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Folate and methionine metabolism is involved in DNA synthesis and methylation processes. Polymorphisms in the genes of folate metabolism enzymes have been associated with some forms of cancer. In a case-control study, we evaluated whether four common polymorphisms in methylenetetrahydrofolate reductase (MTHFR C677T and A1298C), methionine synthase (MS A2756G), and methionine synthase reductase (MTRR A66G) genes may have a role in altering susceptibility to adult acute lymphoblastic leukemia (ALL) and non-Hodgkin's lymphoma (NHL). We analyzed DNA of 120 adult ALL, 200 NHL, and 257 healthy control subjects. Individual carrying the MTHFR 677TT genotype showed a 3.6-fold decreased ALL risk [odds ratio (OR) 0.28, 95% confidence interval (95% CI) 0.12–0.72] than wild-types. Similarly, MS 2756GG individuals showed a 5.0-fold decreased ALL risk (OR 0.20, 95% CI 0.02–1.45) than wild-types. In combined results, subjects with the MTHFR 677CT/TT and MS 2756AG/GG genotypes revealed a 3.6-fold ALL risk reduction (OR 0.28, 95% CI 0.14–0.58) and those with the MTHFR 677TT and MTRR 66AG genotypes revealed a 4.2-fold ALL risk reduction (OR 0.24, 95% CI 0.06–0.81). Finally, those with the MS 2756AG/GG and MTRR 66AG/GG genotypes revealed a 2.2-fold ALL risk reduction (OR 0.45, 95% CI 0.10–0.85). Single analysis for NHL did not show any significant difference for all the polymorphisms investigated, but in the low-grade NHL subgroup, we found a 2.0-fold risk reduction for the MTRR 66GG homozygous genotype (OR 0.50, 95% CI 0.25–0.99), which was higher (OR 0.37, 95% CI 0.14–0.85) when analyzed in combination with MS 2756AA genotype. These data are in accordance with the hypothesis that polymorphisms in the genes for folate and methionine metabolism might play a greater role in the occurrence of ALL than NHL by influencing DNA synthesis and/or DNA methylation.

Gene amplification and copy number changes play a pivotal role in malignant transformation and progression of human tumor cells by mediating the activation of genes and oncogenes, which are involved in many different cellular processes including development of drug resistance. Since doxorubicin (DX) and methotrexate (MTX) are the two most important drugs for high-grade osteosarcoma (OS) treatment, the aim of this study was to identify genes gained or amplified in six DX- and eight MTX-resistant variants of the human OS cell lines U-2OS and Saos-2, and to get insights into the mechanisms underlying the amplification processes. Comparative genomic hybridization techniques identified amplification of MDR1 in all six DX-resistant and of DHFR in three MTX-resistant U-2OS variants. In addition, progressive gain of MLL was detected in the four U-2OS variants with higher resistance levels either to DX or MTX, whereas gain of MYC was found in all Saos-2 MTX-resistant variants and the U-2OS variant with the highest resistance level to DX. Fluorescent in situ hybridization revealed that MDR1 was amplified in U-2OS and Saos-2/DX-resistant variants manifested as homogeneously staining regions and double minutes, respectively. In U-2OS/MTX-resistant variants, DHFR was amplified in homogeneously staining regions, and was coamplified with MLL in relation to the increase of resistance to MTX. Gene amplification was associated with gene overexpression, whereas gene gain resulted in up-regulated gene expression. These results indicate that resistance to DX and MTX in human OS cell lines is a multigenic process involving gene copy number and expression changes.

Purpose: The paucity of new drugs for the treatment of Ewing sarcoma (EWS) limits the cure of these patients. CD99 has a strong membranous expression in EWS cells and, being also necessary for tumor survival, is a suitable target to aim at. In this article, we described a novel human monospecific bivalent single-chain fragment variable diabody (dAbd C7) directed against CD99 of potential clinical application. Experimental Design: In vitro and in vivo evaluation of cell death and of the molecular mechanisms triggered by anti-CD99 agents were performed alone or in combination with doxorubicin to demonstrate efficacy and selectivity of the new dAbd C7. Results: The dAbd C7 induced rapid and massive EWS cell death through Mdm2 degradation and p53 reactivation. Mdm2 overexpression as well as silencing of p53 in p53wt EWS cells decreased CD99-induced EWS cell death, whereas treatment with nutlin-3 enhanced it. Furthermore, cell death was associated with induction of p21, bax, and mitochondrial depolarization together with substantial inhibition of tumor cell proliferation. Combined treatment of anti-CD99 dAbd C7 with doxorubicin was additive both in vitro and in vivo against EWS xenografts. Normal mesenchymal stem cells showed no p53 activation and were resistant to cell death, unless transformed by EWS-FLI, the oncogenic driver of EWS. Conclusions: These results indicate that dAbd C7 is a suitable candidate tool to target CD99 in patients with EWS able to spare normal stem cells from death as it needs an aberrant genetic context for the efficient delivery of CD99-triggered cell death. Clin Cancer Res; 21(1); 146–56. ©2014 AACR.