Patpetra Svasdisant1, Waraporn Glomglao1, Preeyanun Siraprapapat1, Wiyakan Inthararujikul1, Kalaya Tachavanich1, Chetsada Boonthimat1, Sakkarin Ardsiri1, Kochpinchon Chansing1, Suwimon Sriprach1, Sasima Tongsai2, Phakatip Sinlapamongkolkul3, Kleebsabai Sanpakit1 and Jassada Buaboonnam1.
of Hematology and Oncology, Department of Pediatrics, Faculty of
Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand.
2 Research Department, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand.
3 Department of Pediatrics, Faculty of Medicine, Thammasat University, Pathum Thani, Thailand.
Jassada Buaboonnam, MD. Associate Professor of Pediatrics, Division of
Hematology and Oncology, Department of Pediatrics, Faculty of Medicine,
Siriraj Hospital, Mahidol University, 2 Wanglang Road, Bangkoknoi,
Bangkok 10700, Thailand. Tel: +66 2 419 5960; Fax: +66 2 411 3010.
Published: March 1, 2023
Received: January 10, 2023
Accepted: February 28, 2023
Mediterr J Hematol Infect Dis 2023, 15(1): e2023024 DOI 10.4084/MJHID.2023.024
| This is an Open Access article distributed
under the terms of the Creative Commons Attribution License
which permits unrestricted use, distribution, and reproduction in any
medium, provided the original work is properly cited.
(6-MP), a thiopurine agent, is a essential medication for treating
pediatric acute lymphoblastic leukemia (ALL). However, its side effects
of neutropenia and hepatotoxicity might interrupt treatment, resulting
in poor outcomes. Inosine triphosphate pyrophosphatase (ITPA), an enzyme in the thiopurine pathway, may prevent the accumulation of toxic thiopurine metabolites. Studies on ITPA and thiopurine-associated toxicities are scarce.
study retrospectively investigated 1- to 15-year-old children with ALL
who received 6-MP during the maintenance phase of treatment between
2000 and 2020. Toxicity during the first year of maintenance therapy
and the mean dose of 6-MP were analyzed.
Results: The 209 patients had a median age of 4.8 (0.3-14.8) years. Of these, 124 patients (59.3%) had wild-type ITPA, 73 patients (34.9%) had heterozygous ITPA 94C>A (heITPA), and 12 patients (5.7%) had homozygous ITPA 94C>A (homITPA), with an allele frequency of 0.23. The incidence of neutropenia among ITPA polymorphisms did not significantly differ (P = 0.813). In patients harboring homITPA, transaminitis was more frequent than other polymorphisms but without a significant difference (P = 0.063). The mean dose of 6-MP for patients with homITPA was significantly lower than that for patients with hetIITPA or wild-typeITPA (P = 0.016).
Conclusions: HomITPA had a higher incidence of transaminitis and required a significantly larger dose reduction of 6-MP than wild-type ITPA. Further study is warranted to elucidate the effects of ITPA polymorphisms on toxicity in patients with ALL treated with 6-MP.
lymphoblastic leukemia (ALL) is children's most common hematologic
malignancy. With advances in treatment, its event-free survival is
approximately 90%. 6-Mercaptopurine (6-MP) is one
of the key medications used during maintenance therapy, with the most
extended phase of treatment being at least two years. Nevertheless,
6-MP's side effects of neutropenia and hepatotoxicity may interrupt
treatment. Two well-known thiopurine enzymatic
genetic polymorphisms have demonstrated clinical significance for
patients with 6-MP. They are thiopurine methyltransferase (TPMT), prevalent in European populations, and nudix (nucleoside diphosphate linked moiety X) type motif 15 (NUDT15), prevalent in Asian populations.[3,4] Inosine triphosphate pyrophosphatase (ITPA),
another enzyme in the multistep thiopurine pathway, prevents the
accumulation of methylthioinosine triphosphate, a toxic thiopurine
metabolite, thereby catalyzing inosine triphosphate to inosine
monophosphate.[5,6] ITPA 94 C>A genotypes were found to have markedly decreased ITPA activity.
However, the clinical relevance between toxicities and such
polymorphisms still needs to be well elucidated, and clinical
recommendations for dose modifications according to the ITPA polymorphism need to be developed. Such studies on thiopurine enzymatic genetic polymorphisms apart from TPMT and NUDT15
may better guide physicians in prescribing doses of thiopurine drugs,
especially 6-MP, to prevent toxicity. The present investigation aimed
to determine the effects of ITPA polymorphisms on (1) neutropenia and hepatotoxicity, (2) the modification of 6-MP dosage, and (3) survival for childhood ALL.
Patients and Methods
retrospective study included patients aged 1 to 15 years diagnosed with
ALL between January 2000 and December 2020. The patients were
classified into risk groups according to the National Cancer Institute
classifications and Thai Pediatric Oncology Group classification.[8,9] They were treated according to the national protocol of the Thai Pediatric Oncology Group.
during maintenance therapy consisted of monthly pulse intravenous
vincristine and 5-day oral prednisolone, weekly oral methotrexate (20
mg/m2), and daily oral 6-MP (50 mg/m2).
Patients who experienced prolonged neutropenia or severe infection
during previous therapy with 6-MP may have been administered a
decreased 6-MP dosage, depending on the attending physician's
assessment. Complete blood counts and liver function tests were
performed monthly and trimonthly, respectively. The dose of 6-MP was
adjusted to maintain an absolute neutrophil count between 500 and 1500
cells/mm3. Treatment was temporarily interrupted if a patient developed cytopenia (an absolute neutrophil count < 500 cell/mm3 or a platelet count < 50,000/mm3)
or hepatic dysfunction (direct hyperbilirubinemia or an elevated
transaminase > 20 times the upper normal value). Patients with
elevated transaminase > 5 times but < 20 times the upper normal
value may have been prescribed a decreased dose of 6-MP at the
attending physician's discretion. The mean dose of 6-MP at each
interval was calculated using the sum of a 28-day dose (mg) divided by
the product of 28 and the body surface area.
Neutropenia was defined as an absolute neutrophil count of fewer than 500 cells/mm3. Transaminitis was defined as serum alanine aminotransferase more than five times the upper normal value.
a retrospective study, the remaining blood specimens of patients during
the diagnosis procedure were collected and subsequently evaluated for ITPA 94C>A
polymorphisms using an allele-specific polymerase chain reaction
method. The wildtype primer was 5’CGTTCAGATTCTAGGAGATAAGTTCC-3’. The
mutant forward primer was 5’-CGTTCAGATTCTAGGAGATAAGTTCA-3'. For
internal controls, 5'GCTTAGCACAAGCAGAGACCTGACG-3' and
5'TTCCACGAACATGTGTGAATGCAGC-3' were used. Patients harboring TPMT or NUDT15 polymorphisms were excluded from this cohort.
this research began, its protocol was approved by the Siriraj
Institutional Review Board, Faculty of Medicine Siriraj Hospital,
Mahidol University, Bangkok, Thailand (Si-479/2021).
Demographic data were determined using descriptive statistics.
Categorical variables are presented as a number and percentage, and
continuous variables are given as a mean ± standard deviation (SD) or a
median (range), as appropriate. The associations between ITPA
polymorphisms, neutropenia, and transaminitis were determined using
Pearson's chi-squared test. Simple and multiple binary logistic
regression analyses assessed the associations between ITPA
polymorphisms, 6-MP doses, and transaminitis. The odds ratio (OR) with
a corresponding 95% confidence interval (95% CI) was used to evaluate
the strengths and directions of associations. Differences in 6-MP doses
among the heterozygous and homozygous ITPA polymorphisms and wild-type ITPA
at baseline and each follow-up were evaluated using 1-way ANOVA,
followed by Bonferroni corrections for multiple post hoc comparisons of
means. The Kaplan-Meier method was used to estimate the 3-year overall
survival (OS) and event-free survival rates. A log-rank test was
constructed to compare the 3-year OS and event-free survival rates. IBM
SPSS Statistics for Windows, version 23 (IBM Corp, Armonk, NY, USA) was
used for the data analyses. Probability (P) values of less than 0.05 were considered statistically significant.
study cohort consisted of 209 patients with ALL. There were 112 boys
and 97 girls; their median age was 4.8 (0.3-14.8) years. The median
duration of follow-up was 36 (16–36) months. As for risk, 114 patients
were classified as standard risk, 69 patients were high risk, and 26
patients were very high risk.
In terms of genotype, 124 patients (59.3%) had wild-type ITPA 94C>A, 73 patients (34.9%) showed heterozygous ITPA 94C>A (hetITPA), and 12 patients (5.7%) showed homozygous ITPA 94C>A (homITPA). The allele frequency of ITPA 94C>A
was 23%. The percentage of standard risk, high risk, and very high risk
in wild-type ITPA are 55.6%, 30.6%, and 13.7%, respectively. Those in
hetITPA are 53.4%, 37%, and 9.6%, respectively, while those in homITPA are 50%, 33.3%, and 16.7%, respectively.
Univariable analysis was undertaken for ITPA 94C>A, the 1-year mean dose of 6-MP, and side effects. No association was revealed between ITPA 94C>A and neutropenia during one year of maintenance therapy (P = 0.813; Table 1). The incidences of neutropenia during one year of maintenance therapy in patients harboring wild-type ITPA, hetITPA, and homITPA were 33.9%, 37.0%, and 41.7%, respectively. The association between ITPA 94C>A and transaminitis during one year of maintenance therapy (P = 0.063) is demonstrated in Table 2.
Pairwise comparisons using Bonferroni correction showed a significant
difference between patients with and without transaminitis in homITPA (12.2% vs. 3.8%; P = 0.036). The incidences of transaminitis during one year of maintenance therapy in patients harboring wild-type ITPA, hetITPA, and homITPA were 20.2%, 24.7%, and 50.0%, respectively.
||Table 1. Comparison of homozygous ITPA, heterozygous ITPA, and wildtype ITPA for cases with and without neutropenia in Thai pediatric acute lymphoblastic leukemia patients.
||Table 2. Comparison of homozygous ITPA, heterozygous ITPA, and wildtype ITPA for cases with and without transaminitis in Thai pediatric acute lymphoblastic leukemia patients.
The 1-year mean doses of 6-MP for wild-type ITPA, hetITPA, and homITPA were 39.11 ± 14.19 mg/m2, 39.40 ± 14.21 mg/m2, and 27.08 ± 12.58 mg/m2, respectively (P = 0.016). The Bonferroni post hoc test revealed a significantly lower 1-year mean dose of 6-MP for homITPA than for the wild-type and hetITPA groups (P < 0.05).
factors were significantly associated with transaminitis in univariable
binary logistic regression analysis of factors and transaminitis. They
were ITPA 94C>A and a 1-year mean dose of 6-MP. Patients with hetITPA and homITPA developed transaminitis more frequently than those harboring wild-type ITPA (hetITPA: OR, 1.296; 95% CI, 1.650–2.583; P = 0.461; homITPA: OR, 3.960; 95% CI, 1.177–13.328; P = 0.026). Higher doses of 6-MP were associated with no incidence of transaminitis (OR, 0.943; 95% CI, 0.919–0.968; P < 0.001). In multivariable binary logistic regression analysis, there was no statistically significant association between ITPA 94C>A and transaminitis.
there was a significant association between the 1-year mean dose of
6-MP and no incidence of transaminitis (adjusted OR = 0.958; 95% CI,
0.930–0.987; P = 0.005; Table 3).
- Table 3. Univariable and multivariable binary logistic regression analysis of factors and transaminitis.
patients' estimated 3-year OS rate was 95.1%, while the estimated
3-year event-free survival rate was 81.1%. The 3-year OS rates of
patients harboring wild-type ITPA, hetITPA, and homITPA were 90.8%, 92.8%, and 83.3%, respectively (P = 0.370; Figure 1). The 3-year event-free survival rates for wild-type ITPA, hetITPA, and homITPA were 76.9.1%, 87.7%, and 82.5%, respectively (P = 0.375; Figure 2)..
||Figure 1. Comparison of
3-year overall survival rates in Thai children with acute lymphoblastic
leukemia by inosine triphosphate pyrophosphatase (ITPA) polymorphism status.
||Figure 2. Comparison of
3-year event-free survival rates in Thai children with acute
lymphoblastic leukemia by inosine triphosphate pyrophosphatase (ITPA) polymorphism status
polymorphisms in thiopurine metabolism pathways increase the
accumulation of toxic metabolites. The increased accumulation might
account for the varied responses and toxicities among patients
receiving thiopurine drugs such as 6-MP. The polymorphisms vary among
ethnicities. ITPA is a catalytic enzyme that hydrolyzes inosine triphosphate, a toxic noncanonical nucleotide, to less toxic metabolites. The allele frequency of 2 well-known polymorphisms, TPMT and NUDT15, varies among ethnicities, with the latter prevalent in Asian populations. As for ITPA, its allele frequency in this study was 0.23, consistent with other studies on Asian populations.[4,12]
is among the most common complications for those treated with
thiopurine agents. The polymorphisms of the genes involved in
thiopurine metabolism account for the neutropenia of patients treated
with such agents. Currently, thiopurine doses are based on the TPMT and
NUDT15 polymorphisms. However, the recommended thiopurine doses for patients harboring ITPA polymorphisms need to be better established. Additionally, research findings on the association of ITPA
and neutropenia in patients treated with thiopurine are controversial.
Several studies have demonstrated a significant risk of neutropenia or
neutropenic fever with decreased doses of thiopurine[13,14] in patients with ITPA polymorphisms. Conversely, some studies did not identify any association between neutropenia and ITPA polymorphisms.[15,16] In the present study's cohort, the incidences of neutropenia in patients harboring ITPA polymorphisms were not significantly different from those with wild-type ITPA. The heterogeneity of the results might be due to the different 6-MP doses used by studies (range: 50-75 mg/m2) and pharmacogenomic variations related to different ethnic or racial groups.
Transaminitis during treatment with combined 6-MP and MTX is common in pediatric ALL.
The methylated metabolites of 6-MP account for hepatotoxicity.
Therefore, the genetic polymorphisms leading to excessive methylated
metabolites are considered plausible causes of hepatotoxicity. The correlation between ITPA polymorphisms and elevated transaminase is uncertain.[4,6,14] In the univariate analysis of the current investigation, the incidence of transaminitis in patients with homITPA was significantly higher than in those with hetITPA and wild-type ITPA.
Fortunately, this cohort did not exhibit severe transaminitis (> 20
times) or severe hepatic dysfunction, including coagulopathy. Careful
monitoring of liver function tests may be necessary for individuals
The dose of 6-MP in patients harboring homITPA was significantly lower than that for patients with hetITPA or wild-type ITPA in this cohort. This finding may signify a clinically meaningful effect of ITPA
polymorphisms, even though the incidences of neutropenia were not
significantly different. Furthermore, dose reduction of 6-MP did not
appear to worsen ALL treatment outcomes, given that the survival rates
of patients with ITPA and wild-type ITPA
were not significantly different. However, further study with a larger
cohort is warranted to determine whether dose reduction and ITPA do not affect such patients' relapse risk.
limitations need to be mentioned. First, as a retrospective study, some
data might be missing or incomplete. Second, the number of cases with
was low, which might impact the analysis of the outcomes of these
patients. Third, the data were restricted to a single center.
Consequently, the generalizability of our data and findings might be
Patients harboring ITPA, especially homozygous ITPA, seemed to have transaminitis and significantly required a greater dose reduction of 6-MP than patients with wild-type ITPA. Nevertheless, the neutrophil count did not appear to be affected. Further study is needed to elucidate the effects of ITPA polymorphisms on toxicity in patients with ALL treated with 6-MP.
authors are indebted to Mrs. Sam Ormond from the Clinical Research
Centre, Faculty of Medicine, Thammasat University, and Mr. David Park
for editorial assistance.
Conflict of Interest Declaration
authors declare no personal or professional conflicts of interest and
no financial support from companies that produce and/or distribute the
drugs, devices, or materials described in this report.
- Maloney KW, Devidas M, Wang C, Mattano LA,
Friedmann AM, Buckley P, et al. Outcome in children with standard-risk
B-cell acute lymphoblastic leukemia: Results of Children's Oncology
Group trial AALL0331. J Clin Oncol. 2020;38:602-12. https://doi.org/10.1200/JCO.19.01086 PMid:31825704 PMCid:PMC7030893
MV, Schwab M. Clinical pharmacogenetics implementation consortium
guideline for thiopurine dosing based on TPMT and NUDT15 genotypes:
2018 Update. Clin Pharmacol Ther. 2019;105:1095-1105. https://doi.org/10.1002/cpt.1304 PMid:30447069 PMCid:PMC6576267
J, Sripatanatadasakul P, Treesucon A, Glomglao W, Siraprapapat P,
Narkbunnam N, et al. Effect of NUDT15 on incidence of neutropenia in
children with acute lymphoblastic leukemia. Pediatr Int.
2019;61:754-758. https://doi.org/10.1111/ped.13905 PMid:31166660
X, Yin R, Sun G, Zhou Y, Yang C, Fang C, et al. Effects of TPMT,
NUDT15, and ITPA genetic variants on 6-mercaptopurine toxicity for
pediatric patients with acute lymphoblastic leukemia in Yunnan of
China. Front Pediatr. 2021;9:719803. https://doi.org/10.3389/fped.2021.719803 PMid:34660484 PMCid:PMC8518605
S, Marinaki AM, Arenas M, Fairbanks L, Shobowale-Bakre M, Rees DC, et
al. Genetic basis of inosine triphosphate pyrophosphohydrolase
deficiency. Hum Genet. 2002;111:360-7. https://doi.org/10.1007/s00439-002-0798-z PMid:12384777
A, Moulsma M, Gustin M-P, Lachaux A, Boulieu R. ITPA activity in
children treated by azathioprine: Relationship to the occurrence of
adverse drug reactions and inflammatory response. Basic Clin Pharmacol
Toxicol. 2018;122:588-595. https://doi.org/10.1111/bcpt.12958 PMid:29327413
M, Duley J, Sumi S, Sanderson J, Marinaki A. The ITPA c.94C>A and
g.IVS2+21A>C sequence variants contribute to missplicing of the ITPA
gene. Biochim Biophys Acta. 2007;1772:96-102. https://doi.org/10.1016/j.bbadis.2006.10.006 PMid:17113761
MA, Arthur DC, Camitta BM, et al. Uniform approach to risk
classification and treatment assignment for children with acute
lymphoblastic leukemia. J Clin Oncol. 1996;14 1:18-24. https://doi.org/10.1200/JCO.19220.127.116.11 PMid:8558195
Thai Pediatric Oncology Group. Treatment protocol for acute
lymphoblastic leukemia. In: National protocol for treatment of
childhood cancers, 1st ed. Bangkok: M Print Cooperation. 2016:1-83
MA. Inosine triphosphate pyrophosphatase (ITPase): Functions,
mutations, polymorphisms and its impact on cancer therapies. Cells.
2022;11:384. https://doi.org/10.3390/cells11030384 PMid:35159194 PMCid:PMC8833965
JJ, Whirl-Carrillo M, Scott SA. Pharmacogene variation consortium gene
introduction: NUDT15. Clin Pharmacol Ther. 2019;105:1091-4. https://doi.org/10.1002/cpt.1268 PMid:30515762 PMCid:PMC6465081
T, Wiwattanakul S, Tiyasirichokchai R, Prommas S, Sukprasong R, Koomdee
N, et al. TPMT*3C as a predictor of 6-mercaptopurine-induced
myelotoxicity in Thai children with acute lymphoblastic leukemia. J
Pers Med. 2021;11. https://doi.org/10.3390/jpm11080783 PMid:34442427 PMCid:PMC8400562
B, Muwakkit S, Zamani F, Ghaderi E, Mohammadi E, Zgheib NK. ITPA, TPMT,
and NUDT15 genetic polymorphisms predict 6-mercaptopurine toxicity in
middle eastern children with acute lymphoblastic leukemia. Front
Pharmacol. 2019;10. https://doi.org/10.3389/fphar.2019.00916 PMid:31507415 PMCid:PMC6718715
F, Mortazavi Y, Alavi S, Khalili M, Ramazani A. Frequency of ITPA gene
polymorphisms in Iranian patients with acute lymphoblastic leukemia and
prediction of its myelosuppressive effects. Leuk Res.
2015;39:1048-1054. https://doi.org/10.1016/j.leukres.2015.06.016 PMid:26242828
M, Nilsson A, Kahlin AZ, Broliden K, Myrberg IH, Appell ML, et al. The
role of TPMT, ITPA, and NUDT15 variants during mercaptopurine treatment
of Swedish pediatric patients with acute lymphoblastic leukemia. J
Pediatr. 2020;216:150-7.e1. https://doi.org/10.1016/j.jpeds.2019.09.024 PMid:31635813
B, Monsereenusorn C. ITPA:c.94C>A and NUDT15:c.415C>T
polymorphisms and Their relation to mercaptopurine-related
myelotoxicity in childhood leukemia in Thailand. Appl Clin Genet.
2021;14:341-51. https://doi.org/10.2147/TACG.S318912 PMid:34349542 PMCid:PMC8326781
MS, Nygaard U, Rosthøj S, Sørensen D, Nersting J, Vettenranta K, et al.
Hepatotoxicity during maintenance therapy and prognosis in children
with acute lymphoblastic leukemia. J Pediatr Hematol Oncol. 2017;39. https://doi.org/10.1097/MPH.0000000000000733 PMid:28060115
LM, Zanetti RC, Parekh DS, Warwick AB, Lieuw K. A Retrospective review
of mercaptopurine metabolism reveals high rate of patients with
suboptimal metabolites successfully corrected with allopurinol. J
Pediatr Hematol Oncol. 2021;43:e1003-e9. https://doi.org/10.1097/MPH.0000000000001939 PMid:32925411