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B-cell receptor (BCR) signaling and T-cell interactions play a pivotal role in chronic lymphocytic leukemia (CLL) pathogenesis and disease aggressiveness. CLL cells can use microRNAs (miRNAs) and their targets to modulate microenvironmental interactions in the lymph node niches. To identify miRNA expression changes in the CLL microenvironment, we performed complex profiling of short noncoding RNAs in this context by comparing CXCR4/CD5 intraclonal cell subpopulations (CXCR4dimCD5bright vs CXCR4brightCD5dim cells). This identified dozens of differentially expressed miRNAs, including several that have previously been shown to modulate BCR signaling (miR-155, miR-150, and miR-22) but also other candidates for a role in microenvironmental interactions. Notably, all 3 miR-29 family members (miR-29a, miR-29b, miR-29c) were consistently down-modulated in the immune niches, and lower miR-29(a/b/c) levels associated with an increased relative responsiveness of CLL cells to BCR ligation and significantly shorter overall survival of CLL patients. We identified tumor necrosis factor receptor–associated factor 4 (TRAF4) as a novel direct target of miR-29s and revealed that higher TRAF4 levels increase CLL responsiveness to CD40 activation and downstream nuclear factor-κB (NF-κB) signaling. In CLL, BCR represses miR-29 expression via MYC, allowing for concurrent TRAF4 upregulation and stronger CD40–NF-κB signaling. This regulatory loop is disrupted by BCR inhibitors (bruton tyrosine kinase [BTK] inhibitor ibrutinib or phosphatidylinositol 3-kinase [PI3K] inhibitor idelalisib). In summary, we showed for the first time that a miRNA-dependent mechanism acts to activate CD40 signaling/T-cell interactions in a CLL microenvironment and described a novel miR-29–TRAF4–CD40 signaling axis modulated by BCR activity.

Recirculation of chronic lymphocytic leukemia (CLL) cells between the peripheral blood and lymphoid niches plays a critical role in disease pathophysiology, and inhibiting this process is one of the major mechanisms of action for B-cell receptor (BCR) inhibitors such as ibrutinib and idelalisib. Migration is a complex process guided by chemokine receptors and integrins. However, it remains largely unknown how CLL cells integrate multiple migratory signals while balancing survival in the peripheral blood and the decision to return to immune niches. Our study provided evidence that CXCR4/CD5 intraclonal subpopulations can be used to study the regulation of migration of CLL cells. We performed RNA profiling of CXCR4dimCD5bright vs CXCR4brightCD5dim CLL cells and identified differential expression of dozens of molecules with a putative function in cell migration. GRB2–associated binding protein 1 (GAB1) positively regulated CLL cell homing capacity of CXCR4brightCD5dim cells. Gradual GAB1 accumulation in CLL cells outside immune niches was mediated by FoxO1-induced transcriptional GAB1 activation. Upregulation of GAB1 also played an important role in maintaining basal phosphatidylinositol 3-kinase (PI3K) activity and the “tonic” AKT phosphorylation required to sustain the survival of resting CLL B cells. This finding is important during ibrutinib therapy, because CLL cells induce the FoxO1-GAB1-pAKT axis, which represents an adaptation mechanism to the inability to home to immune niches. We have demonstrated that GAB1 can be targeted therapeutically by novel GAB1 inhibitors, alone or in combination with BTK inhibition. GAB1 inhibitors induce CLL cell apoptosis, impair cell migration, inhibit tonic or BCR-induced AKT phosphorylation, and block compensatory AKT activity during ibrutinib therapy.

Follicular lymphoma (FL) is a common indolent B-cell malignancy with a variable clinical course. An unfavorable event in its course is histological transformation to a high-grade lymphoma, typically diffuse large B-cell lymphoma. Recent studies show that genetic aberrations of MYC or its overexpression are associated with FL transformation (tFL). However, the precise molecular mechanisms underlying tFL are unclear. Here we performed the first profiling of expression of microRNAs (miRNAs) in paired samples of FL and tFL and identified 5 miRNAs as being differentially expressed. We focused on one of these miRNAs, namely miR-150, which was uniformly downmodulated in all examined tFLs (∼3.5-fold), and observed that high levels of MYC are responsible for repressing miR-150 in tFL by binding in its upstream region. This MYC-mediated repression of miR-150 in B cells is not dependent on LIN28A/B proteins, which influence the maturation of miR-150 precursor (pri-miR-150) in myeloid cells. We also demonstrated that low miR-150 levels in tFL lead to upregulation of its target, namely FOXP1 protein, which is a known positive regulator of cell survival, as well as B-cell receptor and NF-κB signaling in malignant B cells. We revealed that low levels of miR-150 and high levels of its target, FOXP1, are associated with shorter overall survival in FL and suggest that miR-150 could serve as a good biomarker measurable in formalin-fixed paraffin-embedded tissue. Overall, our study demonstrates the role of the MYC/miR-150/FOXP1 axis in malignant B cells as a determinant of FL aggressiveness and its high-grade transformation.

We and others have shown that deregulation of microRNAs (miRNAs) is associated with the biology of B cell malignancies, including regulation of B cell proliferation and survival (Musilova & Mraz, Leukemia, 2015). We focused on studying miRNAs that associate with the aggressiveness of FL and its transformation to diffuse large B cell lymphoma (DLBCL). First, we analyzed the expression of 380 miRNAs (TaqMan Arrays, ABI) in 8 paired primary samples of FL that subsequently transformed to DLBCL. We identified statistically significant changes (P1.8) in the expression of 5 miRNAs. The most significant change was the down-regulation of miR-150 (~5 fold, P=0.01). Similarly, we observed significantly reduced miR-150 levels in an independent cohort of non-paired samples of FL before vs. after transformation to DLBCL, and miR-150 was significantly less expressed in de novo DLBCL in comparison with FL. MicroRNA miR-150 is of particular interest as we have shown that its expression determines BCR signaling propensity in chronic lymphocytic leukemia (CLL) B cells, and low levels associated with worse survival (Mraz et al., Blood, 2014). Therefore, we analyzed miR-150 expression in a cohort of 89 FL samples. We noticed that miR-150 expression was lower in samples from patients with a FLIPI score ≥3 (P=0.03), and with high Ki67 positivity (>20%; P=0.003). Moreover, FL patients with low miR-150 levels ( To determine the potential reason for variable miR-150 levels in FL B cells, we tested the effect of microenvironmental interactions on its expression. In this experiment, a short term (48hrs) co-culture of B cell lymphoma cells with stromal cells (HS-5) led to down-regulation of miR-150 levels (P CONCLUSION: Low miR-150 levels associate with a shorter overall survival in FL. This could be used as a reasonable prognostic marker since high miRNA stability allows reliable analyses of miR-150 levels from formalin-fixed, paraffin-embedded (FFPE) samples. Interactions with stromal cells and/or soluble microenvironmental factors down-modulate miR-150 levels in B cells, which support their BCR signalling potential. We are further investigating to what extent the miR-150 down-regulation is causally connected with the aggressiveness and/or transformation of FL. This work was supported by: the Ministry of Education, Youth and Sports of the Czech Republic under the project CEITEC 2020 (LQ1601); the European Union's Horizon 2020 research and innovation programme under grant agreement No. 692298; the research grant GACR (16-13334Y); the Ministry of Health of the Czech Republic, grant nr. 16-29622A. All rights reserved. This work was financed from the SoMoPro II Programme (project No. 4SGA8684), co-financed by European Union and the South-Moravian Region. This publication reflects only the author's views and the Union is not liable for any use that may be made of the information contained therein; Masaryk university as part of the project "New approaches in research, diagnostics and therapy of hematological malignancies III", number MUNI/A/1028/2015 with the support of the Specific University Research Grant, as provided by the Ministry of Education, Youth and Sports of the Czech Republic in the year 2016; the Ministry of Health of the Czech Republic - conceptual development of research organization (FNBr, 65269705, Sup 3/16); the Ministry of Education, Youth and Sports of the Czech Republic, grant nr. LD15144 (COST CZ); the research grant TACR (TEO2000058/2014-2019); and EHA Research Fellowship award granted by the European Hematology Association. G.P. is a city of Ostrava scholarship holder. Disclosures No relevant conflicts of interest to declare.

Introduction Results of randomized studies showed benefit of maintenance therapy with monoclonal anti-CD20 antibody (rituximab) in terms of time to progression (PFS) and overall survival (OS) in follicular lymphoma (FL). General recommendation, based on large clinical trial, is to give 2 years of rituximab maintenance á 375mg/m2every 2 months (12 doses) in first line setting. On the other hand, there are various rituximab maintenance schedules; however, the clear comparison of clinical efficacy is missing. Our retrospective analysis compared two different schedule of rituximab maintenance in first-line treatment of FL used in university centers participating on CLG registry. Methods Data were recruited from 1702 FL patients registered in the prospectively maintained multicentric database (Czech Lymphoma Group; CLG). For the analysis, patients with stage II-IV of new diagnosed FL (grade 1-3a) responding (complete or partial remission) to 6-8 cycles first-line RCHOP (rituximab, cyclophosphamide, doxorubicine, vincristine and prednisone) followed with rituximab maintenance (RM) were included. Completed planned maintenance was inclusion criterion. Patients with previous watch and wait or additional first line therapy (radiotherapy, other chemotherapy, transplant therapy) were excluded. Results Totally, 168 evaluable FL patients with median age 57ys (range 28-82) including 70 (41.7%) men treated with RCHOP + RM were found in CLG database. 52/168 patients received totaly 8 doses of rituximab maintenance every 3 months for 2 years (RM8 arm), whereas 47/168 patients were treated with totaly 12 doses (RM12 arm) of rituximab maintenance every 2 months for 2 years. All patients in both subgroups completed planned RM therapy. There was no difference in distribution of age, gender, FLIPI, grade, B-symptoms, bone marrow involvement, performance status, LDH and beta2microglobuline level between both arms. Induction treatment in terms of administered cycles CHOP (6xCHOP in 41/52 and 35/45 pts., for RM8 and RM12 arm) and rituximab doses (8xR in 48/52 and 41/45 pts., for RM8 and RM12 arm) was similar between arms (ns). There were 4/52 (7.7%) and 5/47 (10.6%) relapses in subgroups RM8 and RM12, with no statistical significance. Median PFS was 3.8 (2.1-5.8) years vs. 3.9 (2.4-7.8) years in RM8 and RM12 arms (not significant), and median OS 3.91 (2.2-6.94) years vs. 3.1 (2.48-8.6) years also with no statistical significance. Conclusion Our results show, that rituximab maintenance given every 2 or every 3 months for two years in first line treatment brings similar benefit to the FL patients in terms of remission duration and overall survival. Despite the fact, that presented data are retrospective observation, this is the first report comparing two different rituximab maintenance regimens in FL. Further prospective study and longer follow up are needed to confirm our preliminary data. This work was supported by grant NT/12193-5 and MHCZ-DRO (FNBr 65269705) Disclosures: Mayer: Roche: Consultancy, Research Funding; Glaxo: Consultancy, Research Funding. Trneny:Roche: Honoraria, Research Funding.

Abstract 4138 Background: 18F-FDG-positron emission tomography (PET) is a powerful tool for imaging of various lymphomas. Follicular lymphoma (FL) is the most common indolent lymphoma with usually slow progressive course, where growing is dependent on accumulation of cells with defect in apoptosis. In spite of its indolent nature, FL belongs to tumours with high 18F-FDG avidity. Semi-quantitative evaluation of PET activity can be defined as a standard uptake value (SUV max). Up to now, it is unclear, why FL is so highly 18F-FDG avid and whether SUV max can be of some prognostic value. Aims: We tried to correlate PET activity defined as SUV max with grade, tumor growth activity (Ki67), and prognosis using progression free survival (PFS). FL has, however, generally low proliferative activity. Therefore, other cells are expected to be responsible for 18F-FDG avidity in this disease. We selected suitable cells of “microenvironment”, and tried to correlate their numbers wit SUV max. Methods: Patients with newly diagnosed FL having PET with defined SUV max were included in this retrospective study. Diagnosis of FL including grading (grade 1–3) was confirmed by experienced hematopathologist on original lymph node samples and proliferation activity was evaluated by immunostaining with Ki67. In 25 cases, material was available and tissue microarrays (TMA) were done; populations of CD34 (endothelial marker), CD3 (global T-lymphocytes), CD8 (cytotoxic lymphocytes), FOXP3 (T-regulatory lymphocytes), CD23 (follicular dendritic cells) and CD68 (lymphoma associated macrophages) were evaluated. Ki67 as well as numbers of non-malignant elements were given in % of positive cells. Lymph node samples chosen for TMA were taken in our institution only, to avoid interlaboratory variability. Results: Data from 73 FL patients of stage II-IV were analyzed. Median age was 57 (31-76) years, and grading distribution was as follows: grade 1, 2 and 3 were observed in 40, 21 and 12 patients. Median follow up of the whole group was 45 months (1-73). PET activity defined as SUV max had median 7,8 (0-22,2), proliferation activity measured by Ki67 (n=53) ranged between 1,5-80% with median 25%. SUV max did not seem to correlate with grade or Ki67; moreover, SUV max did not predict course of follicular lymphoma in terms of PFS. No differences in gender, age or FLIPI were observed between subgroups with high and low SUV max. Additionally, 25 samples were available for TMA and subpopulations of microenvironment were evaluated. Although the number of samples was limited, we observed a tendency of positive correlation between amount of CD34+ cells (angiogenesis marker) and CD68+ (lymphoma associated macrophages) with SUV max (using cut off 8,0). Unfortunately, statistical significance could not be reached in these subpopulations (p 0.11 and 0.13). Surprisingly, we could identify strong negative correlation between number of interfolicullary localized CD8+ cells (cytotoxic lymphocytes) and SUV max using cut off 8 and 9 (p 0.02 and 0.003). Conclusions: Our data support the assumption, that proliferation activity, although various in FL, seems have nothing to do with 18F-FDG avidity. On the contrary, we observed strong correlation of CD8+ cells and SUV max and certain tendency that cells involved in angiogenesis (CD34+) and inflammatory response (CD68+) may influence SUV max as well. Based on our pilot results, we suggest that the microenvironment gives global 18F-FDG activity in FL. The microenvironment, however, is a mixture of various cells, and that could be the reason why SUV max does not seem to be a prognostic tool in this disease. Our preliminary results require confirmation by further research. Disclosures: No relevant conflicts of interest to declare.