TPMT and NUDT15 genes are both related to mercaptopurine intolerance in acute lymphoblastic leukaemia patients from Uruguay

Acute lymphoblastic leukaemia (ALL) is the most common childhood cancer in Uruguay and worldwide, comprising approximately 25% of all cancers in children under 15 years (Hunger & Mullighan, 2015). In recent decades, the event-free survival rate of ALL patients in Uruguay has significantly increased, reaching about 80%. This is similar to that observed in developed countries (Castillo et al, 2012). However, adverse events related to drugs such as 6-mercaptopurine (6-MP) remain an important complication of therapy. Accumulation of 6-MP metabolites can cause haematological and hepatic toxicities contributing to the morbi-mortality of ALL. TPMT gene variants have been associated with haematological toxicity in many populations, mainly with European ancestry. However, in Asian-or Asian-derived populations, such as Native Americans, the frequency of TPMT variants is lower and only explains a small element of toxicity. Recent studies have shown that variants in other genes, such as ITPA and NUDT15, can better explain the toxicity observed in these populations (Yang et al, 2015; Moriyama et al, 2016). The Uruguayan population is mainly comprised by individuals with European ancestry (Spaniards, Italians, Portuguese, Basques, etc.) with a minor proportion of Native Americans and African populations (Sans et al, 1997). The tri-hybrid structure of the Uruguayan population, similar to other Latin-American populations, may allow the co-existence of TPMT, ITPA and NUDT15 variants at high frequency. Here, we present data of TPMT, ITPA and NUDT15 genetic variant frequencies and their relationship with haematological toxicity relative to 6-MP maintenance therapy. We analysed a sample of 124 ALL Uruguayan paediatric patients aged 1–15 years, followed up at the Servicio de Hemato-Oncolog ıa Pedi atrica from Pereira-Rosell Hospital, Montevideo, Uruguay. The protocol and procedure employed for this research were undertaken in compliance with the Helsinki Declaration and patient parents gave written informed consent to participate in this research. All patients were treated according to the Berlin-Frankfurt-Munster (BFM) protocol and receive uniform oral maintenance therapy of 6-MP (50 mg/m/day) and methotrexate (20 mg/m/ week). Complete blood count (CBC) was performed weekly during the first 2 months of maintenance and then every two or three weeks. The ITPA c.94C˃A (rs1127354) variant and NUDT15 variants were determined by automated sequencing using previously reported primers (Cao & Hegele, 2002; Moriyama et al, 2016). TPMT*2, *3A, *3B and *3C variants were determined as previously described (Ameyaw et al, 1999). The cumulative 6-MP dose at weeks 8, 16 and 24, and the mean weekly 6-MP dose were compared between individuals with and without these variants by the Kruskal–Wallis test. We also analysed the association between gene variants and the number of cases with leucopenia grade 3-4 (White blood cell count <2 0 9 10/l) by the Mann–Whitney test. Moreover, odd ratios were determined to evaluate the leucopenia risk among patients with and without gene variants. Twenty-two patients were excluded from these analyses due to the presence of germline chromosomal abnormalities or because they were not yet in maintenance phase. Statistical analyses were performed in SPSS 22 0 (IBM Corp. Armonk, NY, USA) with 95% confidence interval. TPMT variants were observed in 13 individuals; 12 were heterozygous for TPMT*3A and one for TPMT*2. The spectrum and frequency of heterozygotes (10 5%) is similar to that observed for other Latin-American and European populations. However, the ITPA 94A variant frequency (5 3%) was higher than that reported for a Chilean population (Silva et al, 2008; Farfan et al, 2014; Ar aoz et al, 2015). Interestingly, 11 of the 115 individuals analysed were heterozygous for NUDT15 variants: five for NUDT15*2, two for *3, one for *4 and three for *6. This frequency (8 8%) is similar to that reported for Guatemalan and other American populations that have a higher Native American influence than in Uruguay (Sans et al, 1997; Moriyama et al, 2016). ITPA c.94C˃A variant was not associated with 6-MP dose, number of leucopenia events or risk of leucopenia (data not shown). For TPMT and NUDT15, we assigned a genetic risk score for each individual based on the number of risk alleles in both genes as previously reported (Yang et al, 2015): zero (no TPMT or NUDT15 variants), one (presence of TPMT or NUDT15 variant) or two risk alleles (presence of TPMT and NUDT15 variants). The 6-MP dose was significantly lower in individuals with one or two risk alleles compared to those without risk alleles in all analysed intervals. Similar results were observed with the mean weekly 6-MP dose. In contrast to previous reports (Yang et al, 2015), the cumulative 6-MP dose for individuals with two risk alleles was higher than those with one risk allele at weeks 8 and 16. However, this was not observed at week 24 or when we analysed the mean correspondence