Keywords
6-MP toxicity - chemotherapy delay - consolidation phase -
NUDT polymorphism -
TPMT polymorphism
Introduction
Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy with a
cure rate of more than 90%. The management of ALL involves a structured treatment
regimen, typically divided into several phases, which include induction, consolidation,
interim maintenance, delayed intensification, and maintenance. Each phase plays a
crucial role in achieving remission, preventing relapse, and ensuring long-term survival.[1]
[2] The induction phase aims to rapidly reduce the leukemia burden and achieve complete
remission. Once remission is achieved, the consolidation phase (CP) follows, which
is designed to eliminate any remaining leukemia cells. This phase is critical in reducing
the risk of relapse and solidifying the gains made during induction therapy. The goal
is to further intensify the treatment to eradicate residual disease, preventing leukemia
cells from surviving and proliferating during remission.[1]
[3]
Consolidation chemotherapy typically involves a combination of drugs, which include
6-mercaptopurine (6-MP), cytarabine, intrathecal methotrexate, cyclophosphamide, and
L-asparaginase if the patient falls in the high-risk category.[2]
[4] 6-MP is often administered over several weeks to months, depending on the risk stratification.
It is a thiopurine drug, which is metabolized by enzymes encoded by genes such as
thiopurine S-methyltransferase (TPMT) and nucleoside diphosphate (NUDT15). Genetic polymorphisms in these genes have been identified as significant factors
influencing drug metabolism, efficacy, and toxicity profiles. Clinical Pharmacogenetics
Implementation Consortium (CPIC) guidelines elaborate 6-MP dosage regimen, frequency
of administration, and dose reduction based on pharmacogenomic impact.[5] The variability in response to thiopurine treatment can lead to severe side effects,
especially myelotoxicity, which is dose-limiting with a narrow therapeutic index.
This side effect may delay chemotherapy regimens and impact patient morbidity, prolong
hospitalization, and impact overall morbidity.[6]
[7]
These variations can result in adverse reactions or insufficient therapeutic response,
which in turn may necessitate dose adjustments. Understanding the impact of chemotherapy
during the CP, alongside genetic factors that affect drug metabolism, is essential
for optimizing treatment protocols. TPMT polymorphisms are more common worldwide and have been established as the main factor
affecting 6-MP metabolism. NUDT15 polymorphisms are the most common in East Asian countries, followed by South Asian
countries.[8]
[9]
[10] There are a limited number of studies from India in this regard. Two studies from
Vellore and Chandigarh have reported 14 and 9.5% prevalence of NUDT15 polymorphism in children with ALL, respectively.[6]
[11]
[12] The effect of these mutations on the delay in the consolidation therapy and treatment
outcomes has not been studied. Moreover, there are no studies from North Karnataka
regarding these polymorphisms in children with ALL and the effect of the polymorphism
on the CP delay in India.
In this study, we aim to study the prevalence of TPMT and NUDT15 gene polymorphisms and assess their impact on chemotherapy delay and morbidities
such as febrile neutropenia (FN) during the CP of treatment in patients with ALL.
This study is an attempt to understand the genetic factors that influence thiopurine
metabolism, which will help optimize therapeutic strategies, minimize adverse effects,
and improvisation of chemotherapy regimens.
Materials and Methods
Objectives
The primary objective of the study was to study the prevalence of TPMT and NUDT15 gene polymorphisms, and the secondary objective was to study the chemotherapy delay
and toxicities due to TPMT and NUDT15 polymorphisms during CP of chemotherapy.
Study Design and Setting
This was a longitudinal study conducted on 106 children aged 1 to 18 years with newly
diagnosed ALL at the pediatric hematology oncology unit in a tertiary care hospital.
All these children were treated as per the Indian Childhood Collaborative Leukemia
Group protocol. The children who had received allopurinol after the induction phase
of chemotherapy and those who did not give consent to participate in the study were
excluded.
The epidemiological and clinical data of the patients were documented in a standardized
proforma. All the patients who were eligible for the study underwent upfront TPMT and NUDT15 variant detection at the time of diagnosis.
Three mL of blood was collected in EDTA vacutainers, following which DNA was extracted
using standard methods.
Genomic DNA was amplified, including regions of the TPMT and NUDT15 genes. Bidirectional sequencing was carried out using 3500/3730 XL Genetic Analyzer
(Applied Biosystems) to detect common variant alleles in these genes. Dose of 6-MP
was noted for the entire CP of chemotherapy. Furthermore, the number of FN, other
cytopenias, the number of days of delay in CP, the number of episodes, and the duration
of FN were noted. They were followed up until the end of the CP of chemotherapy. However,
further research is underway in which patients will be followed up for up to 5 years
postmaintenance chemotherapy, resulting in a total timeline of 8 years.
Expected Outcomes
The primary outcome is the prevalence of TPMT and NUDT15 gene polymorphisms.
The secondary outcome is chemotherapy delay and toxicities due to TPMT and NUDT15 polymorphisms during the CP of chemotherapy.
Ethics
The study was approved by the ethics committee (KAHER/EC/22-23/272) dated November
21, 2022. We obtained written informed consent from the parents of the patients in
their own vernacular language. All procedures involving human participants in this
study were conducted in accordance with the ethical standards of the institutional
and/or national research committee and with the 1964 Declaration of Helsinki and its
later amendments or comparable ethical standards.
Statistical Analysis
All quantitative variables were checked for normal distribution within each category
of explanatory variable by using Shapiro–Wilk's test. The p-value of >0.05 was considered as anormal distribution. For nonnormally distributed
quantitative parameters, the median values were compared between study groups using
Mann–Whitney's U test (two groups). The p-value of < 0.05 was considered statistically significant. IBM SPSS version 22 (IBM
Corp. Released 2013. IBM SPSS Statistics for Windows, Version 22.0. Armonk, New York,
United States: IBM Corp) was used for statistical analysis.
Results
A total of 128 patients were diagnosed with ALL over a period of 2.5 years, out of
which 0.17% (n = 22) of patients did not give consent to participate, so a total of 106 patients
were enrolled in our study. Among them, 60.4% (n = 64) were males and 39.6% (n = 42) were females ([Fig. 1]). The male-to-female ratio was 1.52. The mean age of the study population (n = 106) was 7.19 ± 4.08 years. A total of 87.7% (n = 93) of patients had pre-B ALL and 12.3% (n = 13) had T ALL. Regarding the risk stratification in our study population, 38.68%
(n = 41) of patients belonged to the high-risk category, 58.49% (n = 62) were in the intermediate-risk category, and 2.83% (n = 3) belonged to the standard-risk category. Out of the 106 patients with ALL, 30
(28.3%) were found to have either NUDT15 or TPMT polymorphisms. Among these patients, 24.5% (n = 26) and 3.77% (n = 4) had NUDT15 and TPMT polymorphisms, respectively ([Table 1]). The mean duration of delay in consolidation chemotherapy was 13.5 days for patients
with polymorphisms, and these patients experienced a higher number of FN episodes
compared with those without polymorphisms. This difference was statistically significant,
with a p-value of 0.002 ([Table 2]).
Fig. 1 Bar chart of gender in the study population (n = 106).
Table 1
Descriptive analysis of polymorphism in the study population (n = 106)
|
Polymorphism
|
Frequency
|
Percentage
|
|
Present
|
30
|
28.3%
|
|
Absent
|
76
|
71.6%
|
|
Polymorphism type
|
|
NUDT
|
26
|
24.5%
|
|
TPMT
|
4
|
3.7%
|
|
NUDT polymorphism
|
|
Homozygous
|
1
|
0.9%
|
|
Heterozygous
|
25
|
23.5%
|
|
TPMT polymorphism
|
|
Homozygous
|
1
|
0.9%
|
|
Heterozygous
|
3
|
2.8%
|
Abbreviations: NUDT, nucleoside diphosphate; TPMT, thiopurine S-methyltransferase.
Table 2
Comparison of median of number of FN in consolidation between polymorphism (n = 106), polymorphism type (n = 30), NUDT polymorphism (n = 26), and TPMT polymorphism (n = 4)
|
Parameter
|
Polymorphism (IQR)
|
p-Value
|
|
Present (
n
= 30)
|
Absent (
n
= 76)
|
|
Number of FN in consolidation
|
2 (1–3)
|
1 (1–2)
|
0.007
|
|
Delay in consolidation
|
13.50 (4–16.25)
|
7 (2–12)
|
0.002
|
|
Parameter
|
Polymorphism type (IQR)
|
p
-Value
|
|
NUDT (
n
= 26)
|
TPMT (
n
= 4)
|
|
Number of FN in consolidation
|
2 (1–3)
|
2 (1.25–11)
|
0.975
|
|
Delay in consolidation
|
13.50 (4–16.25)
|
10.50 (3.5–18.25)
|
0.976
|
|
Parameter
|
NUDT polymorphism (IQR)
|
p
-Value
|
|
Homozygous (
n
= 1)
|
Heterozygous (
n
= 25)
|
|
Number of FN in consolidation
|
3 (3–3)
|
2 (1–3)
|
0.692
|
|
Delay in consolidation
|
43 (43–43)
|
13 (4–16)
|
0.077
|
|
Parameter
|
TPMT polymorphism (IQR)
|
p-Value
|
|
Homozygous (
n
= 1)
|
Heterozygous (
n
= 3)
|
|
Number of FN in consolidation
|
1 (1–1)
|
2 (2–2)
|
0.500
|
|
Delay in consolidation
|
5 (5–5)
|
16 (3–16)
|
1.000
|
Abbreviations: FN, febrile neutropenia; IQR, interquartile range; NUDT, nucleoside
diphosphate; TPMT, thiopurine S-methyltransferase.
Discussion
CP is critical in reducing the risk of relapse, but CP chemotherapy in this phase
is often interrupted and delayed due to 6-MP-related toxicity. 6-MP is an important
part of the consolidation and delayed intensification phase of chemotherapy, and is
the backbone of maintenance phase chemotherapy.[2]
[3] Hence, 6-MP-related toxicities have a significant impact on treatment outcome. There
are a few studies from Western world and a few from India, which highlight the impact
of 6-MP-related toxicities due to NUDT and TPMT polymorphisms affecting 6-MP metabolism during the maintenance phase, but there is
a lack of research on these polymorphisms during CP.[7]
[8] Hence, we decided to study TPMT and NUDT-15 polymorphism prevalence and assess their impact on chemotherapy delay and morbidities
such as FN during the CP.
6-MP belongs to the thiopurine group of drugs, with a typical dosage in CP being 50
to 60 mg/m2 per day. The adverse effects of 6-MP include alopecia, hepatotoxicity, pancreatitis,
and myelosuppression. Myelosuppression is a dose-limiting toxicity with a narrow therapeutic
index.[13]
[14] Variation in the degree of myelosuppression is observed between individual children,
especially from different ethnicities. This variation is mostly attributed to genetic
polymorphisms in enzymes involved in the metabolism of 6-MP. It is well established
that TPMT and NUDT15 polymorphisms affect 6-MP dosing and toxicity. Other genetic variants in the ITPA and MRP4 genes may affect 6-MP metabolism and are common in the Asian population.[9]
[11]
[12]
[14] However, their effect on 6-MP-induced myelotoxicity is controversial and requires
further research.
TPMT enzyme converts 6-MP to its inactive metabolite 6-methylmercaptopurine nucleotide.
TPMT genetic polymorphisms, which reduce enzymatic activity, lead to increased levels
of active metabolite of 6-MP, that is, 6-thioguanine (6-TG), which is responsible
for myelosuppression. TPMT 1* is a wild-type allele. TPMT 2*, 3A*, 3B*, and 3C* alleles
account for 95% of significant polymorphisms. These variants are responsible for more
frequent cytopenia, FN, and 6-MP interruption. Hence, dose reduction is advised in
these patients ([Fig. 2]).[6]
Fig. 2 Metabolism of 6-MP. GMPS, GMP-synthase; HPRT, hypoxanthine phosphoribosyl transferase;
IMPDH, inosine-5-monophosphate; ITPA, inosine triphosphate pyrophosphatase; 6-MeTG,
6-methyl thioguanine; 6-MMP, 6-methylmercaptopurine; 6-MP, 6-mercaptopurine; NUDT15,
nucleoside diphosphate–linked moiety X-type motif 15; 6-TGDP, 6-thioguanine diphosphate;
6-TGMP, 6-thioguanine monophosphate; 6-TGNs, 6-thioguanine nucleotides; 6-TGTP, 6-thioguanine
triphosphate; 6-TIDP, 6-thioinosine 5-diphosphate; 6-TIMP, thioinosine 5-monophosphate;
TPMT, thiopurine S-methyltransferase; 6-TU, 6-thiouric acid; XO, xanthine oxidase.
(Adapted from Singh et al.[6])
Guidelines developed by CPIC recommend a normal dose for normal metabolizers. For
intermediate metabolizers, a 30 to 70% reduction is recommended for 6-MP and a 30
to 50% reduction for 6-TG. Poor metabolizers receiving 6-MP or 6-TG should receive
a 90% reduction in dose with drug administration three times per week in order to
avoid adverse drug reactions (ADRs) ([Table 3]). Pre-emptive patient testing is highly recommended either to avoid ADRs in case
of malignant disease or to reduce the time needed for upward titration of drug dosage.
Recent in-depth research has focused on interindividual differences in drug-metabolizing
enzymes to adjust drug dosage and therapy.[15]
Table 3
CPIC guidelines on 6-MP dosage regimen
|
TPMT phenotype/genotype
|
Dosing recommendation 6-MP
|
Dosing recommendation 6-TG
|
|
Normal metabolizer (two functional alleles)
|
Start with normal dose
|
Start with normal dose
|
|
Intermediate metabolizer (one functional alleles)
|
Start with 30–70% reduced dose
|
Start with 90% reduced dose, thrice weekly
|
|
Poor metabolizer (no functional alleles)
|
Start with 90% reduced dose, thrice weekly
|
Start with 90% reduced dose, thrice weekly
|
Abbreviations: CPIC, Clinical Pharmacogenetics Implementation Consortium; 6-MP, 6-mercaptopurine;
6-TG, 6-thioguanine; TPMT, thiopurine S-methyltransferase.
NUDT15 polymorphisms are also known to affect 6-MP dosing and toxicity. NUDT15 dephosphorylates cytotoxic metabolite thioguanine triphosphate into nontoxic monophosphate.
Genetic polymorphisms with low NUDT15 enzyme activity lead to the accumulation of active metabolites, leading to myelosuppression.
It is well known that TPMT polymorphisms are more common in Caucasians as compared with Asians.[8]
[9]
[11] In the Indian population, studies done on the normal population and children with
ALL have reported 1 to 4.5% frequency of TPMT polymorphisms associated with low to intermediate enzyme activity.[5]
[9]
[11] Many studies from Western countries have shown that TPMT polymorphisms are associated with low enzyme activity resulting in increased risk
of cytopenia, FN, and 6-MP interruption. Homozygous variants need a significant (10%)
reduction in the dose of 6-MP. Similarly, Indian studies have also shown the need
for dose reduction in the TPMT polymorphism group.
Interestingly, TPMT polymorphisms protect against hepatotoxicity and mucositis.[11]
[12]
[14]
Apart from TPMT polymorphisms, NUDT15c.415C>T(p.R139C) variant was found to be associated with 6-MP intolerance in Asian children, on a
genome-wide association study in the Children's Oncology Group COG cohort. NUDT15*2(p.V18_V19 insGV and c.415C>T), *3(C.415C>T), and *9 (c.50 dd GAG TCG) polymorphisms are associated with loss-of-function NUDT15 enzyme activity. NUDT15 variants are common in Asian populations and Hispanic populations; higher prevalence
is seen in East Asian countries, followed by South Asian countries. On the contrary,
NUDT15 polymorphisms are rare in the Caucasian population. NUDT*3 is the commonest variant seen in the Asian population.[6]
[7]
[14]
A study from North India reported a NUDT15 polymorphism prevalence of 9.5% in children, all of whom were heterozygous, and a
TPMT polymorphism prevalence of 3.1% during the maintenance phase of chemotherapy.[11] Additionally, a study from Vellore showed a NUDT15 polymorphism prevalence of 14%. This is notably lower than the 24.5% prevalence for
NUDT15 and 3.7% for TPMT observed in our study.[12] The incidence of FN episodes was higher in children with polymorphisms (6.7%) compared
with those without (1.3%) the polymorphism. Further, the mean delay in the CP was
found to be 13.5 days, which was significantly longer in children with polymorphisms
compared with those without it. However, there are no studies regarding the impact
of these mutations on the treatment delay and toxicities during CP.
The strengths of our study include the fact that there are very few studies on TPMT and NUDT15 polymorphisms in India, and those that do exist primarily focus on the maintenance
phase. Our study is the first from India to be conducted during the CP. This study
revealed a very high prevalence rate of the NUDT15 polymorphism (24.5%), which significantly impacted treatment duration and led to
increased toxicities. Therefore, our study is of great importance, as it emphasizes
the need for upfront screening for these polymorphisms and subsequent dose reduction
of 6-MP according to CPIC guidelines. This approach could help reduce toxicities,
minimize delays in the CP, and ultimately lower the relapse rate.
Regarding future research, the impact of TPMT and NUDT15 genetic polymorphisms on 6-MP dosing, toxicity, event-free survival, and relapse
risk in children with ALL warrants further exploration. The limitations of our study
include its single-center design and small sample size, which means that additional
research is needed to establish more accurate prevalence rates during the CP of chemotherapy.
We also could not evaluate single-nucleotide polymorphisms related to ITPA polymorphism, which accounts for the gray areas that need attention for further studies.
Conclusion
The prevalence of NUDT15 and TPMT mutations was found to be 24.5 and 3.8%, respectively. These polymorphisms were also
associated with a significant delay in consolidation chemotherapy, and these patients
experienced a higher number of FN episodes compared with those without polymorphisms.
We propose that TPMT and NUDT polymorphism analysis should be done upfront at diagnosis, so that consolidation
delays can be minimized and toxicities can be reduced, which may improve the overall
outcome.
