Clinical impact of the methotrexate resistance-associated genes C-MYC and dihydrofolate reductase (DHFR) in high-grade osteosarcoma. Aims of this study were the validation of C-MYC involvement in methotrexate (MTX) resistance and the assessment of clinical impact of C-MYC and dihydrofolate reductase (DHFR) in osteosarcoma (OS). Materials and methods: The involvement of C-MYC in MTX resistance was validated with an antisense approach. C-MYC and DHFR protein levels at diagnosis were assessed by immunohistochemistry on series of patients treated with either a MTX-based protocol (IOR/OS-1; 72 patients) or with a standard four-drug regimen (ISG/SSG 1; 61 patients).Down-regulation of C-MYC significantly decreased the MTX resistance level of OS cells, demonstrating its causal involvement in this phenomenon. In clinical samples, a worse outcome was associated with increased levels of DHFR and C-MYC at diagnosis in the IOR/OS-1 patients and of C-MYC in the ISG/SSG 1 patients.Meanwhile the adverse clinical impact of DHFR overexpression appeared to be closely related to the relevance of MTX in the chemotherapeutic protocol, that of C-MYC overexpression was more general and not strictly MTX related. The assessment of C-MYC and DHFR at diagnosis, together with that of other known prognostic markers, can be considered for an early identification of subgroups of OS patients with higher risk of adverse outcome.