ALBERT

Description: Abstract 1680 Individuals with Down syndrome have a 2% cumulative risk for the development of leukemia by the time they reach 30 years of age. In addition, as many as 5–10% of infants with Down syndrome may develop a transient myeloproliferative disorder (TMD). The molecular basis for the increased risk for hematologic disorders and other congenital developmental abnormalities with DS remains unknown. Additional genetic material from the critical Down syndrome locus at 21q22 includes the Cu/Zn superoxide dismutase gene and other genes contributing to high oxidative stress and increased endogenous DNA damage observed in cells from patients with Down syndrome. We hypothesized that functional polymorphic variants in enzymes associated with detoxification of oxidants and repair of oxidant associated DNA damage would be associated with an increased risk for the development of myeloid leukemia (ML) and TMD in children with Down syndrome. We studied the role of functional polymorphisms in the oxidant metabolizing genes, Paraoxonase 1 (PON1) and NADH/NADPH oxidase p22 phox (P22_PHOX) in the potential genetic etiology of ML/TMD in children with Down syndrome. Genotyping was conducted in 192 patients with Down Syndrome ML or TMD, and 251 healthy blood donor controls. All genotype frequencies in patients and control populations were consistent with those expected from Hardy-Weinberg equilibrium. We found that the variant PON1 Gln192Arg allele, associated with reduced free radical metabolism, occurred at a higher frequency in the Down syndrome ML/TMD population compared to healthy controls (OR, 2.71; 95% CI, 1.50–4.97; p=0.0003). Consistent with our hypothesis of increased oxidant sensitivity in these patients, we also observed decreased frequency of the protective variant P22_PHOX His72Tyr allele, purportedly associated with reduced generation of reactive oxygen species, in the Down syndrome ML/TMD population compared to healthy controls (OR, 0.48; 95% CI, 0.23–0.95; p=0.025). Notably, the variant PON1 Gln192Arg allele has also been associated with increased risk for non-Hodgkin's lymphoma in adults (Kerridge et al. Br J Haematol, 2002). Conclusion: These preliminary studies suggest that functional polymorphic variants in the oxidant metabolizing enzymes PON1 and P22_PHOX may influence risk for development of ML/TMD in children with Down syndrome. Studies to confirm these observations in a separate cohort of patients are in progress. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2610 Poster Board II-586 Cytarabine is a key chemotherapeutic agent in the treatment of acute myeloid leukemia (AML). We have taken a pathway based candidate gene approach to select variants likely to be critical to cytarabine metabolism. Single non-synonymous nucleotide polymorphisms and polymorphisms located in promoter regions of genes involved in cytarabine metabolism, with known frequencies 〉 5% in the Caucasian population were identified. We genotyped 479 children treated for de novo AML on the Children's Cancer Group (CCG) 2941 and 2961 protocols for these polymorphisms. All patients received intensively timed induction therapy with IDA-DCTER (idarubicin, dexamethasone, Ara-C (800 mg/m2 total dose), thioguanine, etoposide and daunomycin) given on days 0 to 3 followed by DCTER given on days 10 to 13. On recovery of ANC and platelet counts, patients were randomized to consolidation therapy with Regimen A: a further course of IDA-DCTER/DCTER or Regimen B: IDA-FLAG (idarubicin, fludarabine, Ara-C (7590 mg/m2 total dose Ara-C) and G-CSF). Patients with a matched family donor received intensification with allogeneic transplant; all others received a single course of high dose Ara-C (24,000 mg/m2 total dose) and L-asparaginase. There were no significant associations between genotypes and clinical characteristics of leukemia, although polymorphism frequencies did differ significantly by race. Significant associations with outcome of therapy in a multivariate analysis adjusted for race are listed below: The nucleoside transporter gene hENT promoter polymorphism rs747199 influenced 5 year overall survival (OS) from study entry (46 ± 6% in G/G cases versus 58 ± 9% in other patients (p=0.039). Similarly, 5 year OS from end of two courses was improved in children with a C/C genotype at rs693955 (OS 66 ± 7% vs. 54 ± 11%, p= 0.058). No significant differences were found in disease free survival, relapse rate (RR), or treatment related mortality for these variants. Nucleoside transporter polymorphisms may play a critical role in modulating cytarabine transport and influence the outcome of therapy for AML in children. Disclosures: No relevant conflicts of interest to declare.

Description: Children with Down syndrome can have a wide range of developmental abnormalities related to the presence of additional genetic material from a small region of chromosome 21. Additional copies of the Cu/Zn superoxide dismutase gene and related genes at this locus are believed to contribute to high oxidative stress, and increased endogenous DNA damage and deficient repair observed in cells from patients with Down syndrome. Children with Down syndrome also have a markedly increased risk for the development of serious hematological disorders, including an approximately 20-fold increased risk for the development of acute myeloid leukemia (AML). In addition, in early infancy, as many as 10% may develop a transient myeloproliferative disorder (TMD). The basis for this overall increased risk is unknown, as are the risk factors influencing which specific patients with Down syndrome develop AML/TMD. We sought to test the hypothesis that functional polymorphisms in genes involved in oxidant metabolism and DNA repair contribute to the increased risk of AML and TMD in children with Down syndrome. We studied functional polymorphisms in the oxidant metabolizing enzyme NADPH quinone oxidoreductase 1 (NQO1), and in the DNA repair proteins X-ray repair cross complementing 1 protein (XRCC1) and Fanconi anemia complementation group A protein (FANCA). Genotyping was conducted in 170 patients with Down syndrome AML or TMD, and 202 healthy blood donor controls. All genotype frequencies in the control populations were consistent with those expected from Hardy-Weinberg equilibrium. We found that the variant NQO1 Pro187Ser allele, associated with reduced quinone reductase activity, occurred with a greater frequency in the Down syndrome AML/TMD patient population compared to healthy controls (OR, 1.61; 95% CI, 1.067–2.426; p=0.0231). The FANCA-1501 variant allele frequency was observed more frequently in Down syndrome AML/TMD patients compared to healthy controls (OR, 1.77; 95%CI, 1.220–2.568; p=0.0025). The variant XRCC-1 Arg399Gln allele was observed less frequently in the TMD/DS AML population as compared to controls (OR, 0.536; CI, 0.354–0.811; p=0.0031). These latter findings parallel the observation of a protective effect against AML in non-Down syndrome individuals with at least one copy of the XRCC-1 Arg399Gln allele (Seedhouse et al. Blood2002, 100:3761). Conclusion: These preliminary studies, representing the only reported data in Down Syndrome TMD/AML patients, suggest that functional polymorphic variants of the NQO1, FANCA, and XRCC1 genes involved in oxidant metabolism and DNA repair may influence the risk for developing leukemia. Studies involving future cohorts will be needed to confirm these observations.

Description: Abstract 3425 Impaired anti-oxidant defenses and overproduction of reactive oxygen species (ROS) in individuals with Fanconi anemia (FA) are associated with a pro-oxidant state in this population. Cells derived from individuals with FA demonstrate increased sensitivity to DNA damage by ambient oxygen and increased frequency of chromosomal aberrations. Studies in animals and human subjects indicate that higher levels of and increased sensitivity to both ROS and tumor necrosis factor-alpha in individuals with FA play key roles in the pathogenesis of bone marrow failure (BMF) and neoplastic transformation. Accumulation of somatic DNA damage can be assessed using the Glycophorin A assay (GPA), which is designed to detect potentially inactivating mutations caused by chromosome loss, large deletions, or recombination events at the erythrocyte GPA locus. Previous cross-sectional studies in individuals with FA have shown a markedly increased frequency of GPA variant cells, demonstrating increased DNA damage. Knowledge regarding the timecourse of the accumulation of this somatic DNA damage is lacking. The goal of the current study is to evaluate serum ROS levels and accumulation of somatic DNA damage in individuals with FA to better understand the contribution of oxidative stress to disease phenotype and progression to marrow failure or malignant transformation. ROS levels were assessed ex vivo from peripheral blood and bone marrow of individuals with FA and healthy controls. We used a flow cytometric method to quantify ROS level by incubation of samples with CM-H2DCFDA, a cell-permeable fluorescence dye that reacts to a broad spectrum of ROS. Peripheral blood was also serologically typed on first submission using anti-M and anti-N sera. As only heterozygous (MN) individuals are informative, only these individuals were studied further. We used the GPA assay to quantify the accumulation of somatic cell damage in peripheral blood samples as measured by GPA variant cell frequencies. Peripheral blood and/or bone marrow samples from 19 individuals with FA and normal controls were evaluated for ROS on 23 occasions. ROS levels were variable, with 13 of the FA patients demonstrating high levels of ROS as measured by MFI signal strength. Overall ROS levels in individuals with FA were higher than those of normal control individuals (132% of control ROS levels, p

Description: The glutathione S-transferase (GST) genes are involved in the metabolism of environmental carcinogens and of some classes of chemotherapy drugs. GSTM1 and GSTT1 genotypes are polymorphic in humans, and the phenotypic absence of enzyme activity is caused by a homozygous inherited deletion of the gene. Previous, smaller studies of childhood acute lymphoblastic leukemia (ALL) provided contrasting data on the role of the GST genotype in susceptibility and treatment outcomes. We analyzed GST genotypes in 710 children with ALL treated by the Children's Cancer Group. Frequencies were compared with those of normal controls, and outcomes were analyzed according to genotype. Comparisons of gene frequencies in ALL case and control patients showed similar frequencies (54% vs 53% GSTM1 null in whites,P = .9; 40% versus 32% in blacks,P = .45; 16% versus 15% GSTT1 null in whites,P = .8; 17% versus 28% in blacks,P = .3). ALL was not associated with the GSTM1-null genotype or the double-null genotype in blacks or whites, in contrast to previous reports. Stratification of cases by age at diagnosis, sex, white blood cell count at diagnosis, B or T lineage, or cytogenetics revealed no differences in genotype frequencies. Analysis of treatment outcomes showed no differences in outcome according to GST genotype; in particular, there were no differences in frequencies of relapse at any site. These data, representing a larger series than any reported previously, suggest that GST genotype does not affect etiology or outcome of childhood ALL.

Description: Outcomes for patients with advanced-stage Hodgkin’s lymphoma (HL) remain suboptimal, as dose-intensified regimens produce relatively high cure rates at the cost of increased short- and long-term complications. Prospectively identifying the subset of patients who are at risk of therapy-related complications would allow refinement of treatment regimens. We hypothesized that functional polymorphisms in chemotherapy drug metabolizing enzymes will predict the risk of toxicity for pediatric patients with advanced-stage HL. Glutathione-S-transferase T1 (GSTT1) and glutathione-S-transferase M1 (GSTM1) participate in protection against oxidative damage. Previously, we demonstrated that genetic deletion of these enzymes, a common population polymorphism, increase the risk of regimen-related mortality in pediatric patients with acute myeloid leukemia. The human DNA repair gene XRCC1 is involved in the repair of DNA single-strand breaks, generated in response to alkylating agents and ionizing radiation, and a frequent single nucleotide polymorphism with reduced function (codon 399 arginine to glutamine substitution) has been associated with an increased risk of secondary malignancy. We examined the effects of polymorphic alleles of GSTT1, GSTM1 and XRCC1 on the risk of toxicity in 72 pediatric patients receiving chemotherapy for advanced stage (clinical stage IV, stages IIB/IIIB with bulk disease) Hodgkin’s lymphoma. Therapy consisted of intensified BEACOPP induction (4 cycles of bleomycin, etoposide, adriamycin, cyclophosphamide, vincristine, procarbazine, prednisone) followed by response-adapted continuation therapy. This regimen (Children’s Cancer Group Study 59704) is associated with a 4-year event-free survival rate of 95%. We found that polymorphic low/loss of function alleles were associated with an increased frequency of hematologic toxicities. We observed an increased frequency of therapy-associated thrombocytopenia in patients with absent GSTM1 (79% pts with grade 3/4 thrombocytopenia when GSTM1 absent vs 55% when present, p=0.0491). There was also an increased frequency of infection with neutropenia in patients with absent GSTT1 (57% when GSTT1 absent vs 23% when present, p=0.0522). The XRCC1 variant allele was associated with increased frequency of infection with neutropenia in a dose-dependent fashion (glutamine/glutamine: 67% with toxicity, arginine/glutamine: 27%, arginine/arginine: 13%; p= 0.0308). Similarly XRCC1 genotype was associated with increased frequency of thrombocytopenia (glutamine/glutamine: 67%, glutamine/arginine: 35%, arginine/arginine: 17%; p= 0.0498). Conclusion: These results, representing the only reported data in HL, suggest that deletion of GSTT1 and GSTM1, and variant alleles of XRCC1 may influence risk for toxicity with intensive BEACOPP chemotherapy. Prospective studies in future cohorts are needed to confirm these observations.