ALBERT

Description: Abstract 799 Introduction The accurate prediction and optimal management of transformed follicular lymphoma (t-FL) remains a significant clinical challenge. Investigation of the prognostic significance of genetic factors and the immune microenvironment in FL provides insight into the molecular pathogenesis of transformation and should aid in the development of robust molecular predictors of disease. Patient samples To address this issue, molecular profiling was performed on 91 samples including paired FL/t-FL samples from each of 31 patients (with additional germline (n=19) and relapse (n=10) samples where available). Median age at FL diagnosis was 46 years (range 22–71) with a median time to transformation of 4 years (1-16). Median follow-up of surviving patients was 10 years (2-19). Methods and Results The two most frequently mutated genes detected in FL/t-FL were TNFRSF14 (chr1p36.32) and EZH2 (chr7q36.1). Twelve TNFRSF14 mutations were detected in 10 patients (32%) with 3 arising in t-FL and one rendered homozygous on transformation. All were confined to exons 1–6 with 7/12 mutations causing premature termination of the protein. Heterozygous non-synonymous mutations in codon 641 of EZH2 occurred in 9 patients (29%) with 2 arising upon transformation. In mutated cases, the TNFRSF14 and EZH2 loci were subject to recurrent copy number (CN) loss/uniparental disomy (6/12) and gain (6/9) respectively. Genome-wide profiling was performed using the Affymetrix SNP 6.0 array to search for co-operating genetic events. A total of 1120 CN aberrations (CNAs) were identified across the cohort with a median of 5 gains (range 0–13) and 7 losses (0-42) in the earliest available FL (FL1) samples and 8 gains (0-33) and 10 losses (4-32) in the initial t-FL (t-FL1) samples. Recurrent CNAs included loss of chr1p, 6q, 9p, 17p and gain of 1q, 6p, 18 and × in both FL1 and t-FL1. In 18 cases, FL1 samples had CN changes absent from subsequent relapsed FL/t-FL samples. This is consistent with t-FL evolving from a common progenitor cell (CPC). Divergent changes seen in both FL and t-FL at relapse after t-FL1 further augment this hypothesis. CNAs common among different samples identify early events in FL/t-FL development. Recurrent regions of loss and gain affecting ≥20% of cases ranged in size from 4 kb to 60 Mb. Of these, gain 2p16.1-p15 (including REL and BCL11A) in FL1, predicted worse clinical outcome (survival from FL diagnosis; logrank p = 0.004, hazard ratio = 7). No cases with gain 2p16.1-p15, gain 8q24.13-q24.3, del 9p21.3 or gain 18q21.2-q21.33 were alive. Novel CNAs included gain 16p12.1 (containing IL4-R, IL21-R, NSMCE1) in 5 t-FL1 cases, recurrent deletion of ETS1 (n=4) on chr11q24.3 and further micro-deletions targeting CREBBP (chr16p13.3) and ALK (chr2p23.2). Conclusions The high mutational frequency of TNFRSF14 together with recurrent gain of IL4-R suggests a link between intra-cellular events and B-T cell interactions in the microenvironment. Enrichment of EZH2 mutations, the prognostic impact of gain REL/BCL11a and recurrent CN changes in CPC cases complete a core set of genetic changes that are potential molecular markers in t-FL. Disclosures: No relevant conflicts of interest to declare.

Description: Chronic lymphocytic leukemia (CLL) is associated with profound defects in immune function, resulting in failure of anti-tumor immunity and increased susceptibility to infection. We have previously demonstrated alterations in gene expression profiles of T cells from CLL patients, which translate into functional defects in T-cell immune synapse formation, motility and cytotoxicity (Gorgun et al. JCI 2005; Ramsay et al. JCI 2008, Blood 2013). However a comparison of the transcriptome of natural killer (NK) cells from CLL patients and controls has not been investigated. NK cells were isolated from the peripheral blood of patients with CLL and healthy donors, followed by gene expression profiling using the Affymetrix U133Plus2.0 platform. 117 probes showed a 〉2-fold decrease in expression while only 18 probes showed a 〉2-fold increase in expression (adjusted p-value 〈 0.05) in CLL NK cells compared to healthy donor NK cells. Strikingly, 52 out of the 117 significantly down-regulated probes (44.4%) were for interferon-inducible genes including STAT1 (Signal Transducers and Activator 1), SOCS1 (Suppressor of cytokine signaling 1), interferon regulatory factor genes IRF7 and IRF9, and oligoadenylate synthetase genes OAS1, OAS2, and OAS3. The majority of these genes were inducible by both type 1 and type 2 interferons. Many of these genes have been implicated in host immunity to viral infections, and so it is possible that decreased NK-cell responsiveness to interferon contributes to the increased susceptibility of CLL patients to viruses. Notably, there was also altered expression of signaling pathways in common with T cells from CLL patients, with dysregulation of the cytoskeleton genes RAB3GAP1, RAB38, and EPHA1 and down-regulation of JUN mirroring the dysregulated JNK-signaling and the altered actin cytoskeleton pathways we have found in T cells from CLL patients. These changes were not due to differences in the relative frequencies of CD56DIM and CD56BRIGHTNK cells. Lenalidomide has significant clinical activity in CLL. It is not directly toxic to tumor cells in vitro, but instead is thought to activate anti-tumor immunity and block pro-tumor micro-environmental factors. NK-cell proliferation has been shown to correlate with clinical response to lenalidomide in CLL (Chanan Khan et al. BJ Haem 2011). Therefore, we investigated the effect of lenalidomide treatment on the gene expression profiles of NK cells from CLL patients in comparison to healthy controls. PBMCs from CLL patients or healthy controls were cultured in the presence of 1μM lenalidomide or vehicle control for 48 hours, followed by NK-cell isolation, RNA extraction and gene expression profiling. There were striking differences in the effect of lenalidomide on NK cells from CLL patients compared with healthy NK cells. In CLL NK cells, lenalidomide repaired the down-regulation of interferon-inducible genes, by increasing the expression of genes such as OAS3, IFIT1, IFI44L, IFIT3, OAS1, PDK4, and ACTN1. Pathway analysis highlighted the effect of lenalidomide on inducing interferon signaling, showing significant activation of interferon α, γ, and λ as upstream regulators. While many of the interferon-inducible genes were up-regulated 〉3-fold in CLL NK cells, only OAS3 was significantly up-regulated in healthy NK cells with lenalidomide. Furthermore, the gene for IFNγ, IFNG, was actually significantly down-regulated in healthy NK cells by this agent. Lenalidomide also significantly down-regulated the expression of 5 genes encoding killer-cell immunoglobulin-like receptors (KIRs): KIR2DL1, KIR2DL2, KIR2DS3, KIR2DS4, and KIR3DL2, in healthy NK cells, but did not significantly down-regulate KIR genes in the CLL NK-cell dataset. Lenalidomide treatment did have some overlapping effects on CLL and healthy NK cells, including up-regulation of genes ARL11, CYFIP, and CORO1B that regulate the actin cytoskeleton pathway. In conclusion, NK cells from CLL patients have down-regulation of interferon response genes and pathways known to regulate normal immune function in response to bacteria and viruses. Lenalidomide has a differential effect on CLL and healthy NK cells: in CLL NK cells it repairs defective interferon responses, whereas in healthy NK cells it down-regulates inhibitory pathways. Disclosures: Riches: Celgene: Research Funding. Gribben:Celgene: Research Funding; Pharmacyclics: Honoraria; Roche: Honoraria.

Description: Abstract 141 Patients with follicular lymphoma (FL) have an extremely variable clinical course. Although clinical parameters can be used to define prognostic subgroups, there is a need to identify robust biomarkers that not only aid in prognosis, but also help define the underlying pathophysiology of the disease. Previous gene expression profiling studies demonstrated that among the most important prognostic markers were the molecular features of nonmalignant tumor-infiltrating immune cells present in the tumor at diagnosis (Sandeep et al. NEJM 2004). To investigate the molecular mechanisms whereby T cells are altered in the FL microenvironment we studied highly purified, sorted infiltrating CD4 and CD8 T cells from previously cryopreserved single cell suspensions of lymph node (LN) biopsies at the time of diagnosis in patients with FL (n=12) and compared them to those isolated from reactive tonsils (n=7) as well as from peripheral blood (PB) (n=10) of age matched healthy individuals. These tumor infiltrating T cells (TILs) have impaired proliferative and cytotoxic responses and impaired capacity to form immunologic synapses with antigen presenting cells. Co-culture of FL cells in contact with healthy allogeneic T cells induces similar T cell functional defects, demonstrating that it is the FL cells that drive the altered T cell function. To investigate the molecular mechanisms for this, we performed global gene expression profiling using Affymetrix U133Plus2 chips of highly purified (〉95% purity) CD4 and CD8 cells. For both CD4 and CD8 cells, unsupervised analyses distinguished healthy and FL T cell subsets. Using a fold change cut off 〉 2 and false discovery rate of 5%, 280 genes were differentially expressed for CD4 and 717 genes for CD8, with109 genes overlapped for both subsets. The gene array data was validated for RNA using Real-Time Quantitative-PCR and for protein by flow cytometry and immunohistochemistry. Pathway analysis indicated disruption in multiple pathways including cytokine signaling, T cell differentiation, cell proliferation and, actin-based motility/cytoskeleton formation. In both CD4 and CD8, among the most downregulated genes in FL TILs were ACTN1 and IL17A, and the most upregulated genes were PMCH and ETV1. Using Tissue Microarray (TMA) we demonstrate that the intensity of expression in TILs in FL was significantly higher for PMCH (p

Description: Abstract 769 Previous global gene expression profiling (GEP) studies have demonstrated non-malignant tumor-infiltrating immune cells present in the tumor at diagnosis were among the molecular features of the most important prognostic markers (Sandeep et al. NEJM 2004). We investigated the molecular mechanisms whereby tumor infiltrating T cells (TILs) are altered in the FL microenvironment and examined GEP of highly purified, sorted infiltrating CD4 and CD8 T cells from previously cryopreserved single cell suspensions of lymph node (LN) biopsies at the time of diagnosis in treatment naive patients with FL (n=12) and compared them to those isolated from reactive tonsils (n=7) as well as peripheral blood (PB) (n=10) of age matched healthy donors. In both CD4 and CD8, among the most downregulated genes in FL TILs were ACTN1 and IL17A, and the most upregulated genes were PMCH, ETV1, TNFRSF9 and NAMPT. PMCH is not expressed in PB T cells, but its expression is highly induced when healthy PB T cells are cultured, either in cell contact or in transwell culture, with FL cells. Using well characterized tissue microarrays of diagnostic FL samples (n=172) we now show that the T-cell expressed genes of PMCH, ETV1 and NAMPT have prognostic significance for survival and time to transformation in FL. Patients with higher percentage (p=0.039, median survival 10.59yr vs 19.20yr) and number (p=0.016, median survival 8.67yr vs 19.01yr) of PMCH expressing cells in intrafollicular and higher percentage (p=0.021, median survival 8.18yr vs 16.5yr) in interfollicular area had significantly longer disease specific survival (DSS) compared with patient with low number of PMCH expressing cells. Patients with higher percentage (p=0.014, median survival 19.01yr vs 11.75) and number (p=0.032, median survival 19.01yr vs 12.41yr) of ETV1 expressing cells in intrafollicular area showed significantly shorter DSS comparing to those with lower percentage and number of ETV1 expressing cells. Furthermore, the higher total MI of NAMPT expression showed significantly longer DSS (p=0.003, median survival 7.62yr vs 18.28yr) as well. Pathway analysis indicated disruption in multiple cellular pathways including actin-based motility/cytoskeleton formation which is in keeping with our previous studies where we have shown altered T cell expression of genes regulating actin in CLL (Gorgun et al, JCI 2005) and AML (De Lieu et al, Blood 2009). Using Time-Lapse Imaging we demonstrated both CD4 and CD8 TILs from patients with FL (n=7) have significantly impaired motility compared to those of healthy TILs from reactive tonsil (n=4) (p

Description: Abstract 976 Lenalidomide has recently been demonstrated to have significant activity in chronic lymphocytic leukemia (CLL). Its mechanism of action in this disease is not well understood, but it is thought to act primarily by enhancing anti-tumor immunity and reducing production of pro-tumoral factors in the CLL microenvironment. We have previously demonstrated alterations in the expression of cytoskeletal genes in T-cells from patients with CLL and have subsequently shown that these changes translate into a deficit in T-cell function, due to impaired actin polymerization resulting in defective immunological synapse formation. Treatment of both autologous T-cells and CLL cells with lenalidomide was necessary to repair this defect, suggesting that this may be a key component of this agent's activity in CLL. Therefore we examined the effect of lenalidomide on the global gene expression profiles of isolated B-cells and T-cell subsets from CLL patients and healthy donors. Peripheral blood mononuclear cells from patients with untreated CLL or healthy donors were cultured in the presence of 1 μM lenalidomide or vehicle control for 48 hours. The lymphocyte subsets were isolated, followed by RNA extraction and gene expression profiling using the Affymetrix HGU133Plus2.0 platform. Lenalidomide treatment had similar effects on gene expression in T-cells from both patients with CLL and healthy donors. The most prominent changes in expression were of genes involved in cytoskeletal signaling including a 20-fold increase in WASF1 (Wiskott Aldrich Syndrome protein family, member 1), and greater than 2-fold increases in the expression of Rac-family member RHOC, (Ras homolog gene family, member C), actin binding proteins CORO1B (Coronin 1B), PARVA (Parvin alpha), and the Rho guanine nucleotide exchange factors (GEFs), ARHGEF5 and ARHGEF7. We also observed changes in genes regulating integrin signaling including PXN (Paxilin) and FAK (Focal adhesion kinase), and a shift towards Th1 differentiation with upregulation of TNF, IL-12R, and IL-18R. In addition, we noted increased expression of the transcription factors IKZF1, IKZF4 and IRF4, genes involved in the Ikaros pathways that are essential for hematopoiesis and control of lymphoid proliferation. These changes in gene expression provide further evidence that an important mechanism of action of lenalidomide is the upregulation of the actin cytoskeletal network including Rho-GTPases and integrin activation signaling, and are consistent with our previous observations concerning the functional repair of T-cells in CLL. Initial analysis of the effect of lenalidomide on the gene expression profiles of the CLL B-cells showed similar changes to those previously described in vivo from CLL patients receiving single agent lenalidomide in a clinical trial (Chen et al. JCO 2010). In our system, lenalidomide treatment resulted in a greater than 2-fold upregulation of 189 genes, and a greater than 2-fold downregulation of 85 genes in CLL B-cells. We observed increased expression of several genes belonging to the TNF superfamily including TNF-α, OX40L, and APRIL, and the receptors DR5, DCR2, and OX40. Many of these are known to mediate apoptosis signaling, and we also observed increased expression of pro-apoptotic genes such as FAS, BID (BH3 interacting domain death agonist), HRK (Harakiri), and CFLAR (CASP8 and FADD-like apoptosis regulator), and cell cycle regulators CDKN1A and CDKN1C (Cyclin-dependent kinase inhibitors 1A and 1C). Lenalidomide also upregulated expression of several genes of known importance in the CLL microenvironment, including the chemokines CCL3 and CCL4, CD40, CD274 (PD-L1), CD279 (PD-1), and adhesion molecules LFA3 and ICAM1. The effect of lenalidomide on the gene expression profiles of normal B-cells was less marked, with greater than 2-fold upregulation of 51 genes and downregulation of 12 genes. However, we did observe that lenalidomide treatment induced upregulation of genes involved in cytoskeletal pathways such as RND1 (Rho family GTPase 1), RHOQ (Ras homolog gene family, member Q), and MYO1B (myosin 1B). In conclusion, investigation of the effect of lenalidomide on gene expression profiling in CLL suggests that the drug acts both to enhance T-cell function, and to render the CLL cells more susceptible to immune cell mediated killing. Disclosures: Gribben: Roche: Honoraria; Celgene: Honoraria; GSK: Honoraria; Mundipharma: Honoraria; Gilead: Honoraria; Pharmacyclics: Honoraria.