Haematotoxicity in Maintenance Therapy of Children With Acute Lymphoblastic Leukemia: Focus on Genotyping and Phenotyping of Mercaptopurine
Conditions: Mercaptopurine Adverse Reaction, Leukemia, Lymphoblastic
Sponsor: Indonesia University

 
 
 
 
 
 
 
 
 
 
STUDY PROTOCOL 
Haematotoxicity in Maintenance Therapy of Children With Acute 
Lymphoblastic Leukemia: Focus on Genotyping and Phenotyping of 
Mercaptopurine 
 
 
 
 
 
Dewi Selvina Rosdiana, dr., M.Kes 
                                            Prof. Dr. dr. Rianto Setiabudy, Sp.FK 
Prof. dr. Djajadiman Gatot Sp.A (K) 
Dr. Melva Louisa, S.Si. M. Biomed 
 
 
 
 
 
 
 
 
 
Faculty of Medicine, University of Indonesia 
JAKARTA 2017 
 
 
 

Background 
 
Leukemia is the most common malignant disease in children, of 30% of children under 15 
years diagnosed with cancers caused by leukemia. In children with leukemia, 80% have 
acute lymphoblastic leukemia (ALL). ALL The highest cause of death in children with cancer.  
 
The success rate of LLA patients reaches a remission of more than 90%, but 30-40% of 
them relapse in the maintenance phase.5 In developed countries, the last few years, the 
incidence of relapse to 11% .6 Meanwhile, the relapse rate in RSCM is still 28 , 7% .7 To 
achieve long-term remission, LLA patients require maintenance phase therapy for 2-3 years 
with mercaptopurin daily and low-dose methotrexate once a week.8 Drug toxicity during the 
maintenance phase is one of the main concerns not only because Can be life-threatening, 
but is a major cause of treatment discontinuation, which can increase the risk of relapse 
 
During the maintenance phase, patients received 6-mercaptopurine therapy orally daily and 
low dose methotrexate week sekali. Mercaptopurine (6MP) is the main drug in this phase. 
The efficacy and toxicity of 6MP affected by enzyme activity TPMT.21,22 TPMT enzyme 
activity is influenced by the presence of genetic polymorphisms autosomal co-dominant 
populations variability etnik.23-25 existence of the gene encoding the enzyme 
pemetabolisme medications will cause drug response differences between individuals, 
Which may affect treatment outcomes.26 To date there are 30 known variations of TPMT 
alleles.27 Wild type alleles, TPMT * 1, show full enzyme activity. TPMT * 2, * 3A, * 3B and * 
3C alleles are polymorphic alleles that are found in caucasian populations. All four variations 
of the allele showed decreased TPMT enzyme activity.23-25,27 Based on population 
studies, the distribution of TPMT enzyme activity in erythrocytes was trimodal; The majority 
of the population (89-94%) had a homozygous wild type allele exhibiting high TPMT activity, 
6-11% had heterozygous mutant alleles with moderate TPMT activity, and 0.3% had 
homozygous mutant alleles showing low or undetectable TPMT activity. 
 
Variations in TPMT enzyme activity due to polymorphism are the most important factors 
affecting intracellular 6TGN accumulation after 6MP administration.32 TPMT activity is 
inversely proportional to 6TGN concentrations in erythrocytes in LLA child patients.28,33 In 
homozygous wild type patients, their erythrocyte 6TGN levels are lower Compared with 
heterozygote mutant patients.34,35 Homozygous mutant patients had the highest 6TGN 
levels.35 Compared with homozygous wild type patients, heterozygous mutant patients 
required a lower 6MP dose, 30-50% of the standard dose, 22,31,36 but at risk Experienced 
severe myelosuppression.37 In homozygous mutant patients, the 6MP dose was reduced to 
10-15 times less than the recommended dose, 28,37,38 because of the excessive 
accumulation of 6TGN in hematopoietic tissue, which may have life-threatening side-effects 
of myelosuppression. 
 
The frequency and pattern of TPMT mutant alleles differ among different ethnic populations. 
In the American-Caucasian and African-American populations, all three mutant TPMT * 2, * 
3A, * 3C mutants are found, but the most mutant alleles in the US-Caucasian allele are 
TPMT * 3A (3.2%), and in African-Americans TPMT * 3C (2.4%). In the Southwest Asian 
(India and Pakistan), only 1% of the mutant alleles are TPMT * 3A. In the Southeast Asian 
population, only 1% TPMT * 3C mutant alleles were found. Similarly in populations in 
Thailand, Japan, and China, only TPMT * 3C mutant alleles are found at 1-4.74%. 
23,24,27,31 The frequency and pattern of TPMT mutant alleles in the population of children 
with LLA in Indonesia is currently unknown 
 
 
 

Study Objectives: 
1. Knowing the distribution of TPMT * 3A and * 3C genotypes, 6TGN / 6meMP erythrocyte 
level, risk stratification, nutritional status, and albumin levels in LLA child patients in 
Indonesia. 
2. Analyzing the association of hematotoxicity with TPMT * 3A and * 3C genotypes, 
erythrocyte 6TGN / 6meMP ratio, risk stratification, nutritional status, and albumin levels in 
the LLA child population in Indonesia. 
3. Analyze the genotype associations of TPMT * 3A and * 3C with 6TGN / 6meMP 
erythrocyte ratio in LLA child. 
 
Study Design:  
This study used an observational, cross sectional study to obtain data on patient 
demographics, nutritional status, albumin levels, TPMT genotype, and 6TGN / 6meMP levels 
in LLA children. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Statistical Analysis Plan (SAP) 
Data is presented in the form of narration, tables or graphs. Subject characteristics data, 
TPMT polymorphism frequency, hematology effect, TGN and 6-meMP concentration, and 
TGN/6-meMP ratio in erythrocytes are presented descriptively, in mean (standard deviation), 
or median value (range of values) And in percentages. 
 
To assess the relationship of TPMT polymorphism with 6TGN/6meMP ratio in erythrocytes 
using unpaired t test (normal distribution) or Mann Withney (abnormal distribution): 
 
 
Rasio TGN/6meMP (mean) 
p-
Value 
Genotype 
Wild type 
 
 
Mutant 
 
 
 
To assess the association of hematologic toxicity with factors that may affect it: 
 
Variabels 
Hematotoxicity   
p-
value 
OR 95% CI 
+ 
(Grade 1−4) 
− 
(Grade 0) 
Genotypea 
- Wild type 
 
 
 
 
 
- Mutan  
 
 
 
 
 
Phenotypeb 
6TGN/6meMP 
Ratio   
 
 
 
 
 
Risk 
Stratificationa  
- High risk 
- Standard risk 
 
 
 
 
 
 
Nutritional 
Statusa 
-  poor 
 
 
 
 
 
- normal-over 
weight 
 
 
 
 
 
Albumin 
levelsa 
- Hypo 
 
 
 
 
 
- Normal 
 
 
 
 
OR= odd ratio; CI= confidents interval 
a Statistical method: X2 test or Fischer test 
b Statistical method: unpaired t test (normal distribution) or Mann Withney (abnormal distribution): 
 
 
 
 
 
 
 
 

Informed Consent 
We, a team of researchers at the Pharmacology and Therapeutic Section of the Faculty of 
Medicine, University of Indonesia, Jakarta, are conducting a study to determine the 
incidence of blood toxicity in pediatric patients with acute lymphoblastic leukemia (LLA) 
currently in maintenance phase therapy, and associated with mercaptopurin drug Routine 
given by the doctor during maintenance phase therapy). This study does not increase or 
decrease the drugs given to you. Abroad, the dosage of mercaptopurine given is adjusted to 
the patient's genotype, to avoid the incidence of blood toxicity in the patient. In Indonesia, 
this dose adjustment has not been performed, as there has been no research on genotypic 
data in LLA child patients in Indonesia. This study will include one hundred LLA patients 
aged 1-18 years. You / your child have LLA and are therefore asked to participate in this 
research. 
If you are willing to participate, during routine blood tests, your doctor will take your blood 
sample as much as one tablespoon of blood vessels in your arm by using a syringe. Blood-
collection of arm arms usually causes only mild pain, but sometimes infections and / or 
swelling and blue, which will heal after a few days of treatment. Blood sampling for this study 
was performed only once. You are exempted from the routine blood examination fee, and 
you get a replacement transport of one hundred thousand rupiah (Rp 100.000, -). In addition, 
your doctor will take data from your medical record. This study is of no direct use to you, but 
the results are useful for determining whether or not genotypes are needed to adjust the 
mercaptopurin dosage to avoid the incidence of blood toxicity in LLA patients. You are free 
to refuse to participate in this research without any adverse effects. If you have decided to 
participate, then you are also free to resign at any time without causing any change in the 
quality of service from your doctor. 
All of this research data will be treated in secret so as not to allow others to connect with 
you. You are given the opportunity to ask all the things that are not clear with regard to this 
research. If at any time you need an explanation, you can contact Doctor Dewi Selvina 
Rosdiana in Pharmacology and trapeutic Section of Faculty of Medicine University of 
Indonesia, Jl. Salemba Raya no.6, Central Jakarta, no phone 089517704616. 
 
 
 
 
 

Approval Form 
 
All of the above explanations have been submitted to me and all my questions have been 
answered by the doctor. I understand that if I still need an explanation, I will get an answer 
from Dr. Dewi Selvina Rosdiana. By signing this form, I agree to participate in this research. 
 
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