Citations

Abstract The primary mechanism of action for the chemotherapeutic 5-fluorouracil (5-FU) is inhibition of thymidylate synthase (TS), which leads to nucleotide imbalance and subsequent cellular death. Expression of TS has been suggested to be determined by a variable number of tandem repeats located within the TS enhancer region (TSER). TSER genotype has been inconsistently linked to 5-FU-related adverse events, including severe grade ≥3 drug-related toxicity. Our recent studies demonstrated that the number of repeats within the TSER, as well as the presence or absence of a polymorphism within each repeat, was significantly associated with 5-FU toxicity in 629 patients. Our findings are consistent with a model in which the number of upstream stimulatory factor (USF1) transcription factor binding sites in the TSER, which is determined both by repeat status and genotype within each repeat, defines TYMS expression and contributions to 5-FU toxicity risk. We present an updated nomenclature that concisely and unambiguously identifies both the number of repeats and the number of USF1 binding sites. The difficulty in obtaining clear genotypes within this highly repetitive and variable region limits the ability to integrate this biomarker into predictive tests. We present methodologies for assigning TSER genotypes using conventional and high-throughput sequencing, including whole genome sequence data. Our results are expected to promote the improved study of TSER genotype in future clinical studies and the integration of TSER status into clinical predictive tests for severe 5-FU toxicity. Citation Format: Huixing Huang, Dominic Schaerer, Remington Schmidt, Ting Zhang, Tanja K Froehlich, Kelly Bouchonville, Robert B Diasio, Ursula Amstutz, Carlo Largiadèr, Steven M Offer. Novel approach to thymidylate synthase genotype determination and utility as a biomarker of severe toxicity to 5-FU chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5805.

Abstract Genetic variants in the DPYD gene that are deleterious to dihydropyrimidine dehydrogenase (DPD) enzyme function have demonstrated clinical validity as predictive biomarkers for severe (i.e., CTCAE grade ≥3) and potentially lethal toxicity to fluorouracil (5-FU) and capecitabine chemotherapies. Variants within the coding region only explain 5-10% of 5-FU-related toxicities; and GWAS approaches have been unable to identify validated predictive biomarkers to date. Our previous studies demonstrated that DPD expression was controlled by multiple epigenetic processes; considerable variation was also noted in liver DPYD/DPD levels. Using CRISPR-interference (CRISPRi) and CRISPR-activation (CRISPRa), we identified functional enhancer regions within liver tissues and cells. Epigenetic status at these regions was shown to correlate with DPYD expression in cell models and human liver tissues. Chromosome conformation capture (3C) confirmed direct promoter-enhancer interactions. A multi-omics analysis of >1000 human liver specimens identified a proximal eQTL for DPYD expression within an enhancer, with a global minor allele frequency of 27%. This variant was subsequently shown to directly alter DPYD expression in knock-in cell line models. The variant disrupts transcription factor binding at the DPYD enhancer, leading to reduced enhancer-promoter interaction, reduced DPD expression, and increased cellular sensitivity to 5-FU. Collectively, our data suggest that the variant could represent a novel predictive biomarker for severe 5-FU toxicity. The evaluation of this marker in the clinical setting is currently underway. Citation Format: Ting Zhang, Alisa Ambrodji, Huixing Huang, Kelly Bouchonville, Amy Etheridge, Remington Schmidt, Jose Cardiel Nunez, Zoey Temesgen, Federico Innocenti, Robert Diasio, Carlo Largiadèr, Steven M. Offer. DPYD enhancer variant confers increased risk of severe toxicity in 5-FU chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5420.

Introduction Insulin resistance (IR) plays a key role in the pathogenesis and clinical course of patients with multiple metabolic diseases and diabetes. This study aimed to explore the effect of trimetazidine (TMZ) on skeletal muscle IR in mice fed a high-fat diet (HFD) and explore the possible underlying mechanism. Research design and methods In vivo, a HFD mouse IR model was adopted and TMZ and exercise were used to intervene. Postintervention the following were determined: blood levels of glucose and insulin, homeostasis model assessment of IR index, expression of skeletal muscle insulin signaling-related proteins phosphorylated insulin receptor substrate 1 (p-IRS1/IRS1) and phosphorylated protein kinase B (p-AKT/AKT), nuclear factor erythroid 2 related factor 2 (Nrf2) signaling pathway, and oxidative stress. In vitro, a palmitate-treated C2C12 myotube IR model was constructed. Cellular glucose uptake, p-IRS1/IRS1, and p-AKT/AKT were determined, and reactive oxygen species (ROS) production was analyzed based on treatments with specific small interfering RNA of Nrf2 with or without TMZ. Western blot was used to obtain the protein expression level and ROS production by functional analysis kits. Results In vivo, TMZ and exercise decreased the blood glucose and insulin levels and homeostasis model assessment of IR index, increased skeletal muscle insulin signaling-related protein ratios of p-IRS1/IRS1 and p-AKT/AKT, and both interventions activated Nrf2 signaling and reduced oxidative stress production in HFD mice. In vitro, TMZ reduced the oxidative stress reaction, increased the ratios of p-AKT/AKT and p-IRS1/IRS1, and attenuated the insulin stimulation of PA-induced glucose uptake. However, in the absence of Nrf2, TMZ failed to resist the effects of IR. Conclusions This study showed that TMZ, like exercise, brought about marked improvements to HFD-induced skeletal muscle IR through TMZ, a common pathway with exercise in the form of Nrf2, regulating oxidative stress. We provide new evidence to support the use of TMZ for diabetes treatment.

(1) Introduction: Most studies rely on in-hospital data to predict cardiovascular risk and do not include prehospital information that is substantially important for early decision making. The aim of the study was to define clinical parameters in the prehospital setting, which may affect clinical outcomes. (2) Methods: In this population-based study, we performed a retrospective analysis of emergency calls that were made by patients to the largest private emergency medical services (EMS) in Israel, SHL Telemedicine Ltd., who were treated on-site by the EMS team. Demographics, clinical characteristics, and clinical outcomes were analyzed. Mortality was evaluated at three time points: 1, 3, and 12 months’ follow-up. The first EMS prehospital measurements of the systolic blood pressure (SBP) were recorded and analyzed. Logistic regression analyses were performed. (3) Results: A total of 64,320 emergency calls were included with a follow-up of 12 months post index EMS call. Fifty-five percent of patients were men and the mean age was 70.2 ± 13.1 years. During follow-up of 12 months, 7.6% of patients died. Age above 80 years (OR 3.34; 95% CI 3.03–3.69, p < 0.005), first EMS SBP ≤ 130 mm Hg (OR 2.61; 95% CI 2.36–2.88, p < 0.005), dyspnea at presentation (OR 2.55; 95% CI 2.29–2.83, p < 0001), and chest pain with ischemic ECG changes (OR 1.95; 95% CI 1.71–2.23, p < 0.001) were the highest predictors of 1 month mortality and remained so for mortality at 3 and 12 months. In contrast, history of hypertension and first EMS prehospital SBP ≥ 160 mm Hg were significantly associated with decreased mortality at 1, 3 and 12 months. (4) Conclusions: We identified risk predictors for all-cause mortality in a large cohort of patients during prehospital EMS calls. Age over 80 years, first EMS-documented prehospital SBP < 130 mm Hg, and dyspnea at presentation were the most profound risk predictors for short- and long-term mortality. The current study demonstrates that in prehospital EMS call settings, several parameters can be used to improve prioritization and management of high-risk patients.

Abstract Proximity labeling catalyzed by promiscuous enzymes, such as APEX2, has emerged as a powerful approach to characterize multiprotein complexes and protein–protein interactions. However, current methods depend on the expression of exogenous fusion proteins and cannot be applied to identify proteins surrounding post-translationally modified proteins. To address this limitation, we developed a new method to label proximal proteins of interest by antibody-mediated protein A-ascorbate peroxidase 2 (pA-APEX2) labeling (AMAPEX). In this method, a modified protein is bound in situ by a specific antibody, which then tethers a pA-APEX2 fusion protein. Activation of APEX2 labels the nearby proteins with biotin; the biotinylated proteins are then purified using streptavidin beads and identified by mass spectrometry. We demonstrated the utility of this approach by profiling the proximal proteins of histone modifications including H3K27me3, H3K9me3, H3K4me3, H4K5ac, and H4K12ac, as well as verifying the co-localization of these identified proteins with bait proteins by published ChIP-seq analysis and nucleosome immunoprecipitation. Overall, AMAPEX is an efficient method to identify proteins that are proximal to modified histones.

Approximately 20 drugs have been shown to be effective for the treatment of colorectal cancer (CRC). These drugs are from several classes of agents and include cytotoxic drugs, therapeutics that target cell signaling pathways at the extracellular and/or intracellular levels, and combination therapies that contain multiple targeted agents and/or cytotoxic compounds. Targeted therapeutics can include monoclonal antibodies, fusion proteins, and small molecule drugs. The first introduced into clinical use was 5‐fluorouracil in the early 1960s and remains the foundation for most CRC treatments in both adjuvant therapy and in advanced (metastatic) treatment regimens. As with other cancers, the consideration of biomarkers has the potential to improve CRC therapy through patient stratification. The biomarkers can include germline genetic markers, tumor‐specific genetic markers, immune markers, and other biomarkers that can predict antitumor efficacy or the likelihood of toxicity prior to administration of a specific drug. Consistent with the benefit of considering biomarkers in treatment, many newer targeted therapies are developed and approved simultaneously with a companion diagnostic test to determine efficacy. This review will focus on biomarkers that have demonstrated clinical utility in CRC treatment; however, it is noted that many additional biomarkers have been theorized to contribute to drug response and/or toxicity based on known biological pathways but thus far have not attained widespread use in the clinic. The importance of pretreatment biomarker testing is expected to increase as future drug development will likely continue to focus on the concurrent development of companion diagnostics.

AimsThe aim of this study was to identify risk variants and haplotypes that impair dihydropyrimidine dehydrogenase (DPD) activity and are, therefore, candidate risk variants for severe toxicity to 5‐fluorouracil (5‐FU) chemotherapy.MethodsPlasma dihydrouracil/uracil (UH2/U) ratios were measured as a population marker for DPD activity in a total of 1382 subjects from 4 independent studies. Genotype and haplotype correlations with UH2/U ratios were assessed.ResultsSignificantly lower UH2/U ratios (panova < 2 × 10−16) were observed in carriers of the 4 well‐studied 5‐FU toxicity risk variants with mean differences (MD) of −43.7% for DPYD c.1905 + 1G > A (rs3918290), −46.0% for DPYD c.1679T > G (rs55886062), −37.1%, for DPYD c.2846A > T (rs67376798), and −13.2% for DPYD c.1129‐5923C > G (rs75017182). An additional variant, DPYD c.496A > G (rs2297595), was also associated with lower UH2/U ratios (P < .0001, MD: −12.6%). A haplotype analysis was performed for variants in linkage disequilibrium with c.496A > G, which consisted of the common variant c.85T > C (rs1801265) and the risk variant c.1129‐5923C > G. Both haplotypes carrying c.496A > G were associated with decreased UH2/U ratios (H3, P = .003, MD: −9.6%; H5, P = .002, MD: −16.9%). A haplotype carrying only the variant c.85T > C (H2) was associated with elevated ratios (P = .004, MD: +8.6%).ConclusionsBased on our data, DPYD‐c.496A > G is a strong candidate risk allele for 5‐FU toxicity. Our data suggest that DPYD‐c.85T > C might be protective; however, the deleterious impacts of the linked alleles c.496A > G and c.1129‐5923C > G likely limit this effect in patients. The possible protective effect of c.85T > C and linkage disequilibrium with c.496A > G and c.1129‐5923C > G may have hampered prior association studies and should be considered in future clinical studies.

Inhibition of thymidylate synthase (TS) is the primary mode of action for 5-fluorouracil (5FU) chemotherapy. TS expression is modulated by a variable number of tandem repeats in the TS enhancer region (TSER) located upstream of the TS gene (TYMS). Variability in the TSER has been suggested to contribute to 5FU-induced adverse events. However, the precise genetic associations remain largely undefined due to high polymorphism and ambiguity in defining genotypes. To assess toxicity associations, we sequenced the TSER in 629 cancer patients treated with 5FU. Of the 13 alleles identified, few could be unambiguously named using current TSER-nomenclature. We devised a concise and unambiguous systematic naming approach for TSER-alleles that encompasses all known variants. After applying this comprehensive naming system to our data, we demonstrated that the number of upstream stimulatory factor (USF1-)binding sites in the TSER was significantly associated with gastrointestinal toxicity in 5FU treatment.

Abstract Background It is unclear how the Health Insurance Portability and Accountability Act (HIPAA) should be interpreted in the context of sharing of genomic information between family members. Methods The authors analyzed the HIPAA Privacy Rule, reviewed the literature and constructed a clinical scenario to inform how HIPAA can be interpreted for multiple forms of patient- and provider-mediated genetic risk notification. Results Under HIPAA, healthcare providers can lawfully notify relatives to recommend genetic risk assessment using multiple approaches, including supporting the patient telling their own relatives, contacting relatives directly with the patient’s authorization, or contacting a relative’s provider directly. Conclusions Multiple forms of patient- or provider-mediated contact of relatives are already legally permissible under HIPAA, are consistent with ethical obligations of care to patients and their families, and could result in improved population health through identification of clinically actionable disease risk. Unanswered questions remain about implementation and impacts of provider-mediated programs.