Molecularly Classified High Risk Myeloma Cells with Low ROS Levels and High Nestin Expression Have High Proliferation Index and GEP80 Score,

Abstract 3953

Certain tumor cells have an elevated level of reactive oxygen species (ROS) due to oncogenic signaling and the downregulation of mitochondrial function, known as oxidative stress. Tumor cells with elevated oxidative stress have activated ROS-scavenging systems to cope with this stress and inhibit apoptosis. Lower levels of antioxidants and elevated levels of malondialdehyde, the end-product of lipid peroxidation, an indicator of oxidative stress, were detected in multiple myeloma (MM) patients compared with healthy controls (Sharma et al., 2009). Thus, oxidative stress is increased in patients with MM and may be associated with its pathogenesis. To investigate this, ROS levels were measured by DCF staining and FACS analysis of CD138-selected plasma cells (PCs) immediately after isolation. H₂DCFDA is a cell-permeant indicator that is converted to a polar species intracellularly and fluoresces once oxidized, thus DCF also serves as a vital stain. To verify that ROS levels are detected in viable cells, PCs were stained with DCF or AAD, an apoptosis marker, and were inversely correlated (p < 0.0001). PCs from MGUS (n=5), Smoldering/indolent MM (n=10), newly diagnosed (n=204) and relapsed (n=73) MM were analyzed and shown to have variable levels of oxidative stress within each group independent of stage. Correlation analysis between ROS levels and our molecular classification data reveals that MMPCs of the MF subtype have lowest ROS levels CD2, MS, and CD1 are intermediate, and LB, HY, and PR have highest levels of ROS. To better understand the differences between MMPCs with low (Q1) and high (Q4) ROS in newly diagnosed patients, we examined their GEP by mean ratio analysis (≥ 2-fold) and divided them as follows; low and high ROS PCs from HY and non-HY subsets. Low ROS/non-HY samples overexpress NES, genes of the MF subtype (e.g.MAF, MAFB, SPP1, CX3CR1, SMARCA1)) and CD1/CD2 subtype (e.g. CD20, CCND1, VPREB3). High ROS/non-HY samples overexpress LB subtype genes (e.g. CST6). AREG, EGFR and CCND2 were elevated in low ROS/HY whereas S100A9, CD45, TNFSF10 were elevated in high ROS/HY along with MGST1, a glutathione transferase which may enhance survival of cells with elevated ROS. Several genes were also examined in individual samples to verify differential expression including CCND1 (p <0.001), ITGB7 (p <0.05), TNFSF10 (p =0.001), CD20 (p <0.05) and NES (nestin, p < 0.001). Interestingly, NES, a gene highly expressed in the LB subtype and located on chr1q which is frequently amplified in MM, is elevated 4.2-fold in Low ROS/non-HY MMPCs independent of subtype. Genes that positively correlate with NES are predominantly on chr1q and include S100A4, GPR89A, CACYBP and a low ROS gene, CCND2. TNFSF10 and GNG11 are expressed in the HY subtype (high ROS) and negatively correlate with NES. However, not all low ROS MMPCs have elevated NES. In fact, high NES /Low ROS cases are more likely to have Chr1 and/or 13 cytogenetic abnormalities (15/22) and higher plasma cell percentage in BM aspirates (45%±5.5%) whereas 10/29 low NES /low ROS cases have Chr1 and/or 13 abnormalities and lower percent of plasma cells in BM (25%±3.5%, p < 0.01). In low ROS/high risk cases those with high NES have a higher proliferation index, 80-gene risk score, and have a 3-fold greater level of PRDX1 compared with low NES cases. PRDX1 encodes an antioxidant which may enhance survival of these cells. Taken together these data suggest that, contrary to the paradigm of ROS in tumorigenesis, in MM low ROS samples with elevated NES may be associated with more aggressive disease and should be investigated further.