Genotoxicity of Therapeutic Intervention in Children with Acute Lymphocytic Leukemia

Abstract

The survival rates of children treated for cancer have dramatically increased after the development of standardized multiple-modality treatment protocols. As a result, there is a rapidly growing population of pediatric cancer survivors in which the long-term genotoxic effects of chemotherapeutic intervention is unknown. To study the genotoxic effects of antineoplastic treatment in children, we performed a comparative analysis of the changes in the frequency of somatic mutations (Mfs) at the hypoxanthine-guanine phosphoribosyltransferase (HPRT)-reporter gene in children treated for acute lymphocytic leukemia (ALL). We measured HPRT Mfs from 130 peripheral blood samples from 45 children with ALL (13, low risk; 22, standard risk; and 10, high risk) from the time of diagnosis, as well as during and after the completion of therapy. We observed a significant increase in mean HPRT Mfs during each phase of therapy (diagnosis, 1.4 ؋ 10 ؊6; consolidation, 52.1 ؋ 10 ؊6 ; maintenance, 93.2 ؋ 10 ؊6; and off-therapy, 271.7 ؋ 10 ؊6) that were independent of the risk group treatment protocol used. This 200-fold increase in mean somatic Mf remained elevated years after the completion of therapy. We did not observe a significant difference in the genotoxicity of each risk group treatment modality despite differences in the compositional and clinical toxicity associated with these treatment protocols. These findings suggest that combination chemotherapy used to treat children with ALL is quite genotoxic, resulting in an increased somatic mutational load that may result in an elevated risk for the development of multi-factorial diseases, in particular second malignancies.

Analysis of Genetic Alterations and Clonal Proliferation in Children Treated for Acute Lymphocytic Leukemia

“…Molecular analysis of HPRT mutant isolates. In contrast with our previous study (7), only peripheral blood samples that yielded viable and expandable HPRT mutants were included in this molecular analysis. A total of 562 HPRT mutant T cell isolates, originating from 87 blood samples from 47 children with ALL were characterized for both the type of HPRT mutation by sequence analysis of cDNA and/or genomic DNA, and clonality by characterizing the T cell receptor-h (TCRh) mRNA sequence of each mutant isolate.…”

“…We previously reported that children treated for ALL have significantly elevated somatic mutant frequencies (MFs) at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) reporter gene in their nonmalignant peripheral T cells, compared to children at diagnosis and to healthy controls, and that the MFs remained elevated for years (6,7). The precise genetic mechanisms associated with the dramatic increase in MFs following chemotherapy and the potential consequence of this increased mutational load is unknown.…”

On the malignant potential of thiopurine therapy

“…Children with acute lymphocytic leukemia treated with chemotherapy, including 6-MP, developed a high number of mutations at the hypoxanthine-guanine phosphoribosyltransferase (HGPRT)-reporter gene. 3 In addition, azathioprine therapy in insulin-dependent diabetes mellitus patients resulted in an increase of HGPRT T cell-mutant frequencies that was statistically correlated with duration of therapy. 4 All clinically used thiopurines have to be metabolized by the enzyme HGPRT to become pharmacologically active.…”

“…Thus, during 6MP/methotrexate (MTX) therapy, 1 in 10 3 to 10 4 DNA nucleotides of circulating cells are substituted 6TGN, 1 which may interfere with postreplicative DNA mismatch repair and induce therapy-related malignant clones. 2,3 Such clones may arise very early in therapy, that is, months or years before the therapy-related cancer becomes clinically overt. 4 It is still unknown how thiopurine therapy influences the proliferation and expansion of such clones.…”

Somatic mutant frequency at theHPRT locus in children associated with a pediatric cancer cluster linked to exposure to two superfund sites

“…Although there is strong evidence of elevated Mfs in patients undergoing radiotherapy and chemotherapy [Dempsey et al, 1985;Messing and Bradley, 1985;Seifert et al, 1987;Palmer et al, 1988;Nicklas et al, 1990;Sala-Trepat et al, 1990;Branda et al, 1991;Caggana et al, 1991;Hirota et al, 1993;Rice et al, 2004], results from studies of occupational exposures to genotoxic agents have been inconsistent. HPRT Mf does not appear to be associated with chronic occupational exposure to ionizing radiation, although it may reflect the effects of more recent exposures [Seifert et al, 1993;Cole et al, 1995].…”