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Impact of the Multidrug resistance 1 gene polymorphisms on the outcome of therapy in childhood acute leukemia in Duhok province/Iraq

1 Laboratory Department, Azadi Teaching Hospital, Duhok, Iraq
2 Department of Pathology, College of Medicine, University of Duhok, Duhok, Iraq

Date of Submission09-Mar-2023
Date of Acceptance23-Apr-2023
Date of Web Publication17-May-2023

Correspondence Address:
Adil Abozaid Eissa,
Department of Pathology, College of Medicine, University of Duhok, Duhok
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijh.ijh_27_23


BACKGROUND: The multidrug resistance (MDR1) gene's polymorphism affects the metabolism and pharmacokinetics of chemotherapeutic agents and smooth drug resistance formation in malignancies and the current study aimed to evaluate the probable impact of MDR-1 gene polymorphisms (C3435T, G2677A/T) on the clinical outcome of childhood acute lymphoblastic leukemia (ALL) in Duhok/Iraq.
MATERIALS AND METHODS: All enrolled patients were assessed for MDR-1 (C3435T, G2677A/T) single-nucleotide polymorphisms by means of a polymerase chain reaction followed by enzyme digestion (RFLP-PCR) assay. Response to chemotherapy was assessed by flow cytometry.
RESULTS: Sixty-two patients with ALL enrolled in the current study, with a median age of 7.0 years. The main clinical features at presentations were nonspecific in the form of fatigue and loss of energy. Majority of leukemia were of B-cell origin 88.71%. Majority of patients had low hemoglobin, low platelets, and high white blood cell count mainly of blasts at presentation. Sixty-one percent of patients achieved negative minimal residual disease (MRD) after 1–2 courses of chemotherapy. The alleles frequencies at position of 2677 nucleotide were G: 24/124 (19.35%); A: 52/124 (41.94%); T: 48/124 (38.71%); and for the C3435T, frequency of C and T alleles was 54.84%, 45.16%, respectively. Achievement of negative MRD following 1–2 courses of induction, appeared significantly correlated with the age of patients at presentation. All other parameters including, sex, hematological parameters at presentation; studied polymorphism in the MDR-1 gene; and subtype of ALL were not associated significantly with MRD achievement.
CONCLUSION: Polymorphic variation in MDR-1 gene, in comparison to solid tumors and chronic hematopoietic malignancies, does not have an impact on MRD achievement in ALL.

Keywords: Acute lymphoblastic leukemia, chemotherapeutic resistance, multidrug resistance-1, minimal residual disease

How to cite this URL:
Bathyon SR, Eissa AA. Impact of the Multidrug resistance 1 gene polymorphisms on the outcome of therapy in childhood acute leukemia in Duhok province/Iraq. Iraqi J Hematol [Epub ahead of print] [cited 2023 May 30]. Available from: https://www.ijhonline.org/preprintarticle.asp?id=377213

  Introduction Top

Globally, acute lymphoblastic leukemia (ALL) is one of the most common childhood cancers.[1] Immature lymphoid precursors (lymphoblasts) proliferate continuously in the bone marrow and peripheral circulation, which is one of its hallmarks.[2] Around 80% of patients who follow the current chemotherapy protocols achieve complete remission; however, up to 50% of children experience recurrence, resulting in an ultimate cure rate of 25%–40%.[3] Thus a significant percentage of patients cannot be cured permanently, which is mostly due to the emergence of medication resistance.[4],[5]

P-glycoprotein (P-gp), a transmembrane phosphorylated and glycosylated protein, serves as a membrane efflux pump, is programmed and encrypted by the human (ATP-binding cassette, subfamily B) ABCB1 gene, also known as multidrug resistance (MDR1), and transport several amphipathic molecules through lipid membranes. The ABCB1 gene is found on chromosome 7q21, has been linked to drug resistance and participate in the membrane transport and/or metabolism of a number of chemotherapeutic drugs used to treat malignancies.[6]

Many tissues including the small intestine, colon, small biliary ductules, and proximal tubules of the kidneys are just a few examples of the normal tissues in which the P-gp is produced in addition to tumor cells.[7] Conversely, it has been discovered that MDR-1 expression is up-regulated in a number of chemo-resistant tumor types, including colon, hepatocellular, renal cell, adrenocortical, and hematopoietic malignancies.[8] This suggests that MDR-1 is a poor prognosis signal.

Single-nucleotide polymorphisms that occur every 100–300 bp, are the most frequently inherited genetic variation in humans.[9] The diversity of enzymes functions and, in turn, survival following chemotherapy, may be significantly affected by variation in genes shared in drug metabolism and transportation.[10] The first MDR-l gene mutation linked with a changed pattern of MDR to chemotherapeutic medicines was documented by Choi et al. in 1988.[11] The current study aimed at documenting the effect of such polymorphism upon the achievement of negative minimal residual disease (MRD) following 1–2 courses of induction among children with ALL from Duhok, Iraq.

  Materials and Methods Top

The present work represents a cross-sectional study. Sixty-two children aged <16 years old presented with ALL and attended Jin pediatric hospital in Duhok, Iraq were included. All patients were previously diagnosed by blood film morphology, bone marrow examination, immunophenotyping using multicolor flow cytometry, and cytogenetic studies.[12] Individuals above 16 years of age, those with recurrent cytogenetic anomalies, especially with BCR-ABL transcript, and those with acute myeloid leukemia were excluded from the current study.

At first, the study was approved by the appropriate scientific and ethical committee at Duhok College of Health Science and the Duhok Directorate of Health. The study was explained to the children and their parents and informed permission was taken from all parents. Supportive information and demographic data were collected from all enrolled individuals or their parents. For already diagnosed cases, the results of immunophenotyping and MRD were collected from patients' records, and for newly diagnosed cases, data were collected from flow cytometry unit at Azadi Teaching Hospital.

Two milliliters of blood collected from each patient, then DNA extraction done using a modified salting out method assumed by Iranian scientists, Iranpur-Mubarakeh and Esmailizadeh which produce an ultrapure high quantity DNA.[13],[14] Then genotyping was performed using PCR–RFLP (Restriction fragment length polymorphism) method. The specific sequences of primers used in the current study as well as name of the corresponding restriction enzymes are shown in [Table 1].[15]
Table 1: Primers and enzymes used in the current study

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Using an Applied Biosystems PCR thermal cycler from Hitachi, the amplification process began with the production of the master mix using 2X Hot Start Master Mix (GeNet Bio-South Korea) to create 20 μL that contained the proper primers and DNA template. Exons 21, C2677T/A, and exon 26, C3435T, each underwent PCR under the following conditions: 94°C for 3 min; 35 cycles of denaturation: 94°C for 30 s, annealing: 58°C for 30 s (C2677T/A); 54°C for 30 s (C3435T), and extension: 72°C for 45 s; and final extension: 72°C for 10 min.[15] Then amplicon products were exposed to 2% agarose gel electrophoresis to confirm successful amplification. Later on, amplicon product was subjected to enzyme digestion and gel electrophoresis to confirm appropriate polymorphism. Following DNA extraction, PCR-amplification with specific primers and enzyme digestion, different pattern observed in regard to all 3 studied polymeric variants [Table 2].
Table 2: Different patterns seen following amplification and digestion with specific enzymes

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After the molecular work was finished, the study sample was analyzed statistically using frequency and percentages. For unqualified variables, the Fisher exact test or Chi-square was employed, while the Student's t-test was used for continuous variables. P value was deemed significant if it was lower than 0.05.

  Results Top

The age of the 62 enrolled patients ranged from 1 to 16 years, with a median age of 7.0 years (mean 7.23 ± 4.3). From these, 62.9% (39 cases) were male and the remaining 37.1% (23 cases) were females, with males: females ratio of 1.7:1. By dividing patients into different age groups: no patient was below 1 year of age; 43 (69.35%) patients were aged between 1 and 10 years of age and the remaining 19 (30.65%) patients were aged between 10 and 16 years of age.

The main clinical features at presentations are shown in [Table 3], and reveals that majority of patients presented with nonspecific symptoms in the form of fatigue and loss of energy. Splenomegaly was the most commonly observed sign among enrolled children.
Table 3: Main clinical features at presentations

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Following investigation particularly immunophenotyping by flow cytometry, majority of leukemia were of B-cell origin specifically of common B-ALL constituting 88.71% (55 cases) of all cases. The remaining cases were belonging to T-cell origin: 8.07% (5 cases) belong to Early T-cells and 3.22% (2 cases) of cortical T-ALL. The hematological parameters of all enrolled patients are shown in [Table 4], and show that majority of patients had low hemoglobin (Hb), low platelets, and high white blood cell (WBC) count mainly of blasts at presentation.
Table 4: Main hematological parameters among enrolled patients

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Positive residual disease was detected in 24 (38.71%) patients following at least 2 courses of chemotherapy (UKALL-14) and 38 (61.29%) patients achieve complete remission following 1–2 courses of chemotherapy.

Following DNA extraction, PCR-amplification with specific primers and enzyme digestion, different pattern observed in regard to all 3 studied polymeric variants [Table 5]. Collectively the prevalence of different alleles at position of 2677 nucleotide as follow (G-allele: 24/124 [19.35%]; A-allele: 52/124 [41.94%]; T-allele: 48/124 [38.71%]). From all examined patients for C3435T variant in exon 26, CC genotype observed in 10 patients (16.13%), CT genotype in 48 patients (77.42%) and TT genotype in the remaining 4 patients (6.45%). The allele frequency of C- and T-allele for C3435T was 54.84% and 45.16%, respectively.
Table 5: The prevalence of each gene polymorphism

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Patients' ages at presentation do not have an impact on the hematological parameters (Hb, WBC count, platelet counts, and percentage of blasts in the bone marrow aspirate and the peripheral blood at presentation) with a P value consistently > 0.05. T-cell ALL tends to occur more commonly at older age, though the correlation was not significant with P = 0.082. Younger age group below 10 years of age tends to have a higher chance of achieving negative MRD after 1–2 courses of chemotherapy with P = 0.048.

Females tend to have significantly lower Hb level at presentation in comparison to males (P = 0.035). other parameters including age at presentation, WBC counts, platelet counts, % of blasts, subtype of ALL, MRD achievement, and studied genes polymorphism were not different between both genders with a P value consistently >0.05.

Type of lymphoblastic leukemia does not have an impact on the hematological parameters (Hb, WBC count, platelet counts, and percentage of blasts in the peripheral blood and bone marrow aspirate) at presentation with a P value consistently > 0.05. Also, it does not have a higher chance of achieving negative MRD after 1–2 courses of chemotherapy with P = 0.243. The occurrence of various variants of the studied gene (G2677T/A; C3435T) was not different among different ALL subtypes.

Achievement of negative MRD following 1–2 courses of induction, appeared significantly correlated with age of patients at presentation and with P = 0.048. All other parameters including, gender of patients, hematological parameters at presentation (Hb level, WBC count, platelet counts, and % of blasts), studied polymorphism in the MDR-1 gene, and subtype of ALL were not associated significantly with MRD achievement.

  Discussion Top

Many factors had been linked to the prognosis of childhood ALL. The most important factors include cytogenetic, MRD, and immunophenotyping of leukemia. Achievement of negative MRD at the end of the induction (EOI) was confirmed to be associated with better overall prognosis, as they showed higher chance of remission, lower relapse rate, and longer overall survival. The EOI, according to the Children's Oncology Group, is the ideal starting point for evaluating MRD for risk-based therapy.[16] For this reason, MRD at the EOI is taken as the time-point to evaluate the efficacy of various factors encompassed in the current study including age at presentation, gender, type of ALL, in addition to MDR-1 polymorphism.

The median age was 7.0 years with male predominance and this figure is slightly similar to that reported from Baghdad, Egypt (median age of 6.4), and Lebanon (median age of 7.1).[17],[18],[19] Male predominance in the current study is in concordance with other studies throughout the world.[20],[21]

Majority of childhood ALL shows predilection to B-cell origin, a recent study from Baghdad confirms such predilection with 76.1% of patients being of B-cell origin. This predilection is mainly contributed to the nature of B-cell maturation that makes it more liable to gene re-arrangement.[17],[18],[19]

Polymorphic variations in MDR-1 gene had been linked to intracellular drug concentration as the G2677T/A and C3435T polymorphism was found significantly to correlate with the function of MDR-1 gene and the expression of P-gp.[22] However, this does not bring about alteration in MRD achievement and cure rate in ALL as shown from a meta-analysis conducted by Yue et al., 2015 that included 1462 cases with 1522 control samples from 9 different studies. This might be explained by the complex nature of the disease, doses and intensity of the chemotherapeutic agents used in leukemia, and the duration of therapy in comparison to acute myeloid leukemia, chronic hematopoietic malignancies, and solid tumors.[5],[23],[24],[25] Despite this large meta-analysis, other studies associated these polymorphic variants (C2677T/A; C3435T) with better response, though majorities of them are of small sample size with various time point consideration and different targeted population, as no previous study available from Iraq to make a comparison.

  Conclusion Top

Polymorphic variation in MDR-1 gene, in comparison to solid tumors and chronic hematopoietic malignancies, does not have impact on MRD achievement in ALL. Age is considered to be a significant biological factor in the achievement of negative MRD and response to therapy. Females tend to have lower Hb level at presentation.

Data accessibility

Available on request.

Ethical approval

The ethics committee at the Duhok Directorate of Health and the Scientific Committee at the College of Medical Science at the University of Duhok gave their approval for this work.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

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  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]


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