Largiadèr, Carlo R.

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 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.

Enhancers are critical for regulating tissue-specific gene expression, and genetic variants within enhancer regions have been suggested to contribute to various cancer-related processes, including therapeutic resistance. However, the precise mechanisms remain elusive. Using a well-defined drug-gene pair, we identified an enhancer region for dihydropyrimidine dehydrogenase (DPD, DPYD gene) expression that is relevant to the metabolism of the anti-cancer drug 5-fluorouracil (5-FU). Using reporter systems, CRISPR genome-edited cell models, and human liver specimens, we demonstrated in vitro and vivo that genotype status for the common germline variant (rs4294451; 27% global minor allele frequency) located within this novel enhancer controls DPYD transcription and alters resistance to 5-FU. The variant genotype increases recruitment of the transcription factor CEBPB to the enhancer and alters the level of direct interactions between the enhancer and DPYD promoter. Our data provide insight into the regulatory mechanisms controlling sensitivity and resistance to 5-FU.

Abstract Enhancers are critical for regulating tissue-specific gene expression, and genetic variants within enhancer regions have been suggested to contribute to various cancer-related processes, including therapeutic resistance. However, the precise mechanisms remain elusive. Using a well-defined drug-gene pair, we identified an enhancer region for dihydropyrimidine dehydrogenase (DPD, DPYD gene) expression that is relevant to the metabolism of the anti-cancer drug 5-fluorouracil (5-FU). Using reporter systems, CRISPR genome edited cell models, and human liver specimens, we demonstrated in vitro and vivo that genotype status for the common germline variant (rs4294451; 27% global minor allele frequency) located within this novel enhancer controls DPYD transcription and alters resistance to 5-FU. The variant genotype increases recruitment of the transcription factor CEBPB to the enhancer and alters the level of direct interactions between the enhancer and DPYD promoter. Our data provide insight into the regulatory mechanisms controlling sensitivity and resistance to 5-FU.

Abstract Enhancers are critical for regulating tissue-specific gene expression, and genetic variants within enhancer regions have been suggested to contribute to various cancer-related processes, including therapeutic resistance. However, the precise mechanisms remain elusive. Using a well-defined drug-gene pair, we identified an enhancer region for dihydropyrimidine dehydrogenase (DPD, DPYD gene) expression that is relevant to the metabolism of the anti-cancer drug 5-fluorouracil (5-FU). Using reporter systems, CRISPR genome edited cell models, and human liver specimens, we demonstrated in vitro and vivo that genotype status for the common germline variant (rs4294451; 27% global minor allele frequency) located within this novel enhancer controls DPYD transcription and alters resistance to 5-FU. The variant genotype increases recruitment of the transcription factor CEBPB to the enhancer and alters the level of direct interactions between the enhancer and DPYD promoter. Our data provide insight into the regulatory mechanisms controlling sensitivity and resistance to 5-FU.

Enhancers are critical for regulating tissue-specific gene expression, and genetic variants within enhancer regions have been suggested to contribute to various cancer-related processes, including therapeutic resistance. However, the precise mechanisms remain elusive. Using a well-defined drug-gene pair, we identified an enhancer region for dihydropyrimidine dehydrogenase (DPD, DPYD gene) expression that is relevant to the metabolism of the anti-cancer drug 5-fluorouracil (5-FU). Using reporter systems, CRISPR genome-edited cell models, and human liver specimens, we demonstrated in vitro and vivo that genotype status for the common germline variant (rs4294451; 27% global minor allele frequency) located within this novel enhancer controls DPYD transcription and alters resistance to 5-FU. The variant genotype increases recruitment of the transcription factor CEBPB to the enhancer and alters the level of direct interactions between the enhancer and DPYD promoter. Our data provide insight into the regulatory mechanisms controlling sensitivity and resistance to 5-FU.

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.

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.