ALBERT

Description: Introduction: Micro-RNAs (miRNAs) are 19-24 nucleotide non-coding RNAs that regulate gene expression through the inactivation of their messenger RNA. Previous studies have demonstrated the important role of some miRNAs in the development of cancer. Specifically, in diffuse large B cell lymphoma (DLBCL), miRNAs involved in lymphomagenesis were identified but understanding their biological function continues to be a challenge. Nevertheless, the pathogenesis effects of some miRNAs such as miR-125a, miR-17-92 cluster or mi-R-155 are well characterized. Some studies suggest that miRNAs also possess a prognostic potential role in DLBCL. For this reason, our objective was to analyze the different miRNAs involved in the chemo-sensitivity or resistance to the first line treatment, their correlation with standard prognostic factors at diagnosis and the role of these miRNAs in survival in patients with DLBCL. Material and methods: Patients homogeneously treated with R-CHOP from 1999-2013 were reviewed from 3 Spanish centers. They were retrospectively obtained from Pathology Department registry to avoid selection bias. We included those patients with valid genomic material in formalin-fixed-paraffin-embedded tissue and with available clinical data. Samples were processed using the miRNA 4 Affymetrix microarrays kit in a discovery group that included 2 cohorts (patients with durable complete remission (CR) versus refractory or early relapsing patients). Those miRNAs with differential expression were validated in the whole series with quantitative RT-PCR. We also analyzed the role of these miRNAs as predictors of event-free survival (EFS) and overall survival (OS) and their relationship with standard prognostic factors in DLBCL. Results: We identified 156 patients homogeneously treated with R-CHOP. Finally, 96 samples were obtained with valid material for RNA extraction. To identify those miRNAs with prognostic implication, a discovery cohort of 12 patients was used in which all the cases had poor prognosis with high tumor load and advance disease (III-IV stage and unfavorable R-IPI). On this basis of poor prognosis, 2 groups were defined totally opposed from the point of view of the treatment response and evolution: chemo-resistance group (n=6) including refractory or relapsed (RR) patients (first 12 months) and chemo-sensitive group (n=6) including patients with at least 3 years of CR. A hierarchical clustering was performed in which 26 miRNAs differentially expressed were identified. A screening of miRNAs was carried out based on the fold-change and pathways involved and finally we obtained 10 miRNAs differentially expressed in RR group. The validation of these miRNAs was performed with quantitative RT-PCR in the whole series which finally included 68 samples with valid material. A univariate survival analysis including clinical prognostic factors and the selected 10 miRNAs was performed. We confirmed that 7 of them (miR-20b-5p, miR-1244, miR-6840-3p, miR-1231, miR-193b-5p, miR-6860-5p y miR-199a-5p) significantly influenced EFS and 6 of them (miR-1244, miR-1231, miR-193b-5p, miR-885-3p, miR-182-5p y miR-199a-5p) on OS. From these 10 miRNAs, only 3 had a significant prognosis role not only for EFS but also for OS and progression-free survival (PFS): miR-1244, miR-193b-5p and miR-1231. The overexpression of miR-1244 and miR-193-5p were associated with advance stage and worse clinical prognosis factors (p15% were independently associated with worse SG and EFS (Figure 1). In previously reported studies, these 3 miRNAs have been related with proliferative events such as the overexpression of myc or anti-apoptosis. Also, the miR-1231 may be related with new lymphomagenesis pathways associated to viral infections. Conclusions: Through a discovery group focused on progression/refractoriness, a group of new miRNAs differentially expressed on chemo-resistant patients with DLBCL was identified. The overexpression of miR-1244 and miR-193-5p was associated with more extensive disease and worse clinical prognostic factors. The high R-IPI, reduction of 〉15% RDI and the overexpression of miR-1231 were independently associated with worse EFS and OS. Disclosures Sánchez-González: Amgen: Consultancy, Speakers Bureau; Gilead: Speakers Bureau; Navartis: Consultancy, Speakers Bureau; Shire: Speakers Bureau; Takeda: Consultancy, Speakers Bureau. Salar:Roche: Research Funding, Speakers Bureau; Janssen Pharmaceuticals: Consultancy, Speakers Bureau; Gilead: Consultancy, Speakers Bureau; Celgene: Consultancy.

Description: Introduction: Hodgkin lymphoma (HL) is a hematological malignancy, with an inflammatory majority component of reactive cells and a few (1-2%) Reed-Sternberg cells (RSC). A high percentage of patients are cured with conventional strategies but approximately 15-30% relapse or progress. The standard tool to assess disease burden is the Ann Arbor Stage that classically categorizes HL in early (I-II) and advanced stages (III-IV). However, AA staging lacks accuracy in predicting outcome. New ways to asses tumor burden, such as baseline fluorodeoxyglucose positron emission tomography/computed tomography (FDG PET/CT), detect active disease with higher sensitivity in comparison with computed tomography (CT). Additionally, different volumetric parameters, such as metabolic tumor volume (MTV) and total lesion glycolysis (TLG), may be obtained from FDG PET/TC. We aim to improve disease burden testing using FDG PET/TC-related parameters, to better stratify HL patients at the time of diagnosis. Methods: We retrospectively selected patients with HL, homogeneously treated with ABVD +/- RT, at Son Espases and Son Llatzer University Hospitals in Palma de Mallorca from the databases of Pharmacy, Pathology and Nuclear Medicine Departments, to avoid selection bias. FDG PET/TC was done at baseline. MTV was measured with a semiautomatic method using a 41% maximum standardized uptake value (SUVmax) threshold and represents the sum of metabolic volumes of tumor tissues with increased FDG uptake. TLG was calculated by multiplying MTV and the mean SUV (SUVmean) of the MTV and was representative of the metabolic activity throughout the entire tumor. We used receiver operating curves (ROC) analysis to obtain the optimal cutoff for progression or death of all experimental FDG PET/TC-related variables. Standard clinical prognostic variables were obtained from medical records (age, gender, stage, bulky and ECOG PS) and main prognostic scores (IPS, EORTC and GHSG) were calculated. Progression-free survival (PFS) was considered the time from diagnosis to disease progression or death of any cause. Univariate survival analysis was done using Kaplan-meier plots and comparison between variables with log-rank test. Results: From August-2011 to November-2018, we included 101 patients. Table 1 shows main characteristics of patients. Median age was 37 years (14-83 years), 53% of patients had an advanced stage and 10% had bulky disease. With a median follow-up of 45 months (11-90), median PFS was 78%. The optimal cuttoffs obtained for MTV, TLG and SUVmax were 32.5, 167.8 and 10.4, respectively. In the univariate survival analysis, PFS was significantly influenced by MTV (p=0,007) and TLG (p=0.003), but not AA stage (Table 2). OS was significantly influenced by TLG (p=0.007) and SUVmax (p=0.001). With the three FDG PET/TC-related variables influencing HL survival we designed a FDG PET/TC score as follows: 1 point for high level of MTV, TLG or SUVmax (from 0 to 3). As shown in Table 2, this new FDG PET/TC score had a much better risk assessment that standard AA stage, being able to differentiate three risk groups with 100%, 84% and 65% 4-y PFS and 100%, 84% and 75% OS. Conclusions: The combination of functional 3D measurement of tumor burden (MTV, TLG and SUVmax) obtained from the FDG PET/TC at diagnosis, in HL, could be a valuable tool to better stratify the risk patients from tumor burden at the moment of diagnosis, when compared with standard AA staging. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: Micro-RNAs (miRNAs), have been shown to be one of the main regulators of gene expression and other biological processes. Recently, attention is focused on their potential role as prognostic predictors in different types of cancer, including diffuse large B cell lymphoma (DLBCL). We previously reported several miRNAs associated to progression/refractoriness in DLBCL (miR-1244, miR-193b-5p and miR-1231). The aim of this study was to validate these resultsin vitro, exploring the molecular pathways involved. Material and methods: The DLBCL cell line, U-2932 (activated B-cell (ABC) subtype) (ACC 633) was obtained from DSMZ (Germany). Transient reverse transfection of DLBCL cells for 48 or 72 h with mirVana miRNA mimics or inhibitors (miR-1244, miR-193b-5p and miR-1231) or with a miRNA negative control (100 nM) was performed using Viromer®GREEN reagent (Lipocalyx). In some experiments, after 24 h of transfection cells were treated with vehicle (1% DMSO) or with increasing concentrations of CHOP (cyclophosphamide, adriamycin, vincristine sulfate, and prednisone) (Sigma-Aldrich) for 48 h. The ratio of the four drugs was 80 mg/ 5.33 mg/ 0.16 mg/ 5.77 mg. After 48 h of transfection or treatment, cell viability was measured using the CellTiter 96®AQueous One Solution and total RNA was isolated using the RNeasy Mini Kit from Qiagen. Gene expression profiling (GEP) was performed using GeneChip human Clarion S of Affymetrix-ThermoFisher. Results: Reverse transfection of U-2932 with the miRNA mimic negative control (100 nM) for 48 h did not alter the cell viability compared to non-transfected (NT) cells, showing that the transfection method did not affect viability in these DLBCL cells. Transfection of mimic miR-1244, miR-193b-5p or miR-1231 (100 nM) did not significantly alter the viability compared to cells transfected with the miRNA mimic negative control. However, inhibition of these endogenous miRNA molecules for 48 h significantly decreased by 24.1 ± 5.7%, 28.9 ± 6.5% and 30.9 ± 3.3% cell viability, respectively, suggesting that these miRNAs could be involved in the survival of these tumor cells. In these experiments, the percentage of cell transfection was 89.6%. The effect of miRNA 1244, 193b-5p or 1231 on the response of DLBCL cells to CHOP treatment was also evaluated. Transfection of U-2932 cells with miRNA mimic negative control did not alter its sensibility to CHOP, which dose-dependently decreased its viability with an IC50 of 1.45 μg/ml in NT or transfected cells. Transfection of U-2932 cells with mimic miR-1244 or miR-193b-5p (100 nM) increased cell viability in the presence of vehicle (1% DMSO) and also reverted the inhibitory effect of CHOP (0.3 μg/ml) after 48 h of treatment. However, transfection of U-2932 cells with mimic miR-1231 (100 nM) did not alter the inhibitory effect of CHOP (0.3, 1 and 3 μg/ml) on cell viability at any of the concentrations studied. GEP studies showed that these miRNAs reduced the expression of genes associated to chemosensitivity (MCTP1) in the case of miR-1244 and miR-193-5p or tumoral suppressor genes (NRN1) in the case of miR-1231. Conclusions: We validatedin vitroour previously reported miRNAs about the role of miR-1244, miR-193b-5p and miR-1231 associated to treatment failure. The inhibition of endogenous miR-1244, miR-193b-5p or miR-1231 molecules significantly decreased cell viability. Transfection of U-2932 cells with mimic miR-1244 or miR-193b-5p (100 nM) increased cell viability of cells treated with CHOP. GEP studies showed that these miRNAs were linked to chemoresistance and inhibition of tumoral suppressor genes. Future works will have to translate these results to clinical practice in DLBCL. Disclosures Salar: Celgene:Speakers Bureau;Janssen:Speakers Bureau;Roche:Speakers Bureau.