| Abstract |
Acute lymphoblastic leukemia (ALL) in children represents the most frequent form of malignancy accounting for 25-30% of all cancer cases among pediatric patients. The classification and treatment of the disease is based on known clinical and immunophenotypic characteristics and lately also relies on molecular markers based on methods of molecular analysis. Those methods reveal, besides the role of proto-oncogenes, hyperdiploidy, structural chromosomal defects such as translocations and gene fusions, the importance of gene polymorphism.
Several studies that have been conducted lately, have revealed an association between both the risk and the treatment outcome of ALL, with polymorphisms located in genes encoding for enzymes which are involved in various metabolic pathways. Such an example is the methylenetetrahydrofolate reductase (MTHFR), an enzyme which plays a role in the folic acid metabolism. Two common single nucleotide polymorphisms (SNPs) have been characterized in the MTHFR gene, at nucleotide positions 677 (C—T) and 1298 (A—C). Another example is the reduced folate carrier (RFC), an anion exchanger which transports both folate and methotraxate (MTX) into cells. The gene encoding RFC has been proved polymorphic at nucleotide position 80 (G—A).
Under scrutiny have also been lately, the cytochromes P-450, a family of enzymes which participate in the phase I of the metabolism of precarcinogenic substances and drugs, such as vincristine. CYP3A4 and CYP3A5 enzymes belong to the CYP3A subfamily and have been proved polymorphic at nucleotide positions -392 (CYP3A4*1B: A—G) and 22893 (CYP3A5*3: G—A) respectively. All the polymorphisms mentioned above, affect the catalytic activity of the enzymes described and the interactions between them, as well as their correlation with the occurrence of ALL, have been under investigation, with varying results between different populations.
In our study, the presence of the MTHFR, RFC, CYP3A4 and CYP3A5 SNPs was examined in 35 children with ALL (14 females, 21 males-median age 6.33 years) and in 48 healthy adults (control group), with Cretan origin. A comparison between the children's and the controls' genotype and allele frequency distributions was also performed. Moreover, the association between the MTHFR and RFC SNPs and the presence of MTX-related mucositis, hepatotoxicity and hematological toxicity, as well as the association between the CYP3A4 and CYP3A5 SNPs and neurotoxicity due to vincristine administration, were also studied.
The possible correlation between the SNPs in the genes examined and the susceptibility to ALL was examined using polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) based approaches. The PCR/RFLP reactions were successful for all samples. In order to establish the presence of adverse events in the children treated for ALL, data were used from their medical records. During the consolidation phase of the ALL-BFM 95 protocol, the presence of hematological toxicity and hepatotoxicity was established from the average values of hemoglobin, platelets and white blood cells (HGB, PLTs and WBCs) regarding the former and the average values of aspartate transaminase, alanine transaminase and gamma-glutamyltransferase (AST, ALT and GGT) regarding the latter. The laboratory values were each time calculated one week after the administration of IV methotrexate and the analysis was performed considering only higher grade toxicity. For statistical reasons and using the Common Terminology Criteria for Adverse Events v4 from the National Cancer Institute, children with normal laboratory values and no stomatitis, were labeled as having no presence of an adverse event, while children with toxicity grades 1-4, were labeled as having an adverse event. The same criteria were used for the assessment of the vincristine-related neurotoxicity, during the entire length of the treatment. The clinical records for 3 of the children were incomplete and they were not included in the statistical analysis for the drug-induced toxicities.
In order to examine the possible association of the SNPs mentioned and the susceptibility to ALL, a comparison of the genotype and allele frequency distributions between children with ALL and controls, was performed. The genotype and allele frequencies, regarding both SNPs in cases and controls, did not show any statistical differences. Regarding the MTHFR 677 SNP, the analysis showed that there was a statistically borderline significant association between the carriers of C677T polymorphism and the decrease in the WBC count after the IV administration of methotrexate (p=0.050). Moreover a statistically marginal significant correlation between the A1298C polymorphism and normal AST and ALT values during the course of treatment (p=0.065 and p=0.053 respectively) was also revealed. The remaining adverse events were not associated with the presence of any genotypes. For statistical reasons, we further stratified the patients in 2 categories. The results for the A1298C polymorphism, showed a statistically significant non-predisposing role of the A allele (A1298A+A1298C) regarding hepatotoxicity, as expressed by AST values
(p=0.039).
The frequencies observed in cases and controls were studied, in order to evaluate
the effect of the gene-gene interactions (RFC/MTHFR SNPs and CYP3A4/CYP3A5
SNPs). The results revealed that the MTHFR A1298C / RFC G80A genotype, had a non-predisposing role regarding the susceptibility to ALL (p=0.035), while the differences between the remaining haplotypes were not statistically significant.
The results from our study of gene polymorphisms in children of Cretan origin with ALL may play a role in assessing the risk, both for the ALL susceptibility and the drug-induced toxicity, in our population. Single nucleotide polymorphisms may be a tool in the ongoing effort of individualizing treatment, taking into account during the planning of the treatment, the genetic profile of each patient, the possible gene-gene interactions and the role of environmental factors and dietary habits. Collaboration among institutes in national, European and global level, along with the ongoing study of SNPs in genes encoding for enzymes which participate in various metabolic pathways and also affect the metabolism of commonly used chemotherapeutic agents, could accelerate the achievement of individualized treatment, leading this way to the further increase in the cure rates of ALL
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