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Previous studies have shown that sphingosine kinase interacting protein (SKIP) inhibits sphingosine kinase (SK) function in fibroblasts. SK phosphorylates sphingosine producing the potent signaling molecule sphingosine-1-phosphate (S1P). SKIP gene (SPHKAP) expression is silenced by hypermethylation of its promoter in acute myeloid leukemia (AML). However, why SKIP activity is silenced in primary AML cells is unclear. Here, we investigated the consequences of SKIP down-regulation in AML primary cells and the effects of SKIP re-expression in leukemic cell lines. Using targeted ultra-HPLC-tandem MS (UPLC-MS/MS), we measured sphingolipids (including S1P and ceramides) in AML and control cells. Primary AML cells had significantly lower SK activity and intracellular S1P concentrations than control cells, and SKIP-transfected leukemia cell lines exhibited increased SK activity. These findings show that SKIP re-expression enhances SK activity in leukemia cells. Furthermore, other bioactive sphingolipids such as ceramide were also down-regulated in primary AML cells. Of note, SKIP re-expression in leukemia cells increased ceramide levels 2-fold, inactivated the key signaling protein extracellular signal-regulated kinase, and increased apoptosis following serum deprivation or chemotherapy. These results indicate that SKIP down-regulation in AML reduces SK activity and ceramide levels, an effect that ultimately inhibits apoptosis in leukemia cells. The findings of our study contrast with previous results indicating that SKIP inhibits SK function in fibroblasts and therefore challenge the notion that SKIP always inhibits SK activity.

Endogenous opioid peptides and exogenous opioids modulate immune function, and animal and human studies have shown that some have a depressant immunomodulatory effect. This is potentially of high clinical significance, eg, in cancer patients and surgery. The primary objective of this pilot study was to evaluate the effect of morphine and oxycodone on immune pathways associated with immunosuppression in gynecological laparotomy patients. Gene expression was analyzed in CD4, CD8, and natural killer (NK) cells using the 3' Affymetrix microarray. Patients were randomized to receive morphine, oxycodone, or nonopioid "control" analgesia during and after surgery. Genes demonstrating differential expression were those with a ≥±2-fold difference and P-value ≤0.05 after analysis of variance. Cytometric bead array and NK cell degranulation assay were used to investigate changes in serum cytokine concentration and in NK cell cytotoxicity, respectively. Forty patients had satisfactory RNA which was hybridized to gene chips. Genes were identified (Partek Genomics Suite 6.6) at baseline, 2, 6, and 24 hours and were either ≥2-fold upregulated or downregulated from baseline. At 2 hours, a large number of genes were downregulated with morphine but not with control analgesia or oxycodone. Statistically significant increases in IL-6 concentrations were induced by morphine only; NK cell activity was suppressed with morphine, but maintained with oxycodone and epidural analgesia. Gene expression profiles suggest that at 2 hours, post incision morphine appeared to be immunosuppressive as compared to oxycodone and nonopioid control analgesia.

// Aine McCarthy 1 , Jacek Marzec 2 , Andrew Clear 1 , Robert D. Petty 1 , Rita Coutinho 1 , Janet Matthews 1 , Andrew Wilson 1 , Sameena Iqbal 1 , Maria Calaminici 1 , John G. Gribben 1 and Li Jia 1 1 Centre for Haemato-Oncology, Barts Cancer Institute, Queen Mary University of London, London, United Kingdom 2 Centre for Molecular Oncology2, Barts Cancer Institute, Queen Mary University of London, London, United Kingdom Correspondence: Li Jia, email: // Keywords : Autophagy, BCL-2, follicular lymphoma, autophagy PCR array, and tissue microarray Received : October 09, 2014 Accepted : October 18, 2014 Published : October 18, 2014 Abstract Overexpression of the anti-apoptotic protein BCL-2 is characteristic of human follicular lymphoma (FL) and some cases of diffuse large B cell lymphoma (DLBCL). We aimed to determine autophagy status in primary FL and DLBCL samples and the BCL-2 + /BCL-2 - lymphoma cell lines using both autophagy PCR array and tissue microarray (TMA). A greater number of autophagy machinery genes were up-regulated in the BCL-2 + Su-DHL4 cell line compared with BCL-2 - Su-DHL8 cells, at both the basal level and in response to autophagic stress. The autophagy-related gene expression profiles were determined in purified and unpurified malignant human lymph node biopsies. Seven autophagy machinery genes were up-regulated in purified FL B-cells compared with reactive B-cells. Only 2 autophagy machinery genes were up-regulated in DLBCL B-cells. In unpurified tissue biopsies, 20 of 46 genes in FL and 2 of 5 genes in DLBCL with increased expression were autophagy machinery genes. Expression of autophagy substrates p62 and LC3 were determined by TMAs. FL samples showed significantly decreased levels of both p62 and LC3 compared with reactive and DLBCL, indicative of an increased autophagy activity in FL. In summary, these results demonstrate that FL showed increased basal autophagy activity, regardless of overexpression of BCL-2 in this disease.

Background: Chronic Fatigue Syndrome (CFS/ME) is a complex multisystem disease of unknown aetiology which causes debilitating symptoms in up to 1% of the global population. Although a large cohort of genes have been shown to exhibit altered expression in CFS/ME patients, it is currently unknown whether microRNA (miRNA) molecules which regulate gene translation contribute to disease pathogenesis. We hypothesized that changes in microRNA expression in patient leukocytes contribute to CFS/ME pathology, and may therefore represent useful diagnostic biomarkers that can be detected in the peripheral blood of CFS/ME patients. Methods: miRNA expression in peripheral blood mononuclear cells (PBMC) from CFS/ME patients and healthy controls was analysed using the Ambion Bioarray V1. miRNA demonstrating differential expression were validated by qRT-PCR and then replicated in fractionated blood leukocyte subsets from an independent patient cohort. The CFS/ME associated miRNA identified by these experiments were then transfected into primary NK cells and gene expression analyses conducted to identify their gene targets. Results: Microarray analysis identified differential expression of 34 miRNA, all of which were up-regulated. Four of the 34 miRNA had confirmed expression changes by qRT-PCR. Fractionating PBMC samples by cell type from an independent patient cohort identified changes in miRNA expression in NK-cells, B-cells and monocytes with the most significant abnormalities occurring in NK cells. Transfecting primary NK cells with hsa-miR-99b or hsamiR-330-3p, resulted in gene expression changes consistent with NK cell activation but diminished cytotoxicity, suggesting that defective NK cell function contributes to CFS/ME pathology. Conclusion: This study demonstrates altered microRNA expression in the peripheral blood mononuclear cells of CFS/ME patients, which are potential diagnostic biomarkers. The greatest degree of miRNA deregulation was identified in NK cells with targets consistent with cellular activation and altered effector function. © 2016 Petty et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Background: Human follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) are the most common forms of indolent and aggressive NHL, respectively. The t(14;18) translocation characterizes approximately 85% of FL and 20% of DLBCL and results in constitutive overexpression of the anti-apoptotic protein BCL-2. It was previously reported that BCL-2 plays dual roles in preventing apoptosis and autophagy. Autophagy is a physical and pathological process whereby cells sequester portions of cytoplasm including organelles to form autophagosomes where they are degraded and recycled. Growing evidence demonstrates that autophagy plays important roles in tumorigenesis, tumor progression, and resistance to chemotherapy. Aims: The autophagy status in human B-cell lymphomas is unknown. We hypothesized that overexpression of BCL-2 could change autophagy status and aimed to determined expression of autophagy-related genes and proteins in FL and DLBCL primary samples by PCR array and tissue microarray. We aimed to evaluate whether expression of the autophagy-related proteins p62, Beclin-1 and LC3 individually and in combination with BCL-2 protein expression could risk-stratify FL and DLBCL patients at diagnosis. Patients and methods: Using PCR array, the autophagy-related gene expression profiles were determined in purified and unpurified reactive and malignant human lymph node tissue biopsies. Diagnostic tissues from FL (n=117) and DLBCL (n=109) patients were microarrayed and autophagy protein expression was evaluated using immunohistochemistry. Univariate and multivariate analyses on both continuous and categorical variables were conducted to measure overall survival (OS), disease specific survival (DSS), and progression-free survival (PFS). Results: Seven autophagy machinery genes were up-regulated in purified FL B-cells, namely ATG9A, ATG16L1, MAP1LC3A, GABARAPL1, ULK1, LAMP1 and HDAC6 compared with reactive B-cells. Two autophagy machinery genes, MAP1LC3A and DRAM1, were up-regulated in DLBCL B-cells. In unpurified tissue biopsies, 20 of 46 genes in FL and 2 of 5 genes in DLBCL with increased expression were autophagy machinery genes. CTSD (cathepsin D) and TGM2 (transglutaminase 2) genes and proteins were mainly up-regulated in DLBCL tumor-infiltrating macrophages. These results demonstrate that FL and DLBCL showed increased expression of autophagy-related genes, regardless of the heterogeneity of these diseases. p62, a selective autophagy substrate; LC3, an autophagosome membrane protein; and Beclin-1, an essential autophagy effector, are often used to evaluate autophagy activity in the cell. 91% FL samples were BCL-2 positive but significantly decreased expression of p62, LC3 and Beclin-1 in FL samples was observed in both intra-follicular and non-malignant inter-follicular areas. This suggests that increased basal autophagy activity in both malignant FL cells and surrounding tumor infiltrating cells, indicating that BCL-2 does not inhibit basal autophagy activity. DLBCL samples displayed heterogeneous expression patterns of BCL-2, p62, LC3 and Beclin-1. We found that decreased p62 expression confers worse OS (continuous P=0.015; and categorical P=0.003), DSS (continuous P=0.037; categorical P=0.014) and PFS (categorical P=0.002) in DLBCL patients. Decreased expression of Beclin-1 was also confers poor prognosis in both FL and DLBCL as conducted by categorical analysis, OS (DLBCL, P=0.015; FL, P=0.004), DSS (FL, P=0.006), and PFS (DLBCL, P=0.029). p62 retains prognostic significance after adjustment for the International Prognostic Index (IPI) score and levels of BCL-2, Beclin-1 and LC3 in multivariate analysis. Beclin-1 retains its prognostic significance in FL after adjusting for FLIPI scores. Low p62 plus high BCL-2 expression in DLBCL confers the worst OS (P Conclusions: These results demonstrate that FL has increased basal autophagy activity, while it varies in DLBCL. p62 is a novel, independent prognostic biomarker for DLBCL but not for FL. Combining p62 with BCL-2 provides a more robust and reliable method to risk-stratify DLBCL patients at diagnosis. Importantly, we report for the first time that overexpression of BCL-2 in human NHL does not inhibit basal autophagy activity. We propose that increased autophagy activity could be a therapeutic target for treatment of NHL. Disclosures Gribben: Celgene: Research Funding; Pharmacyclics: Honoraria; Roche: Honoraria.

Introduction IMiDs like lenalidomide have immunostimulatory effects and therefore the potential to reduce relapse after allogeneic haematopoietic cell transplant (AHCT) by increasing graft-versus-tumour (GvT) effects. However, early clinical experience using IMiDs after AHCT has been limited by induction of graft-versus-host disease (GvHD). Although lenalidomide has been shown to augment mitogen-stimulated T cell responses, the effects of this drug on T cell alloresponses that mediate both GvT and GvHD have not been well defined. Better understanding of the immune mechanisms involved would facilitate tracking and manipulation of lenalidomide-potentiated alloresponses and could reveal ways to use the drug to maximise GvT without excess GvHD. Therefore we used an HLA-mismatched in vitro model to analyse in depth the effects of lenalidomide on functional human T cell alloresponses. Materials and Methods We cocultured CFSE-labelled PBMC from healthy donors with irradiated allogeneic PBMC in the presence of 1μM lenalidomide, vehicle control or following pre-incubation with 1μM lenalidomide for 24 hours. Functional alloresponses were quantified after 7-9 days of allo-coculture by flow cytometry. In addition, allo-coculture responders were flow-sorted into alloproliferative or non-proliferative fractions and extracted RNA used for gene expression profiling. Results Addition of lenalidomide to allo-cocultures increased the total number of responder cells (p2-fold altered expression of >500 genes mostly associated with DNA synthesis and cellular proliferation when compared to non-proliferative CD8 cells. Lenalidomide-treated alloCD8 cells showed further increases in expression of many of these genes; however treatment also resulted in significant changes in expression of additional genes in alloCD8 cells compared to untreated alloCD8 cells (Fig 1B). These included >8 fold increases in expression of genes reported to potentiate T cell immune responses in other settings including PFKFB4,Pirin, and SOCS2 (part of the E3 ubiquitin ligase complex with cereblon), and >5 fold decreases in genes which can suppress T cell activation and memory differentiation including FAIM3 and PMCH. Conclusion We have shown for the first time that lenalidomide potentiates human alloresponses primarily by increasing alloproliferation of effector memory CD8 T cells. This likely results from altered expression of (i) multiple genes common to the intrinsic CD8 alloproliferative response and (ii) additional genes involved in the control of T cell activation and differentiation specific to lenalidomide-potentiated CD8 alloresponses. Furthermore treatment enhances the functional capacity of these cells by conferring greater polyfunctional effector potential. These findings could enable tracking of CD8 alloresponses induced by lenalidomide after AHCT and could inform novel clinical strategies for the use of the drug to augment GvT effects. Figure 1 Figure 1. Disclosures Gribben: Celgene: Research Funding; Pharmacyclics: Honoraria; Roche: Honoraria.

Abstract 790 Several investigators have defined gene expression profiling (GEP) signatures in Diffuse Large B-cell Lymphoma (DLBCL) enriched for macrophage and stromal genes, suggesting an active innate immune response against lymphoma. We hypothesize that the malignant B-cells drive tumor-associated macrophage (TAM) dysfunction in a subset of patients with DLBCL which is relevant for their biology and prognosis. We performed GEP on TAM from diagnostic DLBCL in order to recognise key genes and pathways open to functional validation. Single cell suspensions from 8 DLBCL and 8 reactive lymph nodes were used in this study. TAMs were flow sorted using CD36 expression. cDNA synthesis and amplification was performed using the Nugen Ovation Pico WTA system and the Affymetrix GeneChip human gene 1.0 ST platform was used. We used bioinformatics analysis of GEP data from DLBCL whole tumors, DLBCL purified B-cells, in vitro manipulated macrophages and other immune cells in order to define macrophage-enriched genes as well as specific M1/M2 signatures. We identified a 221 gene signature that significantly distinguished DLBCL TAMs from control macrophages, with 165 genes upregulated and 56 genes downregulated. Moreover, a comparative transcriptome analysis of 22 diverse immune cell phenotypes/activation states (IRIS: GSE22886) revealed that 26% of these genes are highly macrophage-enriched. Gene Ontology analysis revealed an over-representation for transcripts involved in inflammatory response (p 6.8×10−23), wound healing (p 1.9×10−20), chemotaxis (p 3.2 ×10−9), and cell motility (p 1.7×10−7). Upregulated genes in TAMs included well known M1 (complement components, CXCL9 or CXCL10) as well as M2 genes (MSR, CD163 or MARCO) (Table 1). In our signature, there was enrichment for M1 compared to M2 genes as defined by bioinformatics analysis. TAMs showed overexpression of the CSF1R gene as well as the chemokines CCL2 and CCL5, suggesting an autocrine feed-back loop of macrophage chemotaxis and survival in DLBCL. Moreover, TAMs showed upregulation of the lymphocyte attractants CCL20, CXCL9 and CXCL10, together with T-cell immunosupressants indoleamine 2,3-dioxygenase 1 and PD-L1, which would support a role for macrophages in T-cell recruitment and dysfunction in DLBCL. We also saw strong upregulation of 7 metallothionein isoforms in TAMs. These are proteins known to be expressed in macrophages and linked to response to oxidative damage, modulation of inflammation and cell proliferation. However their role in cancer microenvironment is unclear. We describe for the first time the GEP from DLBCL TAMs. The TAM transcriptome has partial overlapping genes with both M1 and M2 gene signatures, but also has a characteristic GEP potentially driven by their presence in the DLBCL microenvironment. Although further molecular and functional validation is required, this data provides a platform of genes which serve as excellent candidates for future exploration to understand DLBCL pathogenesis and to define new therapeutic targets. Table 1: Genes of particular interest represented in our TAM signature. Probe collapse was done using the highest expression. The Benjamini-Hochberg multiple hypothesis test was used to determine significance (FDR 0.05). Gene IDs Gene description Log2 Fold Change Adjusted p value MT1E-H, L, M, X, MT2A metallothionein 1E-H, 1M, 1X, 2A 2.3 – 4.4 0.00145 - 0.00017 C1QA, B and C complement component 1, q subcomponent, A, B and C chains 3.1 – 3.6 0.00136 - 0.00255 C2 complement component 2 3.2 0.00136 CCL2, 4, 5, 8, 20 chemokine (C-C motif) ligand 2, 4, 5, 8 and 20 2.4 - 3.7 0.046 - 0.00130 CD163 CD163 molecule 4.6 0.00025 CD14 CD14 molecule 3.4 0.00091 CD274 CD274 molecule 3.7 0.00447 CSF1R colony stimulating factor 1 receptor 2.1 0.00886 CXCL9-11 chemokine (C-X-C motif) ligand 9, 10 and 11 4.2 – 4.4 0.012 - 0.00158 FCGR1B Fc fragment of IgG, high affinity Ib, receptor (CD64) 4.9 0.00091 FCGR2A Fc fragment of IgG, low affinity IIa, receptor (CD32) 3.3 0.00139 FCGR3A Fc fragment of IgG, low affinity IIIa, receptor (CD16a) 5.2 0.00011 IDO1 indoleamine 2,3-dioxygenase 1 4.8 0.00232 MARCO macrophage receptor with collagenous structure 2.6 0.02029 MSR1 macrophage scavenger receptor 1 3.7 0.01467 Disclosures: Gribben: Celgene: Honoraria; Roche: Honoraria; Pharmacyclics: Honoraria; GSK: Honoraria; Mundipharma: Honoraria; Gilead: Honoraria.

Abstract 2699 Transformation from chronic myelomonocytic leukemia (CMML) to acute myeloid leukemia (AML) is associated with a poor prognosis. Clinical features have been used to score those at high risk, e.g. increased marrow blast percentage. Recently, several gene mutations have been identified including TET2, KRAS, NRAS, CBL and RUNX1, of which TET2 is the most frequent (Kohlmann et al 2010, Kosmider et al 2009). Initial descriptions of TET2 mutations in myeloid malignancy suggested that they were early mutations (Delhommeau et al 2009), however subsequent studies have shown in some patients with myeloproliferative neoplasia (MPN) they can be preceded by mutations in JAK2 (Schaub et al 2010). We report a male patient who was diagnosed with CMML aged 43 years. His marrow blast count was 6% and within 2 months he had transformed to AML (FAB M4) with marrow blasts of 50%. He was treated with daunorubicin and AraC (Barts 12 schedule) and obtained a complete morphological remission (CR). However he relapsed 88 days after CR in his skin, central nervous system and marrow. He died with conservative treatment after 6 months. Using this case as a model for transforming CMML, we aimed to identify clonal changes characteristic of the three stages of his disease, chronic leukaemia, transformation to acute leukemia and subsequent relapse. We used cytogenetic analysis, single nucleotide polymorphism (SNP) arrays (Affymetrix SNP 6.0 and 10K; 10K data previously reported by Raghavan et al 2008) to identify the clonal changes. Gene mutation analysis of genomic and cDNA was performed using PCR, cloning and Sanger and amplicon deep-sequencing for the mutational burden of TET2, CBL and KRAS (454 Life Sciences, Branford, CT). At all stages of his disease, he had a normal karyotype, a NPM1 mutation, but no mutations of FLT3, JAK2, KRAS or CBL (Table). Uniparental disomy (UPD) was demonstrated on chromosome 4q at relapse but not present at diagnosis, suggesting an associated TET2 mutation that would be heterozygous at diagnosis and homozygous at relapse. The heterozygous mutation was indentified in the CMML and at transformation, in a region adjacent to the splice site of the end of exon 7. The transcript resulted in a mis-splicing event leading to a stop codon. A second truncated mutant due to a deletion in exon 6 was found expressed at low level. Deep-sequencing of genomic DNA confirmed a deletion in exon 6 consistent with this product at 2.4% (918-fold coverage) in the CMML, and increased to 5.7% (865-fold coverage) burden at transformation. At relapse, the genomic mutation was homozygous. Expression of only the mis-spliced product was observed, with no wild type TET2, and no evidence of the second truncated product by cloning or deep-sequencing (832 reads). Homozygous TET2 mutations are associated with 4qUPD in MPN and myelodysplastic syndromes (Mohamedali et al 2009, Janowska et al 2010), but this is the first description of clonal evolution of a hetero- to homozygous TET2 mutation at disease progression. The case is consistent with the hypothesis that TET2 mutations evolve early in the pathogenesis of myeloid malignancies. However, it also shows there is considerable instability of TET2 mutations, with multiple mutations at various levels, and with mitotic recombination being a late event leading to relapse. CMML Transformed AML Relapsed AML CGN 46,XY 46,XY 46,XY SNP array Not done No somatic copy number abnormalities detected 4qUPD 88201900-qter TET2 c4340+5G>T+10_+21del het result: Cys1298X c4340+5G>T+10_+21del het result: Cys1298X c4340+5G>T+10_+21del hom result: Cys1298X c3732_3733delCT 2.4% result: Tyr1245LeufsX22 c3732_3733delCT 5.7% result: Tyr1245LeufsX22 Absent NPM1 Dup956_959TCTG het Dup956_959TCTG het Dup956_959TCTG het Disclosure: No relevant conflicts of interest to declare.

Immunomodulatory drugs (IMiDs), such as lenalidomide provide a tool to enhance both direct anti-tumor and graft-versus-tumor effects after allogeneic haematopoietic stem-cell transplantation (AHCT). However, early clinical experience with IMiDs after AHCT using adult peripheral blood (APB) as a stem cell source has been limited by induction of graft-versus-host disease. Characterization of the mechanisms by which IMIDs can modulate alloresponses of T-cells and identification of differential effects on T-cells from disparate cell sources could facilitate more effective use of these drugs in the setting of AHCT. We therefore used in vitro modelling, multi-parameter flow cytometry and gene expression analysis to delineate the impact of the widely used IMiD lenalidomide on quantitative and qualitative alloresponses mediated by T-cells derived from APB and umbilical cord blood (UCB). We co-cultured carboxyfluorescein diacetate succinimidyl ester (CFSE)-labelled peripheral blood mononuclear cells (PBMC) from healthy adult donors or donated UCB units with irradiated allogeneic PBMC in the presence of 1μM lenalidomide or vehicle control. In this model, cellular and supernatant concentrations of lenalidomide (determined by mass spectrometry) were similar to in vivo levels achieved at doses used as maintenance after AHSCT in published studies. Functional alloresponses were quantified after 7-9 days of allogeneic co-culture by flow cytometry. In addition, co-culture responders were flow-sorted into alloproliferative or non-proliferative fractions and extracted RNA used for gene expression profiling. We demonstrate that lenalidomide potentiates net alloproliferation of APB derived T-cells cells by selectively enhancing allospecific proliferation of CD8+ T-cells (median 58% (lenalidomide-treated) versus 43% (untreated), n=40, p In common with our findings in APB T-cells, lenalidomide also potentiated proliferation of alloreactive CD8+ T-cells from UCB (median 43% (lenalidomide-treated) vs 24% (untreated), n=17, p=0.0005) with similar polyfunctional effector memory differentiation, immunoregulatory gene expression changes and cellular ikaros depletion. Importantly, lenalidomide reduced allospecific proliferation of UCB CD4+ T-cells (median 58% (untreated) versus 41% (lenalidomide-treated), n=17, p Our findings show that lenalidomide has a qualitatively different impact on alloresponses of T-cells from different cell sources; alloresponses of APB T-cells are increased by lenalidomide via selective expansion of polyfunctional CD8+ effectors while lenalidomide limits alloresponses of UCB T-cells by reducing CD4+ effector expansion and increasing tolerogenic Treg. These findings have important implications for the future use of IMiDs in the setting of AHCT. Disclosures No relevant conflicts of interest to declare.