Amstutz, Ursula

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.

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.

The purpose of this guideline is to provide information for the interpretation of clinical dihydropyrimidine dehydrogenase ( DPYD ) genotype tests so that the results can be used to guide dosing of fluoropyrimidines (5‐fluorouracil and capecitabine). Detailed guidelines for the use of fluoropyrimidines, their clinical pharmacology, as well as analyses of cost‐effectiveness are beyond the scope of this document. The Clinical Pharmacogenetics Implementation Consortium (CPIC ® ) guidelines consider the situation of patients for which genotype data are already available (updates available at https://cpicpgx.org/guidelines/guideline‐for‐fluoropyrimidines‐and‐dpyd/ ).

Abstract Purpose: The microRNA miR-27a was recently shown to directly regulate dihydropyrimidine dehydrogenase (DPD), the key enzyme in fluoropyrimidine catabolism. A common polymorphism (rs895819A&gt;G) in the miR-27a genomic region (MIR27A) was associated with reduced DPD activity in healthy volunteers, but the clinical relevance of this effect is still unknown. Here, we assessed the association of MIR27A germline variants with early-onset fluoropyrimidine toxicity. Experimental Design: MIR27A was sequenced in 514 patients with cancer receiving fluoropyrimidine-based chemotherapy. Associations of MIR27A polymorphisms with early-onset (cycles 1–2) fluoropyrimidine toxicity were assessed in the context of known risk variants in the DPD gene (DPYD) and additional covariates associated with toxicity. Results: The association of rs895819A&gt;G with early-onset fluoropyrimidine toxicity was strongly dependent on DPYD risk variant carrier status (Pinteraction = 0.0025). In patients carrying DPYD risk variants, rs895819G was associated with a strongly increased toxicity risk [OR, 7.6; 95% confidence interval (CI), 1.7–34.7; P = 0.0085]. Overall, 71% (12/17) of patients who carried both rs895819G and a DPYD risk variant experienced severe toxicity. In patients without DPYD risk variants, rs895819G was associated with a modest decrease in toxicity risk (OR, 0.62; 95% CI, 0.43–0.9; P = 0.012). Conclusions: These results indicate that miR-27a and rs895819A&gt;G may be clinically relevant for further toxicity risk stratification in carriers of DPYD risk variants. Our data suggest that direct suppression of DPD by miR-27a is primarily relevant in the context of fluoropyrimidine toxicity in patients with reduced DPD activity. However, miR-27a regulation of additional targets may outweigh its effect on DPD in patients without DPYD risk variants. Clin Cancer Res; 21(9); 2038–44. ©2015 AACR.