Association between SLC19A1 gene polymorphism and high dose methotrexate toxicity in childhood acute lymphoblastic leukaemia and non Hodgkin malignant lymphoma: introducing a haplotype based approach
Article Category: Research Article
Published Online: Sep 18, 2017
Page range: 455 - 462
Received: Feb 14, 2017
Accepted: Aug 18, 2017
DOI: https://doi.org/10.1515/raon-2017-0040
© 2017 Barbara Faganel Kotnik, Janez Jazbec, Petra Bohanec Grabar, Cristina Rodriguez-Antona, Vita Dolzan
This article is distributed under the terms of the Creative Commons Attribution Non-Commercial License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Inter-individual variability in treatment responses and treatment related toxicities is an important issue in clinical practice. It can lead to therapeutic failures or adverse drug events (ADE). Identification and characterization of genetic polymorphisms and haplotypes involved in drug metabolism, transport and mechanism of action would provide important information about individual inherited differences in drug metabolism and treatment response in order to optimize treatment outcome.1
The solute carrier 19A1 (
Several studies so far investigated the association of rs1051266 with high dose MTX (HD-MTX) treatment outcome and treatment related toxicities in ALL patients.910111213 Some of them reported a protective role of rs1051266 AA genotype14,15, while in others either an increased risk of MTX-induced toxicities and fatal outcomes in rs1051266 A carriers9,10, or even no association between
To date all studies but one19 evaluated the role of individual
In the present study we investigated a possible association between
The study group included 88 children and adolescents (41 male, 47 female) with ALL/NHML diagnosed and treated in the period from 1990 to 2008 at the Department of oncology and haematology, University Children’s Hospital in Ljubljana, Slovenia. All the patients were Central European Caucasians and below the age of 18 years at the time of diagnosis. Clinical and demographic data were obtained from the medical records and a questionnaire by a single abstractor blinded to genotypes and are presented in Table 1. Each patient enrolled in the study received a high MTX dose (77.3% of patients received 5 g/m2 and 22.7% received 2 g/ m2 of MTX, according to treatment protocols) on 4 separate occasions and concomitant leucovorin rescue therapy to reduce MTX toxicity. Overall, 352 courses of HD-MTX applications were analysed.
Demographic and clinical characteristics of children with ALL/NHML and MTX treatment toxicity data is missing for one patient data is missing for one patient data was collected after the first cycle of MTX data is missing for one patientCharacteristic ALL patients (N = 88) Gender (%) Male 41 (46.6) Female 47 (53.4) Median age at diagnosis, years (range) 4.58 (0.3–16.6) Median body surface area, m2 (range) 0.73 (0.4–1.9) Leukaemia subtype (%); B-cell 72 (81.8) T-cell 13 (14.7) Undetermined 3 (3.4) Median No. leukocytes at diagnosis, 109 cells/L (range) 9.35 (1–1650) Median No. thrombocytes at diagnosis, 109 cells/L (range) 72 (3–553) Median haemoglobin conc., g/l (range) 84 (43–148) Median % of blasts at diagnosis (range) 23.5 (0–97) Median MTX dose, mg (range) 3300 (540–9200) Relapse 8 (9.2) Exitus 4 (4.6) All MTX-induced ADEs 48 (54.5) Leukopenia 33 (37.5) Thrombocytopenia 16 (18.2) Mucositis 15 (17.0) Neurotoxicity 7 (8.0)
All patients and/or their parents or legal guardians gave their written informed consent to participate in the study. The study was approved by the Republic of Slovenia National Medical Ethics Committee and was carried out according to the Helsinki Declaration.
Adverse drug events (ADE) were recorded retrospectively from patients’ files throughout the course of the HD-MTX treatment. ADEs were classified as (1) leukopenia, defined as white blood cells count less than 5*109 per litre, (2) thrombocytopenia, defined as thrombocyte count less than 140*109 per litre, (3) mucositis, determined and graded according to the WHO oral toxicity scale classification22 or based on physician’s descriptions in medical records, need for analgesia and/ or total parenteral nutrition and (4) neurotoxicity, determined and graded according to National Cancer Institute Common Toxicity Criteria Version 1 and included patients with seizures (regardless of grade) and patients with grade 3 or 4.23 The data on relapse and exitus were also recorded. All the reported ADEs were used for the evaluation of MTX toxicity.
A total of seven SNPs (rs2838951, rs2838956, rs2838958, rs1051266, rs1131596, rs2838451 and rs17004785) in
In total 5 ml of peripheral blood was collected into tubes with sodium citrate and stored for short term at -20°C until DNA isolation. Genomic DNA was isolated from peripheral blood leukocytes using a Qiagen FlexiGene kit (Qiagen GmbH, Germany). Genotyping was performed using a fluorescence-based competitive allele-specific (KASPar) assay.25 Real time PCR was performed on the ABI 7900HT in a 4 μL reaction mix containing 0.055 μL of KASPar allele specific PCR assay, 2 μL of KASPar PCR Master Mix (2x concentration, containing KTaq polymerase and ROX), 2.2 mM MgCl2 and 10 ng of DNA.
Binary logistic regression analysis with the addition of independent variables, such as sex, age at time of diagnosis and body surface area (BSA) was done using SPSS for Windows version 16.0.1 software (Statistical Package for the Social Sciences, Chicago, IL). The risk for toxicity of MTX treatment was expressed as odds ratio (OR) with 95% confidence intervals (CI). The level of significance was set to 0.05. Deviation from Hardy-Weinberg equilibrium (HWE) was calculated for each SNP using Chi-square test. The level of deviation was set to 0.05. Power calculations were done using G*Power software version 3.1.0. Our study had an 80% statistical power to detect medium effect sizes (0.331) with an alpha level of 0.05. The correction for false-positive findings was done using a method for detecting false-positive report probability (FPRP) described by Wacholder
The cut-off for noteworthy results was set as 0.5. The expected ORs were based on the previous reports of an association of SNPs in
Of the 88 patients recruited, 48 (54.5%) patients suffered from MTX-induced adverse events. Specific MTX-induced ADEs after the first cycle of MTX and their frequencies are presented in Table 1.
The characteristics and the minor allele frequencies (MAF) of the investigated SNPs in
With regard to specific ADEs, univariate logistic regression analysis revealed only the association of rs2838958 with the occurrence of mucositis development (p = 0.009). We observed that patients with rs2838958 TT genotype had higher probability for mucositis development as compared to carriers of at least one polymorphic rs2838958 C allele (OR 0.226 (0.071–0.725), p < 0.009) (Table 2).
The influence of rs2838958 genotype on mucositis development - univariate logistic regression analysis Calculation was not possibleDegree of moscositis rs2838958 genotype OR(95%CI) P TT (%) TC + CC (%) 18 (66.7) 53 (89.8) reference 4 (14.8) 4 (6.8) 0.340 (0.077–1.500) 0.154 4 (14.8) 2 (3.4) 0.170 (0.029–1.007) 0.051 1 (3.7) 0 (0.0) - 9 (33.3) 6 (10.2) 0.226 (0.071–0.725) 0.009 0.59 0.14 0.007
We then investigated the possible association between
The impact of The estimates for the odds ratio (OR) were adjusted to the following variables: age, sex, and treatment regimen.SNP ID Any adverse events Leukopenia Thrombocytopenia Mucositis Neurotoxicity OR P OR P OR P OR P OR P (CI 95%) (CI 95%) (CI 95%) (CI 95%) (CI 95%) 0.719 0.360 0.609 0.150 1.170 0.696 1.292 0.579 0.542 0.361 (0.354–1.458) (0.310–1.197) (0.532–2.576) (0.525–3.170) (0.146–2.016) 1.392 0.360 1.643 0.150 0.855 0.696 0.775 0.579 0.542 0.361 (0.686–2.824) (0.836–3.230) (0.388–1.881) (0.316–1.906) (0.146–2.016) 1.221 0.583 1.684 0.136 0.556 0.172 0.476 0.130 0.453 0.236 (0.599–2.490) (0.849–3.339) (0.240–1.291) (0.182–1.243) (0.122–1.677) 1.411 0.349 1.742 0.113 0.689 0.374 0.705 0.454 0.596 0.434 (0.686–2.899) (0.878–3.458) (0.303–1.566) (0.283–1.759) (0.163–2.182) 0.849 0.772 1.393 0.545 0.854 0.821 0.672 0.606 1.226 0.800 (0.280–2.572) (0.476–4.079) (0.218–3.349) (0.148–3.055) (0.255–5.899) 1.475 0.478 1.825 0.238 1.688 0.368 0.930 0.914 1.061 0.939 (0.504–4.318) (0.672–4.956) (0.540–5.280) (0.249–3.473) (0.232–4.861) 1.261 0.483 0.740 0.351 1.652 0.185 2.160 0.081 1.693 0.337 (0.666–2.412) (0.392–1.394) (0.786–3.473) (0.909–5.129) (0.577–4.967)
We also performed haplotype analysis to ascertain whether the combination of polymorphisms that define a specific haplotype impacts the occurrence of ADEs. As shown in Figure 1 the investigated polymorphisms were lying in the same haploblock.
Graphic presentation of haploblocks and selected SNPs in Figure 1
The frequencies of
SNP rs1131596, rs1051266, rs2838958, rs2838956, rs17004785, rs12483377, rs2838951 from 5’ to 3’, respectivelyHaplotype Frequency Standard error H1 = CACCCCG 0.396 0.037 H2 = TGTTCCC 0.334 0.036 H3 = TGTTGTC 0.094 0.022 H4 = TGTTCCG 0.053 0.017 H5 = CATTCCC 0.017 0.010 Other 0.106
H1 was the most frequent haplotype in the patient test group with a frequency of 39.6%. Together, H1, H2, and H3 haplotypes accounted for 82.4% of all haplotypes. In patients with ALL and ML, the haplotypes H4 and H5 were more rarely represented, with the frequency of a single haplotype between 1 to 5%. Therefore haplotype H5 and the other very rare haplotypes have been grouped together as ‘rare’ for further statistical analysis.
We examined whether there is a link between the
The impact of gender, age, treatment regimen and p < 0.05; H2 was the reference haplotype p < 0.05; H2 was the reference haplotypeAll Thrombocytopenia Mucositis Neurotoxicity OR (95% CI) P OR (95% CI) P OR (95% CI) P OR (95% CI) P 2.385 0.078 1.615 0.413 1.523 0.520 0.531 0.475 (0.908–6.259) (5.134–5.076) (0.423–5.472) (0.093–3.024) 1.028 0.588 9.526 0.440 1.054 0.396 1.088 0.289 (0.931–1.134) (8.422–1.078) (0.935–1.190) (0.931–1.272) 2.050 0.031 9.839 0.962 1.676 0.255 1.282 0.678 (1.065–3.943) (4.620–2.095) (0.689–4.075) (0.397–4.139) 1.000 0.943 6.615 0.352 0.419 0.096 0.309 0.120 (0.472–2.117) (2.769–1.579) (0.150–1.167) (0.070–1.364) 1.110 0.844 9.220 0.891 0.593 0.515 0.951 0.934 (0.335–3.618) (2.254–3.750) (0.122–2.877) (0.175–5.150) 0.143 0.030 3.617 0.379 0.005 0.985 0.014 0.992 (0.023–0.852) (4.568–2.541) (0.0–NC) (0.0–NC) 2.002 0.275 9.113 0.871 2.002 0.275 2.002 0.275 (0.580–6.775) (2.607–3.171) (0.580–6.775) (0.580–6.775)
In the present study we have used haplotype based approach to investigate the association of genetic variability in
Among the so far published studies on the role of
Among the investigated
In the haplotype based analysis we found that H4 haplotype (TGTTCCG), which, excluding the last locus (rs2838951), has wild type alleles present, statistically significantly reduces the risk for the occurrence of ADEs during treatment with HD-MTX. Since the frequency is low in our sample it is difficult to ascertain its clinical relevance given the number of patients and the small number of individual toxic events.
Haplotype based approaches have been rarely used when investigating HD-MTX induced adverse events in ALL and NHML patients. One study investigated the influence of
Only study of Lopez-Lopez focused on MTX transporters. This study examined 384 SNPs in 12 transporter genes, including
However, haplotype based approach has been used in analysing the influence of the polymorphisms in the broader region of
The results of the haplotype based analysis indicate, that other polymorphisms that are in linkage disequilibrium with rs1051266 and rs1131596, may impact the functioning of
The results of our study show that
The main limitation of our study, which is probably also one of the reasons for the inconsistent results among studies published so far, is the limited sample size. Since ALL is a rare disease in children, the number of patients that could be as comparable as possible in the entire treatment process is limited. We therefore also included five patients with NHML in the study, who were treated according to the same protocol as patients with ALL.
The great advantage of our study is that all subjects come from a small and very homogeneous population42, that the patient group is clinically well defined and very homogeneous given the type and course of treatment, centralized treatment of patients with an established treatment and supportive regimen. The group was treated by the same medical team in a single centre according to standard criteria.
For genotyping, we used a method that would be suitable for genetic testing in clinical practice, as it is easy to perform, fast, affordable, and enables simultaneous analysis of multiple samples and different polymorphisms if they are run under the same conditions, and also require small amounts of DNA for analysis.
Employing a haplotype based approach we tried to examine the impact of genetic variability in the entire region of the studied gene and also included the possible functional polymorphisms that are in linkage disequilibrium with the selected SNPs. With this approach an even stronger influence of genetic variability of
The observations of our study provide additional information to the ongoing discussion about the most suitable biological markers to be evaluated during treatment with HD-MTX in order to achieve more efficient, safer and more rational treatment of children and adolescents with ALL. Additional prospective pharmacogenetic studies on higher sample sizes are needed to further evaluate a possible impact of genetic variability of the MTX transporters that have an impact on pharmacokinetics of MTX and enzymes involved in MTX metabolism that could help us to identify patients that are threatened by serious ADEs during HD-MTX treatment.