GENETIC POLYMORPHISMS OF THE FOLATE METABOLIC PATHWAY IN CHILDHOOD ACUTE LYMPHOBLASTIC LEUKEMIA. A MOLECULAR STUDY AND A PROPOSAL FOR AN INTERPRETATIVE MODEL.

Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer accounting for 80% of childhood leukemia. The uncontrolled proliferation of lymphoid progenitors in the bone marrow and the accumulation of malignant lymphoblasts in peripheral blood characterize the disease. The molecular analysis of common genetic alterations in lymphoblastic cells has strongly contributed to the comprehension of ALL pathogenesis. Different gene polymorphisms (most of them SNPs) play an important role in the susceptibility to childhood ALL which probably derives from a combination and relation of genetic and environmental factors. Folic acid and the pool of folate of the one carbon-metabolic-pathway are key elements involved in several processes including DNA synthesis and methylation. Polymorphisms in genes coding for enzymes of the folate metabolic pathway can alter the intracellular folate status or distribution and sub-optimal/anomalous folate levels/distribution increase the risk of developing several neoplasias. The two main enzymes involved in cyclization of folate isoforms are DHFR and MTHFR. The first one is responsible for the conversion of dihydrofolate to tetrahydrofolate whilst the second one catalyzes the reduction of 5,10-methylene-THF to 5-methyl-THF. Studies have demonstrated that subjects with the homozygous DD-genotype have higher DHFR mRNA levels that may be responsible for storage of THF and other isoforms within the cell; reduced enzyme activity instead, has been associated to MTHFR 677TT homozygotes. This condition is responsible for an underutilization of methylene-THF in the cell with storage of folate reduced isoforms. Several studies have demonstrated that DHFR and MTHFR polymorphisms may be protective against hematological cancers such as ALL. It is to note that current treatment regimens achieve levels about 80% in overall survival (OS). Unfortunately, the side effects derived from the chemotherapeutic agents used can be severe, especially for high-risk patients. Therefore, the identification of additional markers which can improve risk stratification and individual tailored therapy regimens would be a great goal, in order to avoid over-treatment which can increase long-term adverse side effects. The aim of the present study was, therefore, to investigate whether common polymorphisms (i.e. MTHFR C677T and A1298C in addition to DHFR 19 bp INS/DEL and Bcl-2 -938 C>A) might influence the risk of childhood ALL. After a single analysis we can ascribe to MTHFR C677T gene polymorphism a protective significant role against childhood ALL (P=0.046), whilst Bcl-2 -938 C>A gene polymorphism seems to be a risk factor for the susceptibility to the disease (P=0.049). Then, considering parameters such as disease onset and therapy duration we can observe a significant higher mean age onset disease for homozygotes MTHFR 1298-CC (P=0.05). From the analysis of the therapy duration we found a significant association for MTHFR A1298C and Bcl-2 -938 C>A polymorphisms: MTHFR 1298CC homozygotes showed a slight higher mean therapy duration (P=0.05), as well as Bcl-2 -938AA homozygotes (P=0.03). Finally, in an exploratory way, to validate the proposed model we evaluated among healthy PBL cells harvested from subjects with opposite genotype condition considering DFHR and MTHFR genes (respectively, WW/CC and DD/TT), possible differences in base-line cellular viability and under MTX treatment. The pharmacological induced restriction in folate availability (MTX) yields to results in favor of WW/CC, whilst the base-line cell viability yields comparable results among genotypes. This is in line with the hypothesis that a higher MTX level could be present in DD/TT cells, prone to storage either natural folate isoforms (useful for the cell viability) or synthetic toxic analogue (MTX). This observation argues us into hypothesizing that also MTX being itself a synthetic folate analogue follows the same handling process. Now, inside the cell it should result in an elevated toxicity level, being responsible for an elevated death.