A Polymerase Chain Reaction-Based Method to Detect Gene-Specific Adducts Induced by Anticancer Drugs. Clinical Application in Multiple Myeloma.

Abstract 1879

Poster Board I-903

DNA repair plays an important role in the protection of cells and tissues after exposure to genotoxic agents including chemotherapeutics. We have previously shown that, in peripheral blood mononuclear cells (PBMC) of multiple myeloma (MM) patients treated with melphalan, accumulation of DNA adducts in the p53 gene correlates with better therapeutic response, and that repair in different genes correlated with the gene transcriptional activity and the degree of local chromatin condensation (Dimopoulos et al, J Clin Oncol 2005;23:4381–9; Souliotis et al, DNA Repair 2006;5:972–85; Dimopoulos et al, Haematologica 2007;92:1505–12). However, the assays used are fairly time-consuming, and require complex procedures such as Southern transfer and hybridization. Thus, we now present the development and clinical application in MM of a gene-specific, quantitative method for measuring DNA damage formation/repair following exposure to anticancer drugs inducing bulky adducts. Cell line (HepG2) as well as human whole blood and PBMC from eighteen patients (13M/5F) with MM were in vitro treated with melphalan. These patients underwent high dose melphalan with autologous stem cell support (ASCT) as part of their first line therapy and the whole blood was collected on the day of stem cell mobilization. Ten (55.5%) patients achieved further myeloma reduction after ASCT; 3 patients achieved a stringent complete response (CR), 2 a CR, 2 a very good partial response (vgPR) and 3 a PR. Among 8 non-responders post-ASCT, 6 had a stable disease while 2 experienced disease progression, according to the IMWG criteria. None of the patients had previously received alkylating agent therapy (melphalan-naive patients). Moreover, cell line (HepG2) and PBMC from five healthy volunteers (all females) were treated with platinum-based drugs (cisplatin, carboplatin). Following DNA isolation, gene-specific damage formation/repair was examined using Southern blot as well as a multiplex long quantitative PCR (Q-PCR). The extent of PCR amplification was conversely proportional to the treatment concentrations of all anticancer drugs examined, implying dose-related inhibition by the DNA adducts formed. In the case of melphalan, the adduct levels measured by Q-PCR were identical to the levels of interstrand cross-links (ICL) measured by Southern blot analysis. In addition, monoadducts induced by monofunctional melphalan could not be measured by Q-PCR, suggesting that this assay measures only melphalan-induced ICLs. Application of the Q-PCR assay to in vitro-treated human blood samples from MM patients taken prior to ASCT showed that the levels of DNA damage varied up to 12-fold, which probably reflects inter-individual DNA repair differences. Interestingly, significantly greater gene-specific damage was found in the responders group compared to non-responders [176.8±67.3 adducts/10⁶ nucleotides (range 41.0 to 273.0) for responders and 65.1±39.4 adducts/10⁶ nucleotides (range 22.0 to 135.0) for non-responders, p=0.002]. Similar results were obtained using whole blood from the same MM patients, but differences did not reach statistical significance [84.3±63.0 adducts/10⁶ nucleotides (range 15.0 to 165.0) for responders and 46.5±2.1 adducts/10⁶ nucleotides (range 45.0 to 48.0) for non-responders, p=0.5]. As for the platinum-based drugs, cisplatin-induced intrastrand cross-links levels measured by Southern blot analysis, reached a plateau within ∼3h of treatment, while peak interstrand cross-links was obtained at ∼24h of exposure. Carboplatin-induced maximal levels of both intra- and interstrand cross-links were obtained within 24h of drug incubation. Parallel analysis of the same samples using both Southern blot and Q-PCR showed that the DNA adducts measured by Q-PCR correspond to total platinum-induced lesions. In conclusion, our study suggest that by using the current Q-PCR methodology, it is feasible to measure gene-specific damage formation/repair in a readily accessible biological material such as PBMC from humans exposed to anticancer drugs inducing bulky adducts and to examine, at the level of individual patient, the relationship between the induction/repair of cytotoxic DNA damage and the clinical outcome. Patient accrual is ongoing and updated results will be presented during the meeting.