The Abnormal Expression of B and T Lymphocyte Attenuator in Patients with Myelodysplastic Syndromes

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Poster Presentations – 14th International Symposium on Myelodysplastic Syndromes / Leukemia Research 55 S1 (2017) S45–S167

2016 WHO. The overall series showed a favorable prognosis, consistent with its IPSS-R. Despite patients with SF3B1 mutation have a higher requirement for transfusion and iron overload, further analysis including larger series and follow-up are needed to confirm these observations. 182 IMMUNE CELL DENSITY AS A PREDICTIVE INDEX FOR RESPONSE TO TREATMENT AND OUTCOME OF HIGH RISK MDS TREATED WITH 5-AZACYTIDINE Z. Tsakiraki1, S. Papageorgiou2, A. Spathis3, A. Bouchla2, E. Bazani2, E. Glezou2, P. Karakitsos3, I. Panayiotides1, V. Pappa2, P. Foukas1 1 2nd Department of Pathology, Attikon University Hospital, ChaidariAthens, Greece; 22nd Department of Internal Medicine- Hematology Unit, Attikon University Hospital, Chaidari-Athens, Greece; 3 Department of Cytopathology, Attikon University Hospital, ChaidariAthens, Greece Aim: To investigate the immune landscape in bone marrow (BM) trephine biopsies from patients with intermediate-II and high risk myelodysplastic syndrome (MDS), as well as the possible role of the immune microenvironment in the outcome of 5-Azacytidine (5AZA) treatment. Patients and methods: The study population consisted of 35 (22 male and 13 female) intermediate-II and high risk MDS patients according to IPSS, treated with 5-AZA. Seven patients were diagnosed with RAEB-1, 23 with RAEB-II, 4 with AML and 1 with CMML-2 according to WHO 2008. Sections from BM trephine specimens before and during treatment were immunostained for CD3, CD20 and CD138 for T cells, B cells and plasma cells respectively. The number of positive cells was counted in 5 HPFs (×40, areas with the higher densities for each cell population were chosen). Since BM cellularity differed between specimens, calculations were normalized to 50% cellularity. Results: In BM specimens before treatment, high densities of T-cell infiltrates correlated with complete response ( p = 0.0318, Fisher`s exact test) and high densities of B-cell infiltrates correlated with better overall survival ( p = 0.0093, Log-rank test). Conclusion: Our preliminary results indicate that the density of cells of adaptive immunity in the bone marrow specimens may be construed as another predictive index for response to treatment and outcome of high risk MDS patients treated with 5-AZA. 183 EXPRESSION OF PD-1, PD-L1 AND PD-L2 IN PATIENTS WITH MYELODYSPLASTIC SYNDROMES AND ITS CLINIC RELATIONSHIP S. Geng1, J. Weng1, C. Deng1, M. Li1, Z. Lu1, P. Wu1, X. Huang1, P. Lai1, X. Du1 1 Department of Haematology, Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangzhou, China Immune dysregulation plays an important role in the pathogenesis and progression of MDS. Programmed death receptor 1 (PD-1) is an important immunosuppressive molecule. PD-L1 is a primary PD-1 ligand that induces a coinhibitory signal in activated T cells. Recently studies indicated that PD-1 signaling may be involved in MDS pathogenesis. However, little research has been performed. We aim to investigate PD-1 and its ligands PD-L1 and PD-L2 expression in MDS patients and analyze its clinic relationship. 139 MDS patients were diagnosed in our department including 96 males and 43 females with a median age of 60 (15–84) years. There were 50 RAEB-I patients, 38 RAEB-II patients, 41 RCMD patients, and 10 RAS patients. A total of 64 peripheral blood (PB) and 132 bone marrow (BM) samples were collected. 15 PB and 9 BM samples collected from healthy individuals served as controls. The

expression of PD-1, PD-L1, PD-L2 transcripts were detected by Real-time PCR. In 33 paired PB and BM samples, the PD1, PD-L1 and PD-L2 levels in PB samples were significantly higher than that in BM (P = 0.006, 0.000, 0.003). There was no significant difference of PD-1 expression between RAS and normal controls (P = 0.683). The expression levels of PD-1 in RAEB-I, RAEB-II and RCMD patients increased significantly compared to RAS patients and controls (P < 0.05). The PD-L1 level in PB from RAS patients decreased significantly (P = 0.007) and it increased significantly in RCMD patients (P = 0.008) compared to controls. There was no significant difference in the expression of PB PD-L1 between RAEB-I or RAEB-II and controls (P = 0.468 and 0.661). The expression level of PD-L1 in BM of RAEB-I, RAEB-II and RCMD patients was significantly higher than that of the controls (P = 0.013, 0.02 and 0.002). The differences of PD-L2 in BM and PB samples between RAEB-I, RAEB-II or RCMD patients and controls were not significant (P > 0.05). In 11 patients, BM complete remission was obtained after demethylation treatment and the BM PD-1 level (31.32 ± 15.75%) significantly decreased compared to pre-treatment (59.94 ± 47.44%, P = 0.034). After progression and transformation, the expression of PD-1 (54.72 ± 37.27%) increased and was higher than that for BM complete remission (P = 0.028). The change trend of the expression of PD1 and WT1 was consistent during disease evolution. In conclusion, abnormal PD-1 and PD-L1 expression were found in majority of MDS patients and it was associated with the disease status. However, the role of PD1 and PD-L1in prognosis remains to be further studied in MDS.

184 THE ABNORMAL EXPRESSION OF B AND T LYMPHOCYTE ATTENUATOR IN PATIENTS WITH MYELODYSPLASTIC SYNDROMES S. Geng1, J. Weng1, M. Lin1, P. Wu1, C. Deng1, Z. Lu1, X. Huang1, P. Lai1, H. Zhang1, X. Du1 1 Department of Haematology, Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangzhou, China Immune dysregulation plays an important role in the pathogenesis of Myelodysplastic Syndromes (MDS). B and T lymphocyte attenuator (BTLA) is a novel co-inhibitory molecule that exhibits a critical role in restraining cell-mediated antitumor immunity. BTLA is highly expressed in patients with solid tumors and leukemia. But no results are available regarding the expression of BTLA in patients with MDS. The present study aimed to investigate the expression of BTLA in patients with MDS. 130 MDS patients were diagnosed in our department including 90 males and 40 females with a median age of 60.5 (15–84) years. There were 48 RAEB-I patients, 33 RAEB-II patients, 40 RCMD patients, and 9 CMML patients. A total of 48 peripheral blood (PB) and 123 bone marrow (BM) samples were collected from MDS patients. 13 PB and 8 BM samples collected from healthy individuals served as controls. The expression of BTLA transcripts were detected by real-time PCR. In 31 paired PB and BM samples, BTLA level in PB samples were significantly higher than that in BM (P < 0.01). The median BTLA level in BM samples was 0.034% (range: 0.002–0.272) and 0.027% (range: 0.000–0.275) for RAEB-I and RAEB-II patients respectively, which was lower than that of normal controls (median: 0.065%, range: 0.035–0.108, P = 0.017 and 0.026). But there was no significant difference of BTLA level in BM samples between RCMD (median: 0.045%, range: 0.002–0.279) or CMML patients (median: 0.029%, range: 0.002–0.139) and normal controls (P = 0.36 and 0.074). There was also no significant difference in the PB BTLA level between different types of MDS patients and normal controls (P > 0.05). In conclusion, the expression of BTLA in BM of RAEB patients decreased significantly. The role of BTLA in the

Poster Presentations – 14th International Symposium on Myelodysplastic Syndromes / Leukemia Research 55 S1 (2017) S45–S167

pathogenesis or immune disorders in patients with MDS remains to be explored. 185 A STUDY OF THE RELATIONSHIP BETWEEN TREG LEVELS AND T CELL EXPANSION IN BONE MARROW OF LOW RISK MDS PATIENTS S. Leone1, V. Rubino2, A. Giovazzino2, A.T. Palatucci3, G. Cerciello1, V. Pernice2, F. Pane1, G. Ruggiero2, F. Alfinito1, G. Terrazzano3 1 Department of Clinical Medicine and Surgery, Università “Federico II”, Naples, Italy; 2Department of Translational Medicine, Università “Federico II”, Naples, Italy; 3Department of Science, University of Basilicata, Potenza, Italy Several data have been showing that immune-dependent mechanisms might be relevant for the selection, expansion and dominance of dysplastic clone/s in a subgroup of MDS patients. To date, valuable criteria to identify patients in which a deranged immune response might account for the selection/shaping of the dysplastic precursor/s are still lacking. Immune response is a microsite phenomenon, thus the analysis of the immune effectors in the Bone Marrow (BM) offers a unique possibility to specifically focus the mechanisms underlying the immune-mediated processes likely involved in the selection/expansion of dysplastic clone/s in MDS. We previously described that specific alterations of immune profile, as represented by low Treg levels and high expression of CD54 on CD8 effectors in BM, allow the identification of a subgroup of MDS patients in which an immune-mediated pathogenesis of the disease might be inferred. This study aims to confirm and extend these data trying to correlate tolerance control derangement in BM with pathological expansion of T cell effectors in Low Risk MDS patients. Preliminary data, obtained in 26 Low Risk patients, confirm that Treg, a key element for cell-mediated tolerance control, show a clustered distribution in BM; moreover, their quantitative defect correlates with the recruitment and the activation state of T cell effectors. To investigate the occurrence of antigen-dependent clonal expansion of CD4 and CD8 lymphocytes, we analysed, by flow cytometry, TCR Vb repertoire in BM as compared with peripheral blood in Low Risk patients and healthy donors. The presence of preferential T cell expansion in BM, respect to peripheral blood, was also evaluated. Our data indicate that BM expansion of CD4 and CD8 T cells characterizes a subgroup of Low Risk MDS patients, as compared with controls. We also found that the presence of BM clonal expansion of CD8, but not CD4 T lymphocytes, is significantly related with low Treg level (<2% of BM lymphocytes) and activation of cytotoxic effectors in BM. These data suggest that BM Treg level may represent a valuable criterion to identify the subgroup of Low Risk MDS patients in which immune-mediated mechanisms are relevant for MDS pathogenesis. A more homogeneous grouping of Low Risk patients will improve clinical management of the disease, hopefully allowing a more effective employment of innovative immunemodulating strategies in MDS. 186 MYELODYSPLASTIC SYNDROME: A STUDY OF MUTATIONAL PROFILE AND THE MEDULLAR MICROENVIRONMENT RELATED TO THE PROGRESSION OF THE DISEASE P. Montes Ramos1, M. Bernal Sánchez2, L.N. Campo Blázquez1, M.P. Jiménez Gámiz1, F. Ruiz-Cabello Osuna1, M. Jurado Chacón2, F. Hernández Mohedo2 1 Analisis Clínicos, Complejo Hospitalario Universitario de Granada, Granada, Spain; 2UGC Hematología y Hemoterapia, Complejo Hospitalario Universitario de Granada, Granada, Spain

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Introduction: The progression risk of MDS has been reported to have a correlation with more than 40 mutations. Many of these mutations have been associated with the disease prognosis independently of the percentage of blasts, cytogenetic abnormalities or grade of cytopenia. However, the genetic mechanisms which explain the development of the MDS remain unknown. Some authors believe that there is an immune component in the disease pathogenesis and progression. At early MDS stage, the immune control could take place at the level of malignant clones, while in advanced MDS, immune escape mechanisms prevail. Objectives: To analyse both the mutational status (MDS high/low mutations frequency) and tumour microenvironment to identify factors associated with the disease progression. Material and Methods: The study group includes 32 patients with MDS diagnose, according to WHO-2008 classification: CRDM (10), RAEB-1 and RAEB-2 (9), MDS isolated deletion (5q) (5), MDS/MPN CMML type (6) and 2nd AML (3). Samples were sequenced by Next Generation Sequencing system, NGS (Illumina, San Diego, CA, USA). Cell subpopulations from patients with MDS were analysed by multiparametric flow cytometry. Results: Mutations identified in MDS were localized in 22 different genes. The greater percentage of mutations was observed in the groups RAEB (1 and 2) and MDS/MPN CMML type (29.41%), followed of CRDM (23.52%). No mutation was detected in 5 out of 32 studied patients. The average of mutational variation was 2.32 in each patient. The analyses did not show any correlation between the mutation number and the percentage of blasts in PB and BM samples. In addition, in MDS with greater mutational score we observed a high frequency of CD4+ CD103+ cells, a lower frequency of TH17/TH22 cells, and a reduced frequency of NK cells with cytotoxic phenotype with activating receptor NKp46. In relation to the detected blastosis, both in BM and PB, we observed a positive correlation with CD14+ HLA-DRlow/− monocytic myeloid suppressor cells ( p = 0,002). Conclusions: First, we have found a reverse correlation between mutation frequency and the presence of cells with inflammatory phenotype TH22/TH17, and NKs with activator/cytotoxic phenotype. In addition, a direct correlation between myeloid suppressor cells with monocytic origin CD14+ HLA-DRlow/− was observed. Finally, we did not see any association between the number, the type of mutation and the percentage of blasts.

187 IMMUNE DEREGULATION IN MYELODYSPLASTIC SYNDROMES J. Montoro Gómez1 1 Hematology Department, Vall D´Hebron University Hospital, Barcelona, Spain Introduction: Immune system (IS) abnormalities and MDS are related in a double fashion, as IS seems to play a role not only in the MDS pathogenesis but also in the evolution to acute myeloid leukemia. Moreover, it seems that autoimmune diseases are more frequent in MDS patients. The aim of this study is to describe the prevalence of autoimmune diseases and immune population data among MDS and chronic myelomonocytic leukemia (CMML) patients and compare the characteristics and outcomes of patients with and without autoimmune abnormalities. Methods: We included all MDS and CMML of the Vall d’Hebron Hospital from 2011 to 2016. Patients were divided into 2 groups, those with autoimmune data (MDS-AD) and those without AD (MDS-nonAD). Patients were considered as MDS-AD if clinical abnormalities were present and any positive laboratory test (Coombs test (CT), rheumatoid factor (RF) >14 UL/mL, and antinuclear antibodies (ANAs) ≥1/320). Chi square and t-test were used to compare categorical and continuous variables. Kaplan-Meier