Digestive tumors, especially colorectal cancer, are associated with a high mortality rate throughout the world1. Fluoropyrimidines, particularly 5-fluorouracil and capecitabine, are part of the usual treatment for these types of tumors. Although generally well tolerated, a slight percentage of patients experience severe toxicity (grades 3-4) when treated with these antineoplastic agents2,3, and this has a very significant impact on their quality of life4. Since the preservation of an optimal quality of life is an aspect that patients diagnosed with cancer value very highly, the prevention of severe adverse effects in the context of their treatment is of great importance5.

Several studies have shown that patients with dihydropyrimidine dehydrogenase (DPD) deficiency are at greater risk of experiencing adverse reactions such as diarrhea, mucositis or neutropenia when treated with fluoropyrimidines6–8. A safety alert that was recently published by the Spanish Drug and Healthcare Product Agency (AEMPS) recommends genotype and/or phenotype DPD deficiency testing in candidates to be treated with these drugs9. Specifically, it recommends the genotyping of the most widely studied loss of function variants of the DPYD gene, which are rs3918290 (c.1905+1G>A, DPYD*2A), rs55886062 (c.1679T>G, DPYD*13), rs67376798 (c.2846A>T) and rs56038477 (c.1236G>A/HapB3). Reductions of 25-50% in the initial dose of fluoropyrimidines have already been recommended for patients who are carriers of these variants10–12.

The implementation of these biomarkers in clinical practice can undoubtedly contribute to the prevention of the above-mentioned severe toxicities, at minimal cost to the National Health System13–15. For a year now we have been determining the four variants of the DPYD gene, as part of our center's program of care, in patients who are candidates for treatment with fluoropyrimidines. Few data are as yet available on the prevalence of these variants in the Spanish population and on the clinical importance of genotypic testing in actual practice.

On the basis of the above, the aims of the present study were to determine the prevalence of loss of function variants in the DPYD gene in the population of our reference area and to evaluate the clinical results of genotyping these variants in the daily practice of a level 3 hospital. In addition, variations in the number of DPYD genotyping determinations were evaluated after creating a multidisciplinary team with the hospital pharmacist's active participation.

A total of 115 patients with digestive tumors were included in the study. Most of them (106 patients, 92% of the total number) had been diagnosed with colorectal cancer. One hundred and seven of the patients received fluoropyrimidine-based antineoplastic treatment. The most common drug regimens were FOLFOX (5-fluorouracil, folinic acid and oxaliplatin), XELOX (capecitabine and oxaliplatin) and capecitabine monotherapy. The basal characteristics of the patients included in this study are detailed in Table 1.

The four variants of the DPYD gene described above were genotyped in all the patients in the study. The most frequently identified variant was c.1129-5923C>G (rs75017182) (HapB3), which was found in 6 patients (5.2%). The second most frequent variant was c.2846A>T (rs67376798), identified in 3 patients (2.6%). Finally, the variant c.1905+1G>A (rs3918290) was detected in 2 patients (1.7%) (Table 2). The variants were found in heterozygosis in all cases. None of the genotyped patients carried the c.1679T>G (rs55886062) variant. Thus, the frequency of gene DPYD variant carriers in our cohort was 9.6% (11 patients in a total of 115).

Ten of the 11 patients with a partial DPD deficiency were treated with an antineoplastic protocol that included fluoropyrimidines. Five of them began the first cycle on a standard dose, since their DPYD genotype was not known when the treatment was initiated; in contrast, the DPYD genotype of another 5 patients had been ascertained before their first cycle, and these 5 began the treatment on doses of fluoropyrimidine that were reduced by 25-50% in accordance with each individual case (Table 3). Two of the 5 patients that were given standard doses experienced grade 3-5 toxicity during the first three cycles. One of these patients carried the c.1905+1G>A (rs3918290) variant and was treated with FOLFOX-panitumumab at standard doses as a first line treatment for a metastatic left-sided tumor of the colon. Since this patient developed grade 4 neutropenia during the first treatment cycle, the second cycle was delayed for 15 days and administered with a dose reduction of 20%. The other patient, who had a history of high blood pressure, digestive hemorrhage and anemia, carried the variant c.1129-5923C>G (rs75017182) (HapB3), and suffered a cardiorespiratory arrest 12 days after receiving the first cycle of FOLFOX at a standard dose as adjuvant treatment for a stage III right-sided tumor of the colon. None of the 5 patients treated with reduced doses of fluoropyrimidine from the first cycle developed grade 3-5 toxicity. In 3 of them, given their good tolerance to treatment, it was possible to increase the fluoropyrimidine until it reached 75-100% of the standard dose (Table 3).

In October 2020, a multidisciplinary team was created in order to make sure that all patients who were candidates for antineoplastic protocols including fluoropyrimidines were genotyped for the DPYD gene and given doses that were adjusted on the basis of their test results. This team is made up of medical oncologists, hospital pharmacists specialized in oncology, clinical geneticists and laboratory technicians.

The team's flow of work is organized as follows:

• 1.

  The oncologist requests DPYD genotyping for patients that are to be treated with fluoropyrimidines.

• 2.

  Under the clinical geneticist's supervision, the laboratory technician performs the genetic study and tries to respond in the shortest possible period of time (a week at most). The test's result is entered into the antineoplastic agent prescription application. If the patient is a carrier of any of the loss-of-function variants the geneticist reports the result directly to the multidisciplinary team. In addition, the report with the genotyping results is entered into the patient's medical history. The hospital pharmacists draw up a specific clinical outline in the patient's clinical history, including details regarding the recommended dose adjustments, and the oncologist prescribes the chemotherapy regimen on the basis of such adjustments.

• 3.

  At the time of validating the fluoropyrimidine-based treatment, the hospital pharmacist confirms that the DPYD gene analysis has been requested and performed. If this is the case, the pharmacist reviews the prescribed dose of fluoropyrimidine, to make sure it adjusts correctly to the genotypic profile. If the test has not been performed, the pharmacist will request it directly from the clinical geneticist and assess its results at the time of the next cycle of chemotherapy.

Following the introduction of the above working system, the average monthly number of DPYD analyses has increased from 6.4 (January to October) to 17.5 (November to December) (Figure 1).

Figure 1.

Number of patients genotyped for DPYD in 2020.

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The present study's results indicate that about 10% of patients with digestive tumors in our center are carriers of loss-of-function variants of the DPYD gene. This makes them partially DPD deficient and increases their risk of experiencing severe secondary toxicity when treated with fluoropyrimidines.

The percentage of individuals carrying these loss-of-function variants is slightly higher in our series than in other published studies, which report an incidence of 3-8% among the population11. This might be due to the fact that, in the beginning, genotyping of the DPYD gene was performed mainly on patients who had already experienced severe toxicity when treated with these drugs. Regarding the populational incidence of each of the four variants, the relatively high frequency of the rs75017182 variant stands out in our cohort. As previously explained, this variant is in perfect linkage disequilibrium with the rs56038477 variant, whose reported populational frequency is 2.6-6.3%10. Thus, although the frequency of 5.2% that was found for the rs56038477 variant in the present study comes within the range described in the literature for the Caucasian population, further studies of larger cohorts would be required in order to confirm its high prevalence in the Spanish population. On the other hand, our findings revealed a frequency of 2.6% for the variant rs67376798, which is higher than the allelic frequencies of 0.4-1.4% that have been reported for the general Caucasian population16. Finally, the frequency of 1.7% that was found for variant rs3918290 comes within the range described for Caucasians (0.8-2.2%)16. Unsurprisingly, variant rs55886062 was not found in our cohort; with a reported allelic frequency of 0.06-1.00% in the Caucasian population, this variant has the lowest rate of prevalence of the four variants that were analyzed in the study.

No consensus exists as yet in the scientific community regarding the ideal fluoropyrimidine dosing regimen for patients carrying variants of the DPYD gene. Henricks et al. initially recommended that patients with variants rs3918290 and rs55886062 began treatment with fluoropyrimidines at 50% below the standard dose, while carriers of the rs67376798 and rs75017182 variants did so at doses equaling 75% of the standard dose. However, more recent publications recommend a reduction of 50% in the dose of fluoropyrimidine in patients carrying any of the above-described variants of the DPYD gene16,17. In fact, a prospective study evaluating the safety of dose reduction recommendations determined that reductions of 25% in the case of variants rs67376798 and rs75017182 were not enough, and suggested a reduction of 50%, as subsequent publications have confirmed12.

Five of the patients in our study (four who carried variant rs75017182 and one who carried variant rs3918290) began antineoplastic treatment with a fluoropyrimidine regimen that included standard doses of the drug. Of these five patients, two experienced severe toxicity after the first cycle of chemotherapy, which led to the death of one of them. These results reveal the clinical importance of reducing fluoropyrimidine doses in patients carrying any variants of the DPYD gene. As regards the five carriers of DPYD gene variants in whom the first cycle of chemotherapy was begun at doses that were 25-50% below standard, no grade 3-5 toxicity was observed in any of them during the first three cycles of chemotherapy. These data, preliminary and based on a limited number of cases as they are, seem to support the safety of fluoropyrimidine use in patients carrying variants of the DPYD gene, provided the prescribed doses of the drug are adequately reduced. It should be added that tolerance to treatment, in three of these five patients, made it possible to increase the dose of fluoropyrimidine after the first cycle of chemotherapy, reaching 100% of the standard dose, with excellent levels of tolerance, in one case. This interindividual variability reveals the utility of pharmacokinetic monitoring of fluoropyrimidine during the first cycle of chemotherapy, with a view to adjusting the dosing regimen to optimal levels during the second cycle and beyond.

Our results support the clinical importance of DPYD genotyping in all patients that are to be treated with fluoropyrimidines, firstly because of the relatively high number of individuals who carry gene variants (about 10%), and secondly because of the latter's association with severe, and potentially lethal, toxicity. In this respect, we believe that the creation of a multidisciplinary team guarantees the translation of genotyping test results to the clinical setting. Our results, following the team's creation, are still quite preliminary, but they seem to suggest a higher rate of DPYD genotyping determinations.

The present study has several drawbacks. In the first place, the genotyping tests could not always be performed prior to treatment, as the clinical guidelines recommend, due to the limited amount of time available between DNA sample extractions and the beginning of antineoplastic treatment. This meant that, in five of the patients, treatment was administered at full doses in spite of the fact that they were carriers of DPYD gene variants. In this respect, it should be noted that genotypic test results are delivered in significantly shorter periods of time after the implementation of the multidisciplinary approach. Secondly, we must remember that in the case of therapeutic combinations, such as for instance the FOLFOX protocol, the added drugs also contribute to the occurrence of adverse effects. Thirdly, it was not possible for our study to include all patients diagnosed with digestive tumors in our center. Finally, the study's sample size is not large enough to allow for accurate determination of the prevalence of the different DPYD gene variants in our population, and further studies will be needed to validate these results.

In conclusion, the present study reveals a high prevalence of DPYD gene variant carriers in our population, as well as the clinical significance of these genotypic variants and their impact on the occurrence of adverse effects. It also demonstrates the feasibility of implementing a simple approach to DPYD gene genotyping, based on the collaboration of a multidisciplinary team made up of oncologists, hospital pharmacists and geneticists.

No funding.

No conflict of interests.