Citations

The chemotherapeutic 5‐fluorouracil (5‐FU) is commonly used to treat solid tumors, especially colorectal cancer. Deleterious variants in DPYD, which encodes dihydropyrimidine dehydrogenase (DPD), are strongly associated with severe and potentially lethal 5‐FU toxicity. Phenotypic assays for DPD function have been proposed as alternative or complementary approaches to identify DPD deficiencies with the rationale that they may capture unrecognized causes. These assays include the direct measurement of DPD activity in peripheral blood mononuclear cell (PBMC) lysates and measuring plasma metabolites uracil (U), dihydrouracil (UH 2 ), and the dihydrouracil/uracil (UH 2 :U) ratio. We evaluated correlations between PBMC DPD activity and plasma metabolite measures in matched samples from 203 patients with available DPYD exome sequencing data. We also compared the ability of phenotypic measures to identify carriers of deleterious DPYD variants. In addition to established high‐risk variants, we identified three rare DPYD variants predicted to be deleterious: c.274C>G, c.966C>A, and c.1435G>A. Collectively, these variants were associated with impaired DPD function with effect sizes comparable to well‐studied toxicity‐associated variants. At the population level and within carriers of specific genotypes, weak but significant correlations were observed between PBMC DPD activity and either UH 2 :U ratio or U level. These findings suggest that although phenotypic assays can detect severe DPD deficiency, high variability limits their utility as standalone diagnostic tools. Integration with genetic testing may provide a more robust pretreatment risk assessment strategy.

Abstract Background Dalbavancin is increasingly used off-label for chronic Gram-positive bone and joint infections (BJIs) requiring prolonged antimicrobial therapy, but optimal dosing strategies and the role of therapeutic drug monitoring (TDM) remain unclear. We evaluated the real-world implementation of a proactive TDM-guided dalbavancin workflow within an outpatient parenteral antimicrobial therapy (OPAT) programme. Methods Single-centre prospective observational implementation study including adult patients with BJIs consecutively treated with dalbavancin in OPAT service between July 2022 and August 2025. A multidisciplinary team developed a workflow incorporating TDM-guided pharmacokinetic modelling to individualize dosing intervals after fixed initial administrations (Days 1, 8 and 43). Target Cmin concentrations were ≥8 mg/L, later increased to ≥15 mg/L after European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoint revision. Primary outcomes included implementation metrics (feasibility, fidelity and appropriateness). Secondary outcomes were clinical effectiveness, safety and overall workflow success. Results The workflow was applied to 101 patients accounting for 418 TDM-guided dalbavancin administrations (feasibility). Three patient-driven treatment discontinuations were observed (3/101, 3.0%), and scheduled administrations occurred within the predefined time window in 335/418 (83.5%) of cases (fidelity). Pharmacological target attainment was achieved in 285/342 (89.6%) of administrations without dosing delays (appropriateness). Clinical effectiveness was achieved in 55/73 (75.3%) patients completing therapy, with 28/101 (28%) still on treatment. Adverse events–related discontinuation occurred in 3/101 (3%) patients. Overall workflow success was observed in 74/101 (73.2%) patients. Conclusions A multidisciplinary TDM-guided dalbavancin workflow within an OPAT programme was feasible and safe, achieving high target attainment and favourable clinical outcomes. This approach supports individualized long-acting dalbavancin therapy for complex chronic BJIs.

Abstract 5-Fluorouracil (5FU)-based chemotherapy remains a front-line treatment for most patients with colorectal cancer. However, approximately 30% of patients who receive 5FU experience severe, life-threatening toxicity. Dihydropyrimidine dehydrogenase (DPD, DPYD gene) mediates the rate determining step in 5FU catabolism. Deleterious genetic variations in DPYD have been associated with elevated concentrations of circulating 5FU and significantly increased risk for 5FU toxicity. Five deleterious variants in DPYD have been identified as clinical biomarkers of toxicity and have led to prospective genotyping and differential dosing recommendations for carriers of these variants, as noted on recent updates to the FDA label for the 5-FU prodrug capecitabine. Our previous studies also established the importance of epigenetic control of DPD expression. In this study, we used existing epigenetic repositories to identify candidate enhancer elements for DPYD. We selected 20 novel high-priority putative enhancers for further study using a combination of reporter assays and CRISPRi applied across multiple cell models. Further studies will investigate means to target these regulatory mechanisms as a means to increase localized efficacy of 5-FU-based therapies in colorectal cancer. Citation Format: Hannah Marie Krause, Julia Magee, Kelly Bouchonville, Lauryn Allyn Hahn, Ryan Jonathan Swartz, Brianna Bembenek, Lulu Jiang, Steven M. Offer. Characterizing the critical role of enhancer-gene regulation in 5-fluorouracil toxicity and resistance [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 3140.

Abstract Nearly 300,000 cancer patients are treated with fluoropyrimidine chemotherapy in the U.S. annually. Approximately one-third of patients experience severe and life-threatening toxicities, which can result in death. Deleterious genetic variants in DPYD, which encodes the rate-limiting enzyme of fluoropyrimidine catabolism (dihydropyrimidine dehydrogenase, DPD), have been correlated with severe toxicity in clinical studies. However, these variants explain only 10-30% of toxicity cases. Phenotypic measures of DPD function are attractive biomarkers of fluoropyrimidine toxicity risk with the potential to identify unknown causes of DPD deficiency. In this study, we compared two measures of DPD enzyme activity, blood plasma dihydrouracil to uracil ratio (UH2:U) and direct measurement of DPD activity in peripheral blood mononuclear cells (PBMCs) using 204 subjects with comprehensive DPYD genotype data. DPD PBMC measurements showed higher overall inter-individual variation compared to UH2:U ratios. Low correlation between UH2:U and PBMC measurements were noted for matched samples taken from the same subject. As expected, DPD metabolites (U and UH2) correlated with UH2:U ratio; however, no correlation was noted between U or UH2 levels and the PBMC DPD activity. Furthermore, when data were analyzed by genotype, no correlations were noted between UH2:U and PBMC DPD activity. We observed the expected decrease in UH2:U ratio for carriers of the four clinically tested DPYD variants and identified two novel variants associated with a significantly decreased UH2:U ratio. To further investigate the impact of rare genotypes and previously identified DPYD haplotypes on the UH2:U ratio, additional cohorts of 1994 and 500 subjects, enriched for specific genotypes, were utilized. Our results suggest that phenotyping may have limited validity as a single test when predicting DPD deficiency at an individual level and call into question the often-reported belief that PBMC DPD activity represents the "gold standard" for identifying DPD deficiency. Additional studies are needed to investigate correlations between phenotypic measures and 5-FU toxicity in patients, potentially as secondary biomarkers in conjunction with genetic tests. Citation Format: Brianna Bembenek, Kelly Bouchonville, Carlo Largiadèr, Steven M. Offer. Comparative analysis of phenotypic markers of DPD activity as biomarkers of 5-FU toxicity [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 3135.

Abstract Dihydropyrimidine dehydrogenase (DPD, encoded by the DPYD gene) is the rate-limiting enzyme in the catabolism of the widely used chemotherapeutic drug 5-fluorouracil (5-FU). Genetic variations in DPYD are predictive biomarkers of 5-FU toxicity. One such biomarker, rs75017182 (the causal allele linked to the “HapB3” haplotype), has been suggested to promote alternative splicing of DPYD, leading to expression of an inactive proteoform. However, carriers of rs75017182 exhibit only moderately increased risk of 5-FU toxicity and modestly decreased ex vivo DPD activity compared to carriers of another well-studied splice variant, rs3918290 (*2A). We hypothesized that, unlike *2A, the rs75017182 variant does not cause obligate alternative splicing but instead promotes partial alternative splicing. To test this, we generated dual-fluorescent minigene reporter constructs containing either the G (wildtype) or C (variant) allele. In our minigene system, alternative splicing was elevated in cells transfected with C-containing vectors via flow cytometry, though levels were lower than those observed in a parallel *2A model. To investigate contributions of other variants in LD with rs75017182, we expanded the reporter to include additional relevant genomic regions. The analyses were complemented by the study of in vivo splicing in cells engineered to harbor various combinations of variants and in lymphoblastoid lines carrying specific genotype combinations. Overall, our findings demonstrate that rs75017182-C promotes only partial alternative splicing, eliciting a modest decrease in DPD activity and explaining its comparatively weak association with severe 5-FU toxicity. Additional variants in LD have a minimal impact on splicing and 5-FU sensitivity. Citation Format: Lauryn Allyn Hahn, Ryan Schwartz, Hannah Marie Krause, Lulu Jiang, Brianna Bembenek, Kelly Bouchonville, Steven M. Offer. Partial alternative splicing in carriers of DPYD rs75017182 explains modest association with severe 5-FU toxicity to 5-FU [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 3137.

Abstract 5-Fluorouracil (5-FU) is a widely prescribed chemotherapy for colorectal, breast, gastric, and head and neck cancers. The drug inhibits thymidylate synthase, blocking DNA synthesis and causing cytotoxic stress that halts tumor growth. However, patients carrying deleterious variants in DPYD, the gene encoding the rate-limiting enzyme in 5-FU metabolism (dihydropyrimidine dehydrogenase, DPD), can experience fatal toxicity due to impaired drug clearance and accumulation of 5-FU metabolites. Current pharmacogenetic screening guidelines in the U.S. recommend testing for few of the >2,000 nonsynonymous variants that have been reported for DPYD, leaving patients with rare, uncharacterized mutations at risk. To interpret expanded testing, however, a means to classify DPYD variants of unknown significance is needed. To address this, we developed MPACT-DPD, a random-forest-based classifier that accurately predicts the functional impact of DPYD missense variants. Our model was trained on in-vitro activity of 156 variants and leveraged a feature set of biochemical, evolutionary, and AlphaFold3-derived structural features. We optimized hyperparameters using ten-fold stratified cross-validation and evaluated model performance with Matthews correlation coefficient (MCC) to account for moderate class imbalance (7:3 neutral to deleterious). It achieved exceptional performance, with a Matthews correlation coeffient (MCC) of 0.906 and an accuracy of 95.1% on an independent validation set (n=43). Furthermore, a SHAP (SHapley Additive exPlanations)-based interpretability analysis revealed cofactor proximity and residue conservation as the key drivers of predictions. MPACT-DPD showed superior performance at variant classification against other tools, including a DPYD gene-specific variant classifier, and has the potential to expand pre-treatment genetic screening to improve the safety of personalized 5-FU-based chemotherapy. Citation Format: Lulu Jiang, Ryan Jonathan Swartz, Lauryn Allyn Hahn, Brianna Bembenek, Hannah Marie Krause, Kelly Bouchonville, Steven M. Offer. MPACT-DPD: An interpretable machine learning classifier for predicting the functional impact of DPYD missense variants [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 2712.

Background Bacterial infections remain a major global health burden, causing significant morbidity and mortality. Despite ongoing advances, prompt diagnosis is still hampered by nonspecific host biomarkers and the inherently slow turnaround of traditional microbiological cultures. These limitations often delay the initiation of appropriate treatments. In recent years, affinity-based proteomic approaches have been explored to address this gap. Among them, the Proximity Extension Assay (PEA) has emerged as a promising multiplexed protein quantification tool, capable of simultaneously measuring hundreds of immune and inflammatory proteins with high sensitivity from minimal sample volumes. Such technologies hold the potential to identify novel biomarkers, thereby improving both diagnosis and patient management in bacterial infections. Main body In this systematic scoping review, we examined studies applying PEA-based proteomics to adult bacterial infections. Out of the records screened, ten studies met inclusion criteria. Most were conducted in Europe and North America, relied primarily on plasma samples, and employed commercially available panels enriched for immune and inflammatory mediators. Study quality varied, with some evidence of variability and potential risk of bias. Across the 379 proteins investigated, a subset of proteins were consistently associated with bacterial infections across multiple clinical contexts, whereas others showed limited or no associations. Conclusions Current applications of PEA-based proteomics in adult bacterial infections is limited and largely exploratory. Rather than supporting immediate clinical translation, the available evidence suggests the value of PEA-based approaches for informing biomarker discovery. Future research should prioritize well-designed, longitudinal, and pathogen-stratified studies in clinically relevant settings to strengthen evidence robustness and support the rational development of proteomics-informed diagnostic and translational strategies.

Abstract Background and Aims Celiac disease (CeD) is a chronic digestive autoimmune disorder affecting approximately 1% of the worldwide population. It is driven by T cells activated by specific HLA-DQ2 or HLA-DQ8 molecules leading to the destruction of intestinal villi. We aimed to characterize shared CeD-specific T cells in patients on and off a gluten-free diet (GFD) from a large cohort of cases and controls. Methods We performed bulk TCRβ immune sequencing of the peripheral blood of 1,604 biopsy-confirmed CeD patients (1,339 on GFD, 265 on normal diet) and 1,100 controls. We identified over 300 TCRβs enriched in CeD cases versus controls in an HLA-aware manner, controlling for CeD risk alleles. Results CeD-associated TCRβs were found to be more predictive of disease than previously characterized gliadin and glutenin-binding TCRs in a validation cohort. Furthermore, the clonal breadth of these TCRβs was associated with increased intestinal damage. Immune sequencing of the peripheral blood also uncovered repertoire-level differences between CeD patients and controls. CeD patients displayed significantly higher productive clonality compared to age-matched controls as well as expansion of TCRβs specific to cytomegalovirus (CMV) and Epstein-Barr Virus (EBV). Conclusions These findings underscore the value of unbiased immune repertoire sequencing to identify novel biomarkers for autoimmune disease and to discover new disease mechanisms which can improve both diagnosis and treatment of disease.

Introduction: Adult hepatitis B vaccination coverage in the United States remains low despite updated national recommendations supporting universal adult immunization. Many adults remain unaware of their susceptibility to hepatitis B virus (HBV) infection. This quality improvement (QI) study evaluated a stepwise “Offer–Offer–Test–Vaccinate” workflow designed to improve hepatitis B vaccine uptake among vaccine-hesitant adults in a primary care setting. Methods: This retrospective QI study was conducted at a single primary care clinic in Rochester, Minnesota. Adult patients (≥19 years) seen between October 1, 2022, and October 1, 2024, were routinely offered hepatitis B vaccination during rooming. Two participating physicians implemented an enhanced workflow in which patients who declined vaccination were counseled and then offered serologic screening using the 2023 CDC-recommended triple panel (HBsAg, anti-HBs, and total anti-HBc). Patients with negative serology were notified via the patient portal and reoffered vaccination along with a link to the CDC Vaccine Information Statement. The primary endpoint was initiation of the hepatitis B vaccine series following receipt of negative serology results. Preliminary series-completion data through October 1, 2025, were also assessed. Results: Among 501 eligible adults, 104 (20.8%) accepted vaccination at the initial offer. Of the 397 who declined, 106 (26.7%) agreed to serologic testing; 91 (85.8%) were seronegative. Among seronegative patients, 41 (45.1%) subsequently accepted vaccination. Integrating serologic testing increased overall vaccination uptake from 20.8% to 28.9%, an absolute increase of 8.2 percentage points and a relative increase of 39.4%. Approximately 2.6 serologic tests were required to generate 1 additional first-dose vaccination. Preliminary series-completion rates were 74.0% among those vaccinated at the initial offer and 87.8% among those vaccinated following serologic testing. Discussion: In this primary care setting, incorporating serologic testing into the workflow was associated with modestly improved hepatitis B vaccination uptake among vaccine-hesitant adults. Serologic results may help re-engage patients by providing personalized immunity information. Implementation considerations include staffing needs, laboratory access, patient portal engagement, and insurance coverage. Conclusion: The “Offer–Offer–Test–Vaccinate” strategy was associated with a modest increase in hepatitis B vaccine uptake among hesitant adults. This EMR-integrated, stepwise approach may offer a feasible and scalable strategy to reduce missed vaccination opportunities and support HBV prevention in adult primary care.

Abstract Vaccination is increasingly acknowledged as an effective preventive measure not only against specific infections, but also for the prevention of cardiovascular disease in high-risk patients. Specifically, a growing body of evidence suggests that vaccines against influenza, SARS-CoV-2, respiratory syncytial virus, herpes zoster, and other viruses significantly reduce infection and for influenza the incidence of major adverse cardiovascular events in vaccinated individuals. This clinical consensus statement examines the existing literature and accumulated evidence and offers practical clinical advice on vaccination timing and target demographics, specifically addressing complex clinical scenarios with a focus on cardiovascular conditions. It includes guidelines for vaccinating vulnerable populations such as immunosuppressed individuals, patients with congenital heart disease, and pregnant women as well as safety and potential complications of the procedure.