Background The "double hit" (DH) lymphomas that harbor a c-myc mutation and BCL2 translocation, or "double protein expressor" (DP) lymphomas that over-express c-myc and BCL2 proteins in the absence of a detectable mutation, have amongst the worst clinical outcomes as compared to patients with diffuse large B-cell lymphomas (DLBCL) that lack upregulation of the c-myc oncogene. Metformin can down-regulate translation of c-myc, making it an appropriate anti-cancer drug to explore in c-myc+ lymphomas. Furthermore, amethod to identify DH/DP patients most likely to benefit from metformin treatment has clinical relevance. Methods Within a publicly available gene expression array data set of R-CHOP treated DLBCL (n=232; GSE10846), a subset of DH/DP patients were defined as having above median expression of myc and BCL2 and below median expression of BCL6 as previously published by Horn et al. Survival analysis, significance analysis of microarrays (SAM) and gene set analysis (GSA) were performed characterizing the clinical, individual gene and biological ontology differences between DH/DP and non-DH/DP populations. Expression array data from a study testing metformin effects on THP-1 monocyte cells was reanalyzed using SAM and GSA as well. Changes in individual gene expression and overlapping ontological themes common to both GSA analyses of metformin effects on THP-1 cells and DH/DP characterization were identified. Genes with differential expression (DE) in both groups were evaluated topologically using a protein-protein interaction database to determine if any gene products had previously observed direct interactions. Network community detection identified tightly coupled signaling modules linking co-expression to mechanism. The resulting metformin-DH/DP network metagene was evaluated by k-means, clustering tumor samples into two groups over the metagene members in an independent data set of R-CHOP treated DLBCL patients (n=249; GSE32918) with differences in overall survival (OS) determined by log-rank. Results Of the 232 DLBCL patients treated with R-CHOP, 26 fit the criteria for DH/DP and had significantly lower OS (HR = 2.96; p < 0.001). In DH/DP tumors, 2780 genes had DE (2208 up-regulated; 572 down-regulated), enriched for biological processes related to transcription, metabolism and cytokine production and down-regulated for processes related to immune response, cell signaling, vascular development and proliferation (Fig. 1A). Analysis of metformin treated THP-1 cells relative to control identified 7123 genes with DE. Biological themes common to metformin treatment and DH/DP specific biology were identified including mitochondrial biogenesis, alternate splicing, and hormone secretion (Fig. 1A-B; highlighted in red). The intersection of genes with DE in metformin treated and DH/DP data sets identified 102 genes with direct interaction within a protein interaction network. Of the 19 communities detected by analyzing the resulting network topology, 3 showed significant correlation to survival in the GSE10846 data set (Fig. 2A, in red), forming a metformin-DH/DP metagene (Met-DH/DP-MG; n = 29 genes total). This metagene was validated by applying it to an independent cohort of R-CHOP treated DLBCL patients (n = 249), demonstrating 2 cluster groups (cluster 1, n=178; cluster 2, n=71; Fig. 2B) with differences in OS (HR = 1.61; p < 0.001; Fig. 2C). Conclusion We have identified a metagene of interacting proteins associated with both metformin therapeutic effect and OS in DH/DP patients. This offers a potential method for selecting patients most likely to benefit from metformin therapy and identifies mechanistic avenues by which metformin treatment may specifically benefit DH/DP patients. As such, in vitro studies using DH cell lines and a phase I/II clinical trial exploring chemo-immunotherapy with metformin as an adjunct in DH/DP lymphomas are underway. Disclosures No relevant conflicts of interest to declare.

Smokeless tobacco (ST), an alternative to smoking, has gained wide popularity among tobacco users. This study is conducted to determine the time course of gene expression associated with specific signaling pathways in human oral epithelial cells after exposure to smokeless tobacco extract (STE). A differentiated layer of epithelial cell is created as a way to mimic reasonably similar physiological atmosphere. A dose and time dependent response is observed for cell viability and cell proliferation assays indicating that this model system is responsive to the treatment. Expressions of 84 genes representing 18 different signal transduction pathways are quantitated. This is accomplished by using real-time polymerase chain reaction arrays at 1 h, 3 h, 6 h and 24 h time points following exposure to STE. Changes in gene expression are observed on many cellular processes including cell cycle regulation, cell adhesion, inflammation, apoptosis, and DNA breaksdown including Akt pathway activation. Short time exposure (1 h) leads more genes to down regulate whereas longer incubation time results in more genes up regulation. Most notable differences in the expression of genes during the course of treatment are BCL2A1, BIRC3, CCL20, CDK2, EGR1, FOXA2, HOXA1, IGFBP3, IL1A, IL-8, MMP10, NOS2, NRIP1, PTGS2, SELPLG and TNF-a. This study provides an insight on gene expression on oral epithelial cells as a result of STE exposure. This may also postulate greater understanding on biological effects and the mechanism of action of STE particularly at the transcriptional level.