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Hasta la fecha son pocos los casos descritos de la presencia del alelo *3 del gen NUDT15 en europeos. Se relaciona con un fenotipo metabolizador lento, lo que supone acumulación de, por ejemplo, la 6-mercaptopurina, con los efectos tóxicos que esto conllevaría.

Este caso clínico complementa y completa la evidencia disponible hasta la fecha sobre la presencia del alelo *3 del gen NUDT15 en pacientes de origen no asiático.

La principal implicación práctica de nuestro caso clínico es la necesidad de avanzar en la implementación de la farmacogenética en los hospital españoles, de forma que se consiga predecir la predisposición a eventos adversos graves e impedir su aparición. Del mismo modo que permita individualizar la posología a las condiciones de cada paciente.

Introduction

Acute lymphoblastic leukemia (ALL) accounts for 30 % of all childhood cancers and is the most common malignancy in children of developed countries¹. Higher survival rates and better quality of life have been observed as treatment protocols with cure rates that exceed 80 % have been established. Yet the development of toxicity in some patients remains a cause for concern¹.

6-mercaptopurine (6-MP) functions as an antimetabolite and is normally administered for prolonged periods in the consolidation and maintenance treatment phases (2 to 3 years). It acts through the incorporation into DNA of its metabolite, thioguanosine triphosphate (TGTP), preventing proper repair of the molecule and triggering apoptosis².

Despite its effectiveness, 6-MP has a very narrow therapeutic range and thus, the risk of adverse reactions is high^2,3. Myelotoxicity is one of the most severe toxic effects that may lead to the interruption or even discontinuation of treatment. This toxicity is commonly associated with polymorphisms in the thiopurine S-methyltransferase (TPMT) and Nudix hydrolase 15 (NUDT15) genes³. The main elimination pathway of thiopurines is S-methylation mediated by TPMT. NUDT15, in turn, is responsible for the dephosphorylation of active thiopurine metabolites⁴. Genetic variants involving reduced function of these enzymes result in an accumulation of nucleotides within cells and consequently high toxicity, even with conventional doses⁵.

In this clinical case, we examine the impact of genetic polymorphisms in 6-MP and the importance of knowing the patient’s genetic profile in advance, which will allow us to adjust doses from the start and avoid severe toxicity.

Results

Case Description

A 6-year-old girl was diagnosed with B-cell ALL in April 2017, with intermediate risk due to hyperdiploidy¹, and was treated according to the SEHOP PETHEMA 2013 protocol⁶. After induction phase IB the patient was admitted to the pediatric intensive care unit (PICU) with sepsis as a result of prolonged aplasia. A first pharmacogenetic analysis was ordered, including only the *2, *3B and *3C polymorphisms of the TPMT gene; the results indicated that the patient was a normal metabolizer. During the consolidation phase, which began in August 2017, the patient again developed aplasia and febrile neutropenia, so a reduction to 10 % of the 6-MP total standard dose was performed. However, in the reinduction phase her myelotoxicity reappeared.

In March 2018, the patient started the maintenance phase in month 10, with dose of 6-MP reduced to 10 % (2.5 mg QD) and dose of oral methotrexate (MTX) reduced to 85 % (7.5 mg QW). A month later, she presented with low leukocyte and platelet counts, as well as with hemoglobin level reduction, as shown in Figure 1. Therefore, treatment was temporarily interrupted. During this period, the patient was hospitalized due to febrile neutropenia and the need of platelet transfusion. When her blood parameters had returned to normality, 6-MP at 10 % of dose and MTX at 50 % were resumed.

Figure 1. Evolution of hematologic parameters involved in toxicity: neutrophils (1A), platelets (1B), hemoglobin (1C), and 6-MP doses during the consolidation phase of PETHEMA 2013. 

In month 18, the patient showed grade 4 hematologic toxicity and was admitted to the PICU. Subsequently, the fourth intrathecal dose of MTX was administered and maintenance oral chemotherapy restarted.

Up to the date of the end of treatment, in August 2019, numerous dose adjustments of both drugs were necessary. In addition, many transfusions of packed red blood cells and platelets were needed to avoid further bone marrow aplasia and hospitalization for the resulting complications. Finally, the patient received 6-MP at 5 % of the dose and MTX at 35 % until treatment completion.

An extensive pharmacogenetic study including the TPMT genetic variants (*2, *3B, *3C and *4) and also the variant in NUDT15 (*3), was conducted by real-time polymerase chain reaction (RT-PCR) using TaqMan® probes (ABI Applied Biosystems, 7300 Real-Time PCR System) in accordance with the manufacturer’s instructions. Sanger sequencing (Figure 2) confirmed the NUDT15 results.

Figure 2. Results of the analysis of the NUDT15*3 homozygous rs116855232-TT polymorphism. 

Discussion

The pharmacogenetics of 6-MP has been extensively described. In 2015, Yang et al. first reported the relationship between the development of myelosuppression during treatment with 6-MP and variants of NUDT15⁷. Their observations were based on two prospective clinical trials in an East Asian population. A correlation was observed between the patients with TT genotype at rs116855232 were exquisitely sensitive to 6-MP, with an average dose intensity of 8.3 %, compared with those with TC and CC genotypes, who tolerated 63 % and 83.5 % of the planned dose, respectively. They also observed that this genetic variant was rare in Europeans and absent in Africans⁷. A multiethnic meta-analysis demonstrated that the T allele of rs116855232 contributed to a 7.86-fold (P <0.0001, 95 %CI = 6.13-10.08) higher risk of developing leukopenia⁸.

In accordance with the foregoing, the Clinical Pharmacogenetics Implementation Consortium (CPIC®) developed pharmacogenetic guidelines for TPMT polymorphisms⁹, including determination of allele *3 of the NUDT15 gene, with the main SNPs being at level of evidence of 1A. Consequently, the Spanish Agency for Medicines and Medical Devices (AEMPS) considers performing the genetic test before starting treatment with this drug¹⁰. However, it proves difficult to transfer this into clinical practice, as observed in our clinical case, where the pharmacogenetic analyses were conducted only after the occurrence of severe toxicities.

Our patient manifested prolonged myelosuppression, entailing repeated dose reductions and treatment interruptions, as well as numerous admissions to the PICU. At this point, the first TPMT genotyping was carried out, revealing normal metabolizing activity. Following the CPIC® recommendations⁹, a second analysis, including determination of NUDT15 allele *3, was conducted. The results showed the rs116855232-TT genotype, related to a slow metabolizer phenotype for NUDT15. Thus, explaining the severe toxicity observed when 6-MP was administered at standard and reduced doses. The frequency of these polymorphisms varies between different populations. For the NUDT15 gene, allele *3 is most common in Asians (6.7 %). In those of European and Latin origin, however, its frequency is markedly lower: 0.2 % and 0.8 %, respectively⁹.

Conclusion

Pharmacogenetics could be an important factor in responding to this patient’s high-grade myelotoxicity. If testing results had been known prior to 6-mercaptopurine administration, a dose tailored to the patient’s genetic profile would have been started, which might have prevented the multiple dose adjustments and toxic reactions. Future actions should be focused on facilitating the introduction of pharmacogenetics into everyday clinical practice to validate our results on a larger sample of patients.