Poster Presentations

151 Table CD3 P ≥ 50 CD4 P ≥ 50 CD8 P ≥ 50 CD3/B-cell P ≥ 50 CD4/CD8 P ≥ 50 CD4 TFH/CD4 + P ≥ 75 Age (%) <60 years 39 34 29 21 55 21 ≥60 years 59 56 57 43 43 39 Histological grade (%) 1–2 45 43 35 27 58 29 3 89 67 89 67 0 11 Bulky disease (%) No 57 53 46 60 46 23 Yes 22 17 33 11 56 29 Extranodal (%) ≤1 58 52 51 58 47 23 >1 21 21 21 26 53 29 Leukemic phase (%) No 56 50 48 32 60 22 Yes 10 10 10 0 46 25 182 Poster Presentations resistance. Immune cells in the lymphoma tumour microenvironment have been implicated in disease progression (Dave et al., NEJM 2004) and may play a role in transformation. We recently discovered that expression of the inhibitory receptor PD-1 was associated with suppressed cytokine signalling in FL tumour-infiltrating T cells (Myklebust et al., Blood 2013). Furthermore, it has been suggested that PD-1 int cells are the truly exhausted T cells and that PD-1 high cells are normal T follicular helper cells (TFH) (Yong et al., Blood Cancer J 2015). Antibody immunotherapy targeting PD-1 has shown significant promise in aggressive malignancies (Topalian et al., NEJM 2012), suggesting that exhausted T cells can regain functionality, including anti-tumour effects. Methods: Three mass cytometry (CyTOF) panels were designed to detect 30 markers per cell and used to characterize FL tumour biopsies (n = 9) and human tonsils. In the first panel, inhibitory and co-stimulatory receptors were quantified across 5 major T-cell subsets and maturation stages (Nicholas and Greenplate et al., manuscript in preparation). In the second panel, key surface markers were combined with antibodies to detect 12 phosphoproteins to correlate signalling responses with inhibitory receptor profiles. The last panel was designed to characterize healthy and malignant B cells. The dimensionality-reduction tool viSNE was used to analyse the high-dimensional mass cytometry data. Fluorescent flow panels were used in parallel to measure 9 inhibitory receptors in selected T-cell subsets. Results: viSNE analysis of 23 surface markers revealed a high degree of phenotypic similarity between T cells infiltrating FL and T cells in tonsils. In contrast, viSNE characterized the significant phenotypic differences between malignant B cells and healthy B cells within the same tumour. PD1 + T cells from FL samples displayed reduced cytokine signalling compared to PD1 cells, confirming previous results. TFH cells were identified as CXCR5 hi ICOS + CD4 memory T cells. Among the ICOS + cells in tonsils, a distinct CXCR5 population was identified with intermediate PD1 expression, suggesting an exhausted phenotype. These cells expressed less TIGIT, BTLA and LAIR1 than TFH but contained a subpopulation of TIM3 + cells that was not seen within the TFH population. Conclusions: Striking similarities in phenotype and signalling response of the T cells infiltrating FL tumours and T cells from healthy tonsil observed by mass cytometry suggest active immune responses in these tissues. These results provide further support for characterizing relationships between receptor signalling and T cell function and for researching into combination immunotherapies for FL focused on modulating adaptive immune responses. 151 T-CELL SUBPOPULATIONS QUANTIFIED BY FLOW CYTOMETRY IN LYMPH NODE CELL SUSPENSIONS IDENTIFY A GROUP OF PATIENTS WITH FOLLICULAR LYMPHOMA WITH FAVORABLE OUTCOME L. Magnano 1 , J. Carreras 2 , A. Martinez 2 , A. Martinez-Trillos 1 , J. Rovira 1 , I. Dlouhy 1 , E. Gine 1 , T. Bauman 1 , O. Balague 2 , E. Campo 2 , N. Villamor 3 , A. López-Guillermo 1 . 1 Hematología, Hospital Clinic de Barcelona, Barcelona, Spain, 2 Pathology Department, Hospital Clinic de Barcelona, Barcelona, Spain, 3 Hematopathology Unit, Hospital Clinic de Barcelona, Barcelona, Spain. Introduction: Tumour microenvironment plays an important role in the behaviour of follicular lymphoma (FL). By gene expression and immunohistochemistry, an increase in macrophages has been associated with poor outcome, while an increase in T cells is associated with good prognosis. The aim of the study was to explore the prognostic impact of subpopulations of T cells using flow cytometry and to identify different groups of risk in FL patients. Methods: Seventy-five patients (36 men/39 women, median age 60 years) diagnosed of FL (grades 1–2, 87%; grade 3, 13%) between 1984 and 2009 (median follow-up of 6.5 years) with sample at diagnosis were included in the present study. In 41 cases, T-cell staining were semiquantitatively analysed by immunohistochemical (IHC), including their distribution (intra, inter or perifollicular). T-cell populations from lymph node were quantified by multiparametric flow cytometry in cell suspensions in all cases. The percentage of B-cells, CD3 + , CD4 + , CD8 + , CD57 + , CD4TFH cells (double staining CD4 + CD57 + ), as well as the ratio T/B-cells, CD3 + /CD4 + , CD3 + /CD8 + , CD4 + /CD8 + and CD4TFH/CD4 + were analysed and correlated with initial features and outcome. Copyright © 2015 John Wiley & Sons, Ltd. Results: CD57 expression by IHC was mainly intrafollicular, while CD8 was inter/perifollicular, with these patterns correlating with grades 1–2. The mean (± SD) percentage of B-cells, CD3 + , CD4 + , CD8 + , CD57 + and CD4 + TFH cells were 59% (±15.7), 35.8% (±15.9), 27.1% (±13), 8.7% (±5.4), 6.3% (±4.6) and 3.6% (±2.9), respectively. Main associations with clinical characteristics and outcome are listed in the table. CD4TFH/CD4 and CD4 + /CD8 + ratios were divided in 4 percentiles. Patients with CD4TFH/CD4 + ratio in the 4th percentile had a better 10-year OS (92% vs 47%; p = 0.01) and PFS (66% vs 36%; p = 0.04) than the remainder. Moreover, patients with CD4 + /CD8 + ratio over median had a better 10-year OS (70% vs 48%; p = 0.03) but with no differences in PFS (37% vs 44%; p = NS). There was no correlation of lymphocyte subpopulations with type of therapy, overall response or complete response rate. A multivariate analysis was performed including CD4TFH/CD4 + ratio, CD4 + /CD8 + ratio and FLIPI, with CD4TFH/CD4 ratio being the most important variable to predict OS in the Cox model with 52 patients (relative risk: 32; p = 0.01). Conclusion: Flow cytometry allows the identification of T-cell subpopulation in FL, showing that a high percentage of CD4TFH cells is associated with more favourable prognosis. 152 TXN OVEREXPRESSION IN DIFFUSE LARGE B-CELL LYMPHOMA CELLS ATTENUATES OXIDATIVE STRESS-INDUCED PROAPOPTOTIC ACTIVITY OF FOXO1 TRANSCRIPTION FACTOR T. Sewastianik 1 , M. Szydlowski 1 , E. Bialopiotrowicz 1 , E. Jablonska 1 , P. Kiliszek 1 , P. Gorniak 1 , A. Polak 1 , M. Prochorec-Sobieszek 1 , A. Szumera-Cieckiewicz 1 , TS Kaminski 2 , S. Markowicz 3 , E. Nowak 3 , MA Grygorowicz 3 , K. Warzocha 4 , P. Juszczynski 1 . 1 Dept. of Diagnostic Hematology, Institute of Hematology and Transfusion Medicine, Warsaw, Poland, 2 Dept. of Soft Condensed Matter and Fluids, Institute of Physical Chemistry, Polish Academy of Sciences, Warsaw, Poland, 3 Dept. of Immunology, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Warsaw, Poland, 4 Dept. of Hematology, Institute of Hematology and Transfusion Medicine, Warsaw, Poland. Introduction: Diffuse large B-cell lymphoma (DLBCL) is a highly heterogeneous disease. Unsupervised gene expression profiling has led to the identification of a subset of DLBCLs characterized by enhanced oxidative phosphorylation. Since enhanced oxidative metabolism leads to overproduction of reactive oxygen species (ROS), which can be toxic to cells, we investigated the role of TXN system in the pathogenesis of DLBCL. Methods: Clinical consequences of TXN expression in DLBCLs were assessed with publically available gene expression datasets. BCL6 impact on TXN expression was P, percentile. *p = <0.05. **p = <0.01. Hematol Oncol 2015; 33: 181–243 DOI: 10.1002/hon 183 Poster Presentations assessed with bioinformatic approaches, luciferase reporter assays and shRNAmediated BCL6 silencing. TXN and FOXO1 knock-down was achieved with shRNA. TXN impact on FOXO1 acetylation, FOXO1 gene expression, cell cycle and apoptosis were studied in cells transfected with WT or mutant FOXO1, TXN and p300 vectors. The consequences of TXN inhibition on ROS-induced FOXO1 subcellular localization were determined with confocal microscopy. The consequences of FOXO1 acetylation were assessed in cells transduced with either WT FOXO1 or its acetylation-deficient mutants. Results: TXN expression was significantly higher in DLBCLs classified as OxPhos subtype compared to BCR subtype. The OS of patients with high TXN mRNA expression was significantly shorter than of those with low TXN mRNA expression, regardless of treatment regimen. Consistent with our previous findings indicating that BCL6 does not exhibit repressor activity in OxPhos tumours, we demonstrated that relative differences in TXN expression between different DLBCL subsets are at least in part caused by the lack of BCL6 transcription repressor activity. We found that OxPhos cells lacking TXN were uniformly more sensitive to ROS production than control cells. Since TXN reduces disulfide bonds between FOXO4 and p300, which results in decreased FOXO4 acetylation and attenuated proapoptotic signalling, we next assessed whether p300 and TXN are involved in acetylation of FOXO1, a major FOXO member expressed in DLBCLs. TXN decreased p300mediated FOXO1 acetylation and reduced its proapoptotic activity and expression of FOXO1-dependent genes (TRAIL, p27, Bim and GADD45A). Furthermore, TXN inhibited FOXO1 nuclear translocation in response to oxidative stress in OxPhos DLBCLs. Finally, knock-down of FOXO1 in OxPhos cells with silenced TXN expression markedly reduced DLBCL cell line apoptosis in response to ROS, demonstrating that FOXO1 is a major mediator of DLBCL cells’ responses to oxidative stress. Conclusion: Taken together, these results indicate that TXN is overexpressed in a subset of DLBCLs, and TXN overex