In this study, we have analyzed the frequency of polymorphisms in DNA repair genes previously linked to susceptibility to AML in adults to address the effects of these variants on susceptibility to childhood AML.18
In addition, we investigated the influence of these polymorphisms on the outcome of therapy for childhood AML, which has not previously been studied. Our data show similar genotype frequencies in control and patient populations for the RAD51 G135C, XRCC3 Thr241Met and GSTM1 polymorphisms, suggesting that these variants, when assessed singly, do not play a role in the etiology of childhood AML. Though the control population was dissimilar with respect to age, genotype frequencies were in Hardy-Weinberg equilibrium and corresponded with previously reported genotype frequencies for each of these polymorphisms, suggesting that these subsets are truly representative. These data contrast with our previous observation of increased risk of AML in children with GSTM1 deletion, demonstrating the importance of replicating positive findings in genetic association studies in independent datasets for confirmation.41
It should be noted that our patient population included only de novo
childhood AML, not therapy-related AML.
A similar study in adults by Seedhouse et al.18
showed that the presence of the variant genotype for both RAD51 and XRCC3 significantly increased the risk of developing both de novo
and therapy-related AML (OR 3.77 and 8.11, respectively). These authors also showed that with the addition of the GSTM1 deletion polymorphism, the risk of developing AML was notably increased (OR 15.26). Our study showed a doubling of risk of AML in children with a RAD 51 G135C variant allele and a wild-type XRCC3 Thre241Met genotype. In addition risk of AML was significantly increased in children with at least one variant XRCC3 Thr241Met allele. In contrast, risk was not significantly elevated in children with variant alleles at both wild-type XRCC3 Thre241Met and RAD51 G135C. Addition of GSTM1 genotype to the model did not further increase risk of AML. These data indicate the importance of examining multiple genes in the same pathway to identify the role of genotype. Our study is in agreement with the findings of Seedhouse et al.
to the extent that we demonstrated interaction between genotypes at different loci. Our study differs, however, in that the largest effect was seen in children with a variant allele at one locus and a wild-type allele at the second locus. These findings may be a consequence of biological differences in the etiology of childhood AML, compared with adult AML. The difference in the spectrum of cytogenetic abnormalities seen in childhood AML compared with adult AML supports the hypothesis that the biological mechanisms resulting in childhood AML may be somewhat distinct from those causing AML in adults.42
It is also possible that these are chance observations and replication in an independent pediatric AML dataset will be important to determine the reliability of this finding.
In addition to studying the influence of variant genotypes involved in homologous recombination on susceptibility to AML, we examined the effect of genotype on the outcome of therapy, as one of the cytotoxic effects of AML chemotherapy is the generation of DNA DSBs. The data show that the XRCC3 Thr241Met polymorphism has a significant influence on the post-induction outcomes for our patient population. Surprisingly, the best outcome was found in heterozygotes with improved 5-year DFS for C/T vs CC + TT genotypes (P = 0.05). An increased frequency of relapse in the homozygotes led to inferior outcomes in these children while, TRM was similar in homozygotes and heterozygotes.
Our patient population was randomized to two different post-induction chemotherapy regimens–Regimen A consisted of IDA-DCTER and Regimen B consisted of IDA-FLAG. When outcomes of children with AML treated on CCG2961 and CCG2941 were examined as a whole, without consideration of genotype, no significant difference was observed among the patients treated on Regimen A (IDA-DCTER) and Regimen B (IDA-FLAG). While IDA-FLAG consists primarily of anti-metabolite-based therapy, IDA-DCTER includes etoposide and daunomycin therapy that induces double-strand DNA breaks, requiring homologous recombination for accurate repair. Since XRCC3 plays a key role in homologous recombination, we examined the outcomes of patients treated on Regimen A and B separately with respect to XRCC3 Thr241Met genotype. The heterozygous patients had improved survival in both treatment arms compared to the homozygotes. However, this difference in outcome was statistically significant only in the patients who were treated on Regimen A (IDA-DCTER).
While it is believed that XRCC3 is a key player in the initial strand invasion and nucleoprotein filament during the process of homologous recombination, its precise mechanism of action is still not clear. Previous epidemiological and in vitro
biological studies have suggested that XRCC3 Thr241Met is a functionally important polymorphism.30,43-45
A number of studies of adult malignancies have shown associations of XRCC3 Thr241Met genotype with cancer susceptibility, including melanoma,20
MDS, chronic gastritis and gastric cancers,46
and aerodigestive cancers.47
Functional studies examining the role of the variant allele have also been reported in the literature. Matullo et al.21,25
identified that XRCC3 Thr241Met polymorphism resulted in reduced DNA repair activity when using p32-labelling to measure DNA adduct levels as a measure of DNA repair capacity. In addition, Lindh et al.48
reported an increase in mitotic defects in cells expressing only XRCC3 Thr241Met. In contrast to these findings, Araujo et al.49
were not able to detect a significant difference between wild type and variant proteins in their ability to correct the hypersensitivity of the irsISF cell line to DNA damage using Mitomycin C or to complement the homologous recombination defect in XRCC3-deficient cells.
One possible hypothesis to explain why the outcome was statistically different only in the patients who were treated on Regimen A (IDA-DCTER) could be that DSB repair is inferior in heterozygous children. This would potentially allow superior killing of blasts when homologous recombination is required to repair damage, and the effect of genotype is most evident in children receiving therapy that requires homologous recombination for repair. Unfortunately, in the studies mentioned before, homozygous and heterozygous genotypes were not compared. It might be expected that if the variant allele is truly associated with reduced homologous recombination, the effect of the polymorphism would be most evident in children homozygous for the variant allele. It is surprising, therefore, that the heterozygous group has improved survival as compared to the wild type and the variant homozygotes.
In summary, in our study, we observed a significant interaction between XRCC3 and RAD51 genotypes in susceptibility to childhood AML. In addition, we have shown a difference in the post-induction outcome of childhood AML with respect to the XRCC3 Thr241Met genotype with heterozygous children showing superior survival. The survival difference was most significant in children receiving chemotherapy that we would expect to generate DSBs. While these findings are of interest, like all such studies, these observations need to be replicated in additional independent datasets to validate the importance of this polymorphism in predicting response to chemotherapy. Additionally, molecular and biochemical studies are needed to clarify the functional significance of the XRCC3 Thr241Met polymorphism, particularly in heterozygotes.