Capecitabine belongs to the group of medications known as fluoropyrimidines. It is an anticancer drug used in the treatment of breast and colon cancer, and causes severe toxicity in 25.5% of treated patients1 and even death in 1.6% of such patients2. Interindividual variability in its toxic effects is partly caused by genetic variants in the enzymes involved in its metabolism and in its target genes.

The gene encoding the enzyme thymidylate synthase is a potential marker of response and toxicity to fluoropyrimidines, because the main mechanism of action of these drugs is folate cycle inhibition through the blockade of this enzyme. Several variants in this gene have been studied, and a significant association has been found between the 2R/2R genotype of the rs45445694 variant and severe toxicity to treatment (P = 0.0014; odds ratio [OR] = 5.21). Furthermore, the results of gene expression analysis in tumor tissues suggest a correlation between this genotype and low thymidylate synthase expression3.

Dihydropyrimidine dehydrogenase (DPD) is the rate-limiting enzyme in fluoropyrimidine catabolism. There is ample evidence of an association between DPD deficiency and increased levels of the active metabolite in blood, leading to severe toxicity after drug administration that can be life-threatening4. The four most relevant variants with decreased function in the gene encoding DPD (DPYD) are as follows: DPYD*2A or c.1905 + 1G > A (rs3918290); DPYD*13 or c.1679 T > G (rs55886062); c.2846A > T (rs67376798) and c.1129-5923C > G or HapB3 (rs75017182)5,6. The recommendation of the Spanish Agency of Medicines and Medical Products (AEMPS) on the dosage adjustment of fluoropyrimidines is supported by the guideline of the Clinical Pharmacogenetics Implementation Consortium7, the guideline of the Dutch Pharmacogenetics Working Group8, and the Consensus of Experts from the Spanish Pharmacogenetics and Pharmacogenomics Society and the Spanish Society of Medical Oncology9. In these guidelines, a metabolic status is established for each genotype with the corresponding recommendation supported by level I evidence (i.e. high-quality evidence).

Associations have also been found between several variants in the gene encoding the enzyme cytidine deaminase (CDA), which is essential for drug activation, and increased capecitabine toxicity. Specifically, associations have been found between the CDA variants 79 A > C (rs2072671)10–12, 451 A > G (rs532545)10,13–15, −33 ins/delC13, 92 A > G (rs602950)15, and 435 C > T (rs1048977)11 and an increased risk of treatment toxicity, with abnormal enzymatic activity being observed in some of them16.

However, despite all the published evidence, there are no clinical guidelines on its dosage according to CDA genetic variants: thus, the real prognostic value of this biomarker needs to be established through genotype–phenotype association studies. Given that almost 60% of the European population are carriers of one of these genetic variants11,16, a high percentage of the population would benefit from genetic screening for this biomarker prior to initiating treatment with capecitabine.

Regarding economic justification, a study conducted in the United Kingdom found that the average cost of consultations, hospitalization, and treatment due to the adverse effects of capecitabine was approximately €500 per patient17. These data can be extrapolated to the Spanish population, because both the ethnic group and type of healthcare model are the same. It has also been estimated that screening for DPYD and CDA genotyping in routine clinical practice could halve these costs18. Given that approximately 80 patients per year are treated with capecitabine in the participating centres, the implementation of DPYD and CDA genotyping would result in savings of about €20,000 per year per centre. If we extrapolate these data to the Spanish population as a whole, the saving would be €5,000,000 per year.

The relevance of including CDA in the dosage guideline lies in the high mutation rate of this gene. According to the dbSNP database of the National Centre for Biotechnology Information, the four most relevant DPYD variants have an average allele frequency of 0.005 in the European population, whereas the average frequency of the five selected CDA variants is 0.355. Thus, CDA screening could imply savings of 98.6% (€4,930,000) of the estimated annual savings when compared with those of the single DPYD screening currently performed, which would significantly enhance the sustainability of the Spanish Health System.

The aim of this study is to analyze the association between the presence of CDA enzyme genetic variants, its enzymatic activity, and the development of severe toxicity (grade 3–4 diarrhea, nausea/vomiting, mucositis, palmoplantar erythrodysesthesia syndrome, hepatotoxicity, and/or liver toxicity) in patients initiating treatment with capecitabine.

The study also includes the analysis of the four DPYD genetic variants recommended by the AEMPS: DPYD*2A or c.1905 + 1G > A (rs3918290); DPYD*13 or c.1679 T > G (rs55886062); c.2846A > T (rs67376798): and c.1129-5923C > G or HapB3 (rs75017182)19. This genotyping will be performed prior to treatment with capecitabine, allowing the initial dosage to be adjusted according to the patient's DPYD genotype. Subsequently, studies will be conducted on the allelic frequencies of the variants analyzed, the frequency and degree of toxicity among the participants, and on the dosage adjustment needed according to the CDA genetic variants for the reduction of toxicity to treatment. After the results have been validated in an independent cohort, a clinical dosage guideline for capecitabine based on DPYD and CDA genetic variants will be created and a bioinformatics tool will be developed that will match the patient's DPYD and CDA genotype with the dosage adjustment recommended in the clinical dosage guideline. Fig. 1 shows the flow chart of the different stages of the research protocol.

Fig. 1.

Chronological flowchart of the procedures to be followed in the different phases of the protocol: recruitment phase, phase after initiating treatment, and final phase after statistical data analysis. CPIC, Clinical Pharmacogenetics Implementation Consortium.

(0,13MB).

The genotype–phenotype association project presented is novel in that, through the categorization of the CDA enzyme as a biomarker, it recommends personalized pharmacological prescription based on the DPYD and CDA genotype of each patient, rather than on DPYD alone, as is currently recommended.

After the proposed clinical guideline has been validated in an independent cohort, this approach is expected to reduce the risk of toxicity of capecitabine treatment due to overdosage in the case of patients carrying ultrarapidly metabolizing CDA genetic variants and/or in those carrying poorly metabolizing DPYD genetic variants.

One of the main differences between this project and those already published lies in its prospective cohort design. The great majority of studies are retrospective, are more limited, and only associate polymorphisms with toxicity, without performing dosage adjustments11,20,21. The present study will prospectively perform determinations and pharmacogenetic counseling, by taking advantage of the resources and clinical benefit of performing preventive genotyping. In addition, statistical analysis will provide us with the RR of the presence of CDA variants, which is a more powerful statistical measure than those obtained in case–control studies. It should also be noted that, thanks to the development of the bioinformatics tool, it will be possible to implement the Pharmacogenetic Analysis Service, thus facilitating the interpretation of the results in the clinical practice of hospital centres.

Finally, the categorization of CDA as a biomarker could also be useful in other drug treatments, given that this enzyme is involved in the inactivation of gemcitabine and cytarabine, which are antineoplastic drugs used for the treatment of various types of cancer. Thus, the results obtained in this project could be used in subsequent studies with the aim of developing clinical dosage guidelines for such drugs and reducing chemoresistance to treatment and consequent disease progression.

The fact that the metabolic phenotype is established by enzymatic activity alone may be a limitation of this study, given that it is also relevant to know each patient's pharmacokinetics in relation to their capecitabine metabolism in order to describe a more complete phenotype. This aspect could be a future research line, extending the characterization of CDA as a biomarker to the phenotype by including pharmacokinetic analysis during the period in which each patient receives their first dosage of capecitabine. In this regard, a population pharmacokinetic model has recently been used to study the relationship between the metabolites of capecitabine (5-FU, 5’-DFCR, and 5’-DFUR) and toxicity or clinical response to the drug22. The data obtained identify the CDA enzyme as a statistically significant covariate, thus highlighting the usefulness of simultaneously analyzing genetic, enzymatic, and pharmacokinetic data for this biomarker.

This project has been classified by the Spanish Agency of Medicines and Medical Products (AEMPS), with code ATB-CAP-2020-01, as a “Post-authorization prospective follow-up study” (EPA-SP). It has been authorized by the Autonomous Committee for the Evaluation of Observational Post-authorization Studies, for Prospective Follow-up Studies with Medicines (CAEPO). It has also been approved by the Drug Research Ethics Committee (CEIM) of the General University Hospital of Elda (Spain) and the General University Hospital of Elche (Spain) in accordance with current legislation: Royal Decree 223/2004 of February 6, 2004.

• - V Call for Grants for the Promotion of Research Activity of the Foundation for the Promotion of Health and Biomedical Research of the Community of Valencia, Spain (FISABIO) 2021.

• - Grants for Preparatory Actions to support the exploration and formulation of future joint research and innovation projects between the Universidad Miguel Hernández de Elche (Spain) and FISABIO 2021.

• - VIII Call for grants for the Support and Promotion of Research of the Instituto de Investigación Sanitaria y Biomédica de Alicante, Spain (ISABIAL) 2021, modality B.

• - Roche sponsorship with code CPRES00254.

The following authors contributed to the development of the article and its preparation:

Paula Castro-Sánchez: promotion of the study, development of the protocol, request for classification and authorizations needed for its approval in the participating centres, general coordination of the study, and drafting the article.

Andrés Corno-Caparrós: design, organization, and execution of the genetic analysis of the study, planning and development of the bioinformatics tool, critical review of the article, and approval of the final version for publication.

M. Amparo Talens-Bolós: design and organization of sample logistics between the participating hospitals and the university, issuing the pharmacogenetic reports, critical review of the article, and approval of the final version for publication.

María José Prieto-Castelló: application for public funding for the study, data management for statistical analysis, critical review of the article, and approval of the final version for publication.

Loreto Pitaluga-Poveda: validation of the enzyme activity analysis method, management and preparation of reagents, critical review of the article, and approval of the final version for publication.

Juan Antonio Barrera-Ramírez: organizing and conducting patient recruitment and clinical data collection, critical review of the article, and approval of the final version for publication.