emergency presentation.

Abstract Lung cancer is the most frequently diagnosed malignancy and remains the leading cause of cancer-related deaths worldwide. Non-small cell lung cancer (NSCLC) is the most prevalent subtype, accounting for the majority of these fatalities. Despite numerous approved therapies, the five-year survival rate for NSCLC patients remains poor, largely due to drug resistance and early relapse. Three-dimensional (3D) tumor models that better recapitulate the in vivo conditions of primary NSCLC hold significant potential for advancing both drug discovery and development. In this study, we developed a hydrogel-based, 3D-bioprinted NSCLC model in a tumor-slice format, in which tumor spheroids or organoids of NSCLC and primary CAFs were directly assembled to create a model that mimics the structure and biochemical properties of the tumor microenvironment (TME). Within this bioprinted approach the spatial distribution and compartment ratios can be defined, enhancing reproducibility and enabling a customizable TME reconstruction. A composite bioink consisting of alginate, collagen, and Matrigel was developed to generate a bio-functionalized hydrogel matrix that supports the structural stability and enables the co-culture of different cell types. Several drugs used for first-line treatment of NSCLC were applied to the system to evaluate the treatment efficiency. The printed 3D tumor slices can be cultured for up to 14 days while maintaining their architecture and proliferative capacity. The process is highly reproducible and supports consistent generation of tumor-like constructs suitable for downstream analyses and drug testing. Functional assays including cytotoxicity assays, live imaging, multiplex immunofluorescence staining and 3D imaging were established to assess cell viability, tumor-stromal cell interaction, and treatment response within the printed 3D tumor slices. The culture can also be expanded to include PBMCs to investigate immune cell behavior within the TME, making the model suitable for evaluating immunotherapies. In conclusion, we established a standardized 3D hydrogel-printed model incorporating NSCLC tumor spheroids or organoids and primary CAFs. By reconstructing the TME, this system provides a reproducible and physiologically relevant platform for preclinical drug screening and cancer research. Together with the established analytical methods, this platform serves as a valuable tool for drug discovery and development. Citation Format: Jan A. Schlegel, Julia Thiel, Kanstantsin Lashuk, Schueler Julia, Thomas E. Mürdter, Matthias Schwab, Meng Dong. A 3D bioprinted hydrogel-based tumor model of non-small cell lung cancer for preclinical drug testing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3400.

Thiopurine methyltransferase (TPMT) and Nudix hydrolase 15 (NUDT15) are key enzymes that catabolize thiopurines. Decreased or no-function alleles in TPMT and NUDT15 are associated with reduced or no enzyme activity and predictive of pronounced adverse effects, including severe myelosuppression, that may occur among individuals treated with standard doses of thiopurines. Genetic variants in these genes are present in all world populations; however, their frequency varies by ancestry. In this updated guideline, we provide recommendations for adjusting starting doses of mercaptopurine, thioguanine, and azathioprine based on TPMT and NUDT15 genotypes, including for individuals with variants in both genes (updates on www.clinpgx.org).

Efforts to understand microbiome-host interactions in disease development and progression are growing, with short-chain fatty acids (SCFAs) produced by gut bacteria recognized as key metabolic mediators. To facilitate studies on the effect of bacterially secreted metabolites on human cells, we have developed a new LC-MS(/MS) method that allows quantitative analysis of underivatised SCFAs and simultaneous non-targeted screening for additional microbial or endogenous metabolites from cell culture media. Comprehensive method optimization resulted in methanolic gradient elution using a Kinetex XB-C18 column allowing for quantitative QQQ-MS analysis of propionate, butyrate, isobutyrate, valerate, isovalerate, and caproate, using deuterated internal standards. The method was validated in terms of linearity, selectivity, stability as well as inter-and intra-day accuracy and precision. To integrate non-targeted metabolomics, the assay was subsequently transferred to an LC-QTOF-MS platform and cross-validated to ensure accuracy and precision of SCFA analysis. Applying this method to study the effects of the breast cancer drug tamoxifen and 10 human tamoxifen metabolites (desmethyltamoxifen, Z-endoxifen, (E/Z)-4 '-hydroxy-desmethyltamoxifen, (E/Z)-4-hydroxytamoxifen, tamoxifen-Nglucuronide, (E/Z)-tamoxifen-4-glucuronid, (E/Z)-DM-tamoxifen-4-O-glucuronide, (E/Z)-4-hydroxytamoxifen-N-glucuronide, (E/Z)-endoxifen-4-sulfate, (E/Z)-tamoxifen-4-sulfate) on the secretome of faecal bacterial communities revealed a pronounced impact of the parent drug and non-sulfated metabolites. The omission of derivatisation leads to fast and simple sample preparation. Together with the short LC run time (10 min) and the comprehensive information obtained by combining targeted and non-targeted metabolomics in a single run, the new method represents a valuable tool for the in vitro investigation of metabolite-mediated microbiome-host interactions.

Purpose:Brimonidine, a selective α2-adrenergic agonist, is widely used in topical therapy for ocular hypertension or primary open-angle glaucoma. However, effective delivery is hindered by multiple ocular surface barriers. Because brimonidine is a cationic drug, we hypothesized that organic cation transporters (OCTs) and multidrug and toxin extrusion proteins (MATEs) are involved in brimonidine uptake in the human eye. This study aimed to determine if brimonidine is transported by OCT1, OCT2, OCT3, MATE1, and MATE2K and if the identified transporters are localized in anterior eye structures. Methods:Uptake studies were performed using HEK293 cells stably expressing OCT1, OCT2, OCT3, MATE1, and MATE2K. Intracellular brimonidine accumulation was analyzed by mass spectrometry. Immunohistochemistry of glaucomatous human eyes was used to localize relevant transporters in anterior ocular structures. Results:Brimonidine was transported by OCT2 and MATE1 but not by OCT1, OCT3, or MATE2K. Uptake was time- and concentration-dependent. Both OCT2 and MATE1 were expressed in the cornea, the conjunctiva, and the ciliary body. Conclusions:These results imply that OCT2 and MATE1 may play a role in brimonidine uptake into the human eye and may contribute to the interindividual variability of brimonidine concentrations and effects.

The selective estrogen receptor modulator tamoxifen is a mainstay of endocrine breast cancer therapy. However, the clinical response rates of tamoxifen are inferior to those of aromatase inhibitors, which may be partially explained by variable drug exposure due to the pharmacogenetics of the drug-metabolizing enzyme cytochrome P450 (CYP) 2D6. Clinical trials investigating the association between CYP2D6 impairment and tamoxifen outcomes have yielded conflicting results. The results of a comprehensive meta-analysis of 33 single-center tamoxifen trials reported here address this inconsistency by adjusting for two biases that may affect the validity of previous association studies: allele coverage of CYP2D6 genotyping and loss of heterozygosity of the CYP2D6 locus in tumor-derived DNA. After adjustment for bias, meta-analyses show significantly reduced study heterogeneity and a higher risk of recurrence or death in patients with impaired CYP2D6 metabolism compared with those with normal activity. These data may support the use of pharmacogenetics-guided tamoxifen therapy to improve outcomes in patients with CYP2D6-compromised breast cancer. Prospective studies should be considered. See related article by MacLehose et al., p. 224.

Immune checkpoint inhibitors can lead to ‘exceptional’, durable responses in a subset of persons. However, the molecular basis of exceptional response (ER) to immunotherapy in metastatic clear cell renal cell carcinoma (mccRCC) has not been well characterized. Here we analyzed pretherapy genomic and transcriptomic data in treatment-naive persons with mccRCC treated with standard-of-care immunotherapies: (1) combination of programmed cell death protein and ligand 1 (PD1/PDL1) and cytotoxic T lymphocyte-associated protein 4 inhibitors (IO/IO) or (2) combination of PD1/PDL1 and vascular endothelial growth factor (VEGF) receptor inhibitors (IO/VEGF). In the IO/IO cohort, clonal neoantigen load was significantly higher in persons with ER. In the IO/VEGF cohort, ER participants displayed strong enrichment of B cell receptor signaling-related pathways, tertiary lymphoid structure (TLS) signatures and evidence of increased metabolic activity. Our results suggest that ER may be related to clonal neoantigen-driven cytotoxic T cell responses and TLS formation in tumor microenvironments. Therapeutic combinations that elicit both T cell-directed and B cell-directed antitumor immunity may be important to achieve exceptional benefit to IO-based treatment in ccRCC. Shukla and colleagues study the genomic and transcriptomic data of exceptional responders to immunotherapy in renal cell carcinoma and find that such responses could be related to tertiary lymphoid structures, clonal neoantigen load and altered metabolism.

Leukemic stem cells (LSC) are well recognized for their essential roles in acute myeloid leukemia (AML) initiation and relapse. LSC can be distinguished from non-LSC AML cells by the expression of specific cell surface markers, but there is considerable phenotypic heterogeneity among LSC in AML. Here, using primary patient samples, we report that mannose receptor C-type 2 (MRC2) can be used to enrich for LSC across various AML subtypes. When compared to MRC2- AML cells isolated from the same patient samples, MRC2+ leukemic subpopulations show increased in vitro clonogenic capacity, a stemness transcriptomic signature, and enhanced leukemic capacity in mouse xenograft models. Further, we find that MRC2 is functional on AML cells, and enables their robust uptake of collagen, which supports their glycolytic metabolism. In sum these data highlight the use of functional surface markers to distinguish LSC in AML, and how they can yield insight into their unique characteristics.

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.

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.

Thiopurines are effective drugs for inflammatory bowel disease, but their use is limited by side effects such as pancreatitis, whose mechanism remains unknown and may be more severe in children. This study investigated in a personalized way thiopurine-induced pancreatitis mechanism using induced pluripotent stem cells from pediatric inflammatory bowel disease patients. Ten pediatric patients, five developing pancreatitis (cases) and five without it (controls), were enrolled. Patient-specific stem cells and their pancreatic differentiated counterparts were used to evaluate thiopurine cytotoxicity, to quantify metabolites levels by liquid chromatography-tandem mass spectrometry, and to assess thiopurine pharmacodynamics by western-blot assay. Statistical analyses were performed applying Student's t-test or two-way ANOVA followed by Bonferroni's post-hoc test for multiple comparisons. Cytotoxicity assays revealed higher thioguanine cytotoxicity in stem and pancreatic cells from cases; pancreatic cells from cases were also more sensitive to mercaptopurine. Moreover, thioguanine treatment on stem cells produced thioguanosine monophosphate and its methylated form, but their concentration did not differ significantly between the groups. In addition, higher TPMT gene expression was observed in stem cells from cases, but no differences were observed in pancreatic cells. No significant differences were detected in HPRT, NUDT15, ITPA, or PACSIN2 expression. Lastly, Rac1 protein concentration was similar in stem cells from cases and controls, but pancreatic cells from cases exhibited significantly higher Rac1 expression. These findings suggest that thiopurine cytotoxicity differences might be linked to pharmacokinetics in stem cells, while altered Rac1 expression in pancreatic cells might contribute to pancreatitis, implicating distinct mechanisms between stem and differentiated cells.