Differences In DNA Damage Response Pathways In the PBMCs of Patients with MGUS, Asymptomatic Myeloma and Symptomatic Multiple Myeloma

Abstract 2974

Multiple myeloma (MM) is a plasma cell malignancy which progresses from a “pre-malignant” precursor condition, the monoclonal gammopathy of undetermined significance (MGUS). Asymptomatic MM (AMM) is characterized by the absence of any myeloma related end-organ damage. The risk of progression of AMM to symptomatic MM is approximately 10% per year, while the risk of progression of MGUS is 1% per year. Previous work from our group showed that melphalan-induced DNA damage, formation/repair in the peripheral blood mononuclear cells (PBMCs) was associated with response and progression-free survival in MM patients who underwent high dose melphalan (HDM) with autologous stem cell transplantation (ASCT). The aim of this study was to compare DNA damage response pathways in the PBMCs of patients with MGUS, AMM, symptomatic MM and healthy controls. Three molecular end-points (chromatin condensation, transcription activity, melphalan-induced DNA damage formation/repair) were measured in PBMCs in four genomic loci (beta-actin, p53, N-ras and delta-globin genes). Furthermore, accumulation of p53 protein, recovery of both total RNA and poly(A) mRNA synthesis as well as induction of apoptosis were also studied in the PBMCs from 12 healthy volunteers (7M/5F; median age: 41 years), 10 patients with MGUS (5M/5F; 68.5 years), 10 with AMM (4M/5F; 64 years), and 32 patients with symptomatic MM (14M/18F; 59 years) who underwent HDM and ASCT as part of their first line therapy. In all subjects, beta-actin, p53 and N-ras genes were transcriptionally active. Importantly, delta-globin gene was silent in all healthy volunteers and MGUS patients, while an induction of the transcription activity of this gene was found in all MM patients and in 90% of MM patients (29/32). In all subjects, more relaxed chromatin structure and faster repair were observed in regions inside beta-actin, p53 and N-ras genes, compared to regions on both sides of the genes, while for delta-globin such a difference was observed only in AMM (10/10) and symptomatic MM patients (29/32). In all subjects, 5′- to 3′-end gradients of chromatin condensation and repair efficiency were observed along the active genes, with higher looseness of chromatin structure and faster repair at the 5′-end. Interestingly, inside all genes analyzed, repair was much slower in healthy volunteers relative to MM patients; the difference was greater and statistically significant at the 5′-end of the genes (p<0.001). The repair efficiency in MGUS and AMM patients were intermediate between healthy volunteers and symptomatic MM patients in all genomic loci studied. Moreover, cellular DNA damage response pathways were also investigated. After exposure to melphalan for 5 minutes, PBMCs from all healthy volunteers showed evidence of p53 protein accumulation at doses as low as 10μ g/ml, while for MGUS patients at 25μ g/ml (8/10), for AMM patients at 50μ g/ml (9/10), and for all symptomatic MM patients melphalan doses of at least 75μ g/ml were required. It has been suggested that melphalan adducts, when located in the transcribed strand of active genes, can act as blocks for the elongation of RNA polymerases. In this study, PBMCs from all symptomatic MM patients had almost fully recovered both poly(A) mRNA and total RNA synthesis to control levels by 6 hours following exposure to 100μ g/ml of melphalan for 5 minutes, while PBMCs from healthy volunteers showed a more extensive and prolonged inhibition of RNA synthesis (by 24h), PBMCs from MGUS patients showed full RNA recovery by 18h, and from AMM patients by 12h. Significant differences in the induction of melphalan-induced apoptosis were found between PBMCs from healthy volunteers and symptomatic MM patients, while samples from MGUS and AMM patients showed intermediate characteristics: induction of apoptosis was evident following 10μ g/ml melphalan in PBMCs from all healthy volunteers, 25μ g/ml in MGUS, 50 μ g/ml in AMM and 100μ g/ml in all symptomatic MM patients. In conclusion, our study provides evidence that “intermediate” level abnormalities in the DNA damage response pathways can be detected in PBMCs taken from MGUS and AMM patients. These results warrant further investigation in order to identify possible links with the molecular mechanisms that drive malignant evolution in plasma cell disorders. Furthermore, our findings may help to identify molecular markers that may be used as predictors of progression in patients with MGUS or AMM.