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e18567 Background: Argininoosuccinate synthetase-1 (ASS1) catalyses the rate-limiting step in arginine biosynthesis, the conversion of citruline to arginine. Argininosuccinate lyase (ASL) is an enzyme that catalyses the reversible breakdown of arginosuccinate producing arginine and fumerate. Arginine deprivation using arginine deiminase (ADI-PEG20) is currently considered as a novel therapeutic intervention for cancer. In this perspective, we investigated the methylation status of the ASS1 and ASL CpG islands in multiple myeloma (MM) and analyzed for clinical relevance. Methods: Genomic DNA was extracted from bone marrow aspirate samples from 46 MM patients (28 male, 18 female, median age 64 years) obtained at diagnosis. Methylation-specific PCR was employed to study the methylation in the ASS1 and ASL CpG islands. DNA was isolated and bisulphite modified using commercially available kits. Logistic regression analyses were used to measure the association between gene methylation and sex, age>65, ISS stage, presence of extramedullary disease, renal failure and bone disease. Kaplan-Meier curves were used to estimate the probabilities of survival and the Log-rank test to assess the statistical significance of differences in event rates. Results: Methylation in the CpG island of ASS1 was detected in 71.7% patients and of ASL in 37% patients, while simultaneous methylation in both genes was present in 10 patients. None of the two genes was detectably methylated in the control group. Patients with ASS1 methylation were less likely to have bone disease (p=0.04, OR=0.22) and extramedullary disease (p=0.05, OR=0.22) and a trend was also noted that these patients were less likely to be >65 years of age (p=0.2, OR=0.37). We did not detect any statistically significant difference in overall survival by methylation status of the studied genes in this small study size. Conclusions: We demonstrate for the first time that arginine biosynthesis genes ASS1 and ASL are methylated in MM.Methylation of ASS1 was found to be more frequent and negatively associated with bone or extramedullary disease. Further evaluation of ASS1 and ASL is warranted in MM and supports the expansion of arginine deprivation trials in these patients

8093 Background: Arginine is a semi-essential amino acid, critical to the growth of human cancers. Downregulation of the enzyme argininosuccinate synthetase (ASS1), a key rate-limiting enzyme involved in arginine metabolism, results in an intrinsic dependence on extracellular arginine with arginine deprivation offering a potential treatment option for patients with arginine auxotrophic tumors. Several early phase clinical trials of the arginine lowering drug, pegylated arginine deiminase (ADI-PEG20), have demonstrated clinical efficacy in patients with solid tumors. Methods: We assessed ASS1 expression (ASS1 promoter methylation, ASS1 mRNA and protein levels) in normal and malignant lymphoid cell lines and tissues, and tested the effect of ADI-PEG20. Results: The ASS1 promoter is hypermethylated in lymphomas using the Illumina Infinium methylation platform and provided a discriminator between lymphoma (cell lines and primary tumor samples, n=20) and B cell controls (benign lymph nodes and lymphoblastoid c...

8118 Background: BIK (bcl2-interacting killer) is the founding member of the BH3-only family proapoptotic proteins. Genomic deletions, epigenetic silencing or post-transcriptional downregulation of...

e19537 Background: Multiple myeloma (MM) is an incurable interleukin (IL)-6 dependent plasma-cell malignancy. Transforming growth factor-β (TGF-β) is the major inducer of IL-6 secretion by bone marrow stromal cells. The signaling responses to TGF-b are mediated by the Smad proteins. The Smurf2 gene (Smad ubiqitiniation regulatory factor 2) encodes a Smad-specific E3 ubiquitin ligase and targets Smad2 and Smad3 for proteasome-dependent degradation. Methods: Bone marrow samples from individuals with MM were obtained at diagnosis and in 5 cases at disease progression as well. Genomic DNA was isolated and bisulphite modification was performed using commercially available kits. The methylation-specific polymerase chain reaction was employed to study the methylation status of the CpG island. Logistic regression analysis was used to measure the association between gene methylation and the development of advanced disease (DS≥ II), extramedullary disease, bone disease, anemia (Hb 10 mg/dl), serum albumin and beta 2 microglobulin levels. Results: We analysed the methylation of Smurf2 in 45 cases of MM (24 male, 21 female, mean age 66.4 years). No sample from the control population was found methylated. The Smurf2 gene promoter was found to be methylated in 11/45 MM patients (24%). Interesting trends were noted where patients with methylated Smurf2 promoter had an increased risk of death (HR = 1.3; p = 0.68), anemia (OR=2.1, p=0.2) and advanced stage (OR=1.3, p=0.6) and a reduced risk of extramedullary disease (OR= 0.2, p=0.2). No association was found between Smurf2 methylation status and bone lytic lesions, serum albumin levels or beta-2 microglobulin levels. Conclusions: Interesting associations between Smurf2 methylation and some relevant clinical parameters in patients with MM were suggested by the data. These findings warrant further evaluation in a larger sample of patients in order to enhance our statistical power and better define the prognostic and clinical value of Smurf2 methylation in MM. No significant financial relationships to disclose.

e19532 Background: We previously showed that polo like kinase 2 (Snk/Plk2) is subject to methylation-dependent transcriptional silencing in a very high frequency in Burkitt lymphomas. Here, we have examined CpG methylation in Snk/Plk2 in a well-characterized series of multiple myeloma (MM) patients. Methods: Bone marrow samples from individuals with MM were obtained at diagnosis and in 5 cases at disease progression as well. Genomic DNA was isolated and bisulphite modification was performed using commercially available kits. The methylation-specific polymerase chain reaction (MSP) was employed to study its methylation status. Control methylated and unmethylated genomic DNAs were included in each experiment. Ten bone marrow samples from individuals proven to have no haematological malignancy, served as negative controls. Logistic regression analyses were used to measure the association between gene methylation and the development of advanced disease (DS≥II), extramedullary disease, bone disease, anemia (Hb 10 mg/dl), serum albumin and beta 2 microglobulin levels. Results: We analyzed the methylation of Snk/Plk2 in 45 cases of MM (24 male, 21 female, mean age 66.4 years ±12.4). Classical cytogenetic analysis was available in 30/45 patients and none of them was found to have chromosome 13 abnormalities. No sample from the control population was found methylated. The Snk/Plk2 promoter was found to be methylated in 27/45 patients (60%). Median survival of patients in whom the Snk/Plk2 CpG island was methylated was 7.1 years compared to a median survival of 8.8 years for unmethylated. However Snk/Plk2 methylation was not a predictor of excess mortality (HR=0.6, p=0.5), bone lytic lesions (OR=0.56, p=0.3), anemia (OR=0.5, p=0.3) or advanced stage as defined above (OR=0.8, p=0.7). Conclusions: Snk/Plk2 DNA Methylation is a frequent event in patients with multiple myeloma. There was no association between the methylation status of the gene and relevant clinical parameters. Further evaluation in a larger sample of patients is needed in order to better define the prognostic and clinical value if any of Snk/Plk2 methylation in MM. No significant financial relationships to disclose.