Proliferation Centers Research Articles

Artificial Intelligence-Assisted Mapping of Proliferation Centers in Chronic Lymphocytic Leukemia/ Small Lymphocytic Lymphoma Identifies Patterns That Reliably Distinguish Accelerated Phase and Large Cell Transformation

BackgroundChronic lymphocytic leukemia (CLL) involving tissues is characterized by sheets of small lymphocytes and vague nodules of larger cells, known as proliferation centers (PCs). CLL can undergo progression to accelerated CLL (aCLL) or further progress to diffuse large B-cell lymphoma, also known as Richter transformation (RT). Distinguishing CLL with many PCs from aCLL or RT can be challenging, particularly in small needle-biopsy specimens. In this study, we sought to design an artificial intelligence (AI)-based tool to automate and enhance the delineation of PCs and provide an objective approach to CLL/SLL acceleration/transformation.Material & MethodsWe manually annotated 25, 28 and 21 regions of interest (ROIs) encompassing small round PCs and confluent/ expanded PCs of 10 CLL, 12 aCLL, and 8 RT digitized hematoxylin & eosin stained slides, respectively (Figure A1). We analyzed the ROIs with both length and width larger than 2,000 pixels and set the tile length and stride as 1,000 and 100 pixels, respectively (Figure A1), and were able to extract sufficient tiles from each ROI (Figure A2). To recreate PCs, after performing nuclear segmentation via convolutional neural network and quality control, we quantified the nuclear size/intensity of cells occupying each tile (Figure A3). Nuclear size varied from 8 to 108 square micrometers, and nuclear mean intensity varied from 0 to 255. We normalized both the values of nuclear size and mean intensity to 0.0 and 1.0, by subtracting the minimum value and dividing it by the value range length. Nuclear size and mean intensity were represented as S(celli) and Imean(celli), respectively. We estimated the heat value of one tile integrating nuclear and mean intensity using the equationin Figure A2.ResultsWe generated heatmaps based on the heat values per tile inside each ROI from the 3 disease entities (CLL, aCLL and RT), as illustrated in Figures B1-4. Areas with high heat values (in the yellow spectrum) correspond to tiles harboring cells with increased nuclear size and mean intensity (PCs in CLL cases and expanded/ confluent PCs in aCLL and RT cases). In contrast, areas with low heat values (in the blue spectrum) correspond to tiles with decreased nuclear size and mean intensity (small neoplastic lymphocytes surrounding PCs) (Figure B4). We then generated a heat value histogram per tile for each ROI (Figure B5). Based on these results, the two optimal thresholds isolated to obtain the highest separation value among the three disease entities based on the optimal F-score were: 0.228, below which the case was most likely to be CLL, and 0.288, above which the case was most likely to be RT. Cases with heat values ranging between 0.228 and 0.288 were most likely aCLL cases. We then plotted the mean heat value frequencies of the 3 entities: There was a significant difference in the ranges of mean heat value frequencies for CLL, aCLL, and RT, which were 0.168 to 0.233, 0.212 to 0.307, and 0.261 to 0.353, respectively (Figure C). The accuracy and area under the curve diagnostic predictive values using data from nuclear size alone were 0.658 (+/-0.115) and 0.771 (+/-0.096), respectively; and using mean nuclear intensity, 0.679 (+/-0.094) and 0.841 (+/-0.052), respectively; with a noticeable increase using heat value frequencies (integrating the nuclear size and mean nuclear intensity) reaching 0.813 (+/-0.0630) and 0.885 (+/-0.109), respectively.ConclusionWe describe a novel AI-based heatmap technique to objectively assess the extent of PCs in CLL, based on the integrative analysis of cell nuclear size and mean nuclear intensity. We suggest that an ROI mean heat value less than 0.228 is predictive of CLL, and more than 0.288 is predictive of RT. aCLL cases demonstrate a mean heat value ranging from 0.228 to 0.288. Using the mean heat value of all cases, we were able to reliably separate the three entities in question with robust diagnostic predictive values. [Display omitted] DisclosuresKhoury: Stemline Therapeutics: Research Funding; Kiromic: Research Funding; Angle: Research Funding.

Phosphoglycerate Dehydrogenase Is Required for Germinal Center Formation and Is a Therapeutic Target in MYC-driven Lymphoma

The fields of cancer- and immuno-metabolism have re-emerged as areas of significant translational potential. Even though the upregulation of glycolysis by proliferating lymphocytes is the basis for widely used clinical tests such as FDG-PET, little is known about which metabolic pathways are involved in the utilization of glucose to support B-cell proliferation. The synthesis of serine from glucose has been demonstrated to be a key metabolic pathway supporting cellular proliferation in some healthy and malignant cell types. Importantly, this pathway is regulated by MYC, which is known to be essential for germinal centre formation and is commonly dysregulated in lymphoma. Despite this, the role that the serine synthesis pathway (SSP) plays in germinal center biology and pathology has not been previously investigated. We performed a comprehensive characterization of the role of the SSP in germinal center B cells and lymphomas derived from these cells.We demonstrate that upregulation of a functional SSP is a metabolic hallmark of B-cell activation and the germinal center reaction. We show that both human and murine resting naïve B cells lack expression of the first two enzymes in this pathway, phosphoglycerate dehydrogenase (PHGDH) and phosphoserine aminotransferase (PSAT1) enzymes. However, B-cell activation, predominantly through the B-cell receptor, robustly induces the expression of these enzymes in vitro, resulting in an acquired ability to synthesize serine, glycine and the purine nucleotides adenosine and guanosine from glucose. This is reflected in striking expression of PHGDH and PSAT1 within germinal centers but not in marginal zones confirming that this upregulation is occurring in germinal B cells activated in vivo. We then proceeded to investigate the impact of inhibiting PHGDH on germinal center formation and high-affinity antibody production in vivo. This was done both genetically, using a conditional B-cell knockout mouse model, and pharmacologically using a specific inhibitor of PHGDH, PH-755. Importantly, we show that PHGDH inhibition impairs germinal center formation with a resultant reduction in high-affinity antibody production. Mechanistic experiments demonstrate that PHGDH inhibition effectively blocks cells from synthesising serine and glycine from glucose, making them unable to proliferate in environments that lack these amino acids.We then investigated role of PHDGH and PSAT1 in Burkitt Lymphoma (BL), Diffuse Large B Cell Lymphoma (DLBCL) and Chronic Lymphocytic Leukemia (CLL). Notably, very high expression of these two proteins was observed in BL, with intermediate-to-high expression in DLBCL and relatively low expression in CLL, where expression was restricted to proliferation centers. Given the heterogeneity of expression in DLBCL patients, we next interrogated a published GSE database (Lenz et al. NEJM 2008) to investigate the impact on outcome. Notably high expression of PSAT1 was significantly associated with a poorer overall survival rate in DLBCL.We then investigated whether the SSP could be a therapeutic target in lymphoma. We demonstrate that PHGDH inhibition effectively inhibits de novo serine, glycine and purine nucleotide synthesis from glucose, resulting in impaired proliferation and increased apoptosis in a panel of human BL cell lines in vitro. We then analyzed the impact of PHGDH inhibition on lymphoma development in vivo using Eµ-myc mice, which harbour Myc coupled to the IgH enhancer characteristic of BL. Importantly we confirm the role of MYC by demonstrating that Eµ-Myc B-cells show significantly higher expression of PHGDH and PSAT1 expression resulting in increased serine and glycine synthesis when compared to control cells. We demonstrate that pharmacological inhibition of PHGDH using PH-755 impairs lymphoma progression in this model. We confirm the importance of PHGDH by showing that genetic ablation of Phgdh in Eµ-myc cells in a tamoxifen inducible system (using Eµ-myc/+;Rosa26-CreER T2/+;Phgdh fl/fl mice) also results in a significant reduction in lymphoma progression.Taken together, this work represents the first report of the role of the SSP in the biology of the germinal centre response and lymphomas derived from these cells. These findings establish PHGDH as a critical player in humoral immunity and a clinically relevant target in MYC-driven lymphoma, which is an area of significant unmet need. DisclosuresGribben: Abbvie: Honoraria; AZ: Honoraria, Research Funding; BMS: Honoraria; Gilead/Kite: Honoraria; Janssen: Honoraria, Research Funding; Morphosys: Honoraria; Novartis: Honoraria; Takeda: Honoraria; TG Therapeutis: Honoraria. Calado: Myricx Pharma: Consultancy, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company, Patents & Royalties: Cancer Treatments. WO patent WO 2020/128475 A1 (2020).

Expansion of T Follicular Helper Cells in NLC Co-Cultures Reinforces the Concept of Co-Evolution of CLL and Supportive T Helper Cell Clones

Expansion of T Follicular Helper Cells in NLC Co-Cultures Reinforces the Concept of Co-Evolution of CLL and Supportive T Helper Cell Clones

Recurrent Chromosomal Abnormalities in Tissues Involved by Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma.

Prognostically relevant chromosomal abnormalities in chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) are routinely identified by fluorescence in situ hybridization (FISH) on peripheral blood or bone marrow specimens. We studied the prevalence of chromosomal abnormalities on extramedullary tissues involved by CLL/SLL and evaluated their association with prominent proliferation centers (PPCs). FISH for recurrent abnormalities in CLL/SLL was performed on formalin-fixed, paraffin-embedded biopsy sections. PPCs were identified on H&E-stained sections. Available FISH results on peripheral blood or bone marrow specimens were also reviewed. Recurrent FISH abnormalities were detected in 69% of 320 CLL/SLL biopsy specimens studied, including +12 (35%), 13q- (24%), 11q- (15%), 17p- (6%), 6q- (2%), and IGH/BCL2 (0.9%). Forty-three patients had abnormal blood or bone marrow FISH analyses, of whom 7 (16%) had discordant +12 and/or 13q-, and 3 (7%) had discordant 17p- or 11q-. Morphology was positive (17%), negative (78%), or equivocal (6%) for PPCs on 247 evaluable biopsy specimens, a finding not significantly associated with FISH results (P = .7). Trisomy 12 is overrepresented in tumoral CLL/SLL involvement, compared with the known predominance of 13q- in blood. Discrepancies between leukemic and tissue FISH findings are occasionally encountered. FISH results do not correlate with the presence of PPCs.

Brg1 Supports B Cell Proliferation and Germinal Center Formation Through Enhancer Activation.

Activation and differentiation of B cells depend on extensive rewiring of gene expression networks through changes in chromatin structure and accessibility. The chromatin remodeling complex BAF with its catalytic subunit Brg1 was previously identified as an essential regulator of early B cell development, however, how Brg1 orchestrates gene expression during mature B cell activation is less clear. Here, we find that Brg1 is required for B cell proliferation and germinal center formation through selective interactions with enhancers. Brg1 recruitment to enhancers following B cell activation was associated with increased chromatin accessibility and transcriptional activation of their coupled promoters, thereby regulating the expression of cell cycle-associated genes. Accordingly, Brg1-deficient B cells were unable to mount germinal center reactions and support the formation of class-switched plasma cells. Our findings show that changes in B cell transcriptomes that support B cell proliferation and GC formation depend on enhancer activation by Brg1. Thus, the BAF complex plays a critical role during the onset of the humoral immune response.

Metformin as a Treatment Strategy for Sjögren's Syndrome.

Sjögren’s syndrome (SS), a chronic inflammatory disease involving the salivary and lacrimal glands, presents symptoms of sicca as well as systemic manifestations such as fatigue and musculoskeletal pain. Only a few treatments have been successful in management of SS; thus treatment of the disease is challenging. Metformin is the first-line agent for type 2 diabetes and has anti-inflammatory potential. Its immunomodulatory capacity is exerted via activation of 5’ adenosine monophosphate-activated protein kinase (AMPK). Metformin inhibits mitochondrial respiratory chain complex I which leads to change in adenosine mono-phosphate (AMP) to adenosine tri-phosphate (ATP) ratio. This results in AMPK activation and causes inhibition of mammalian target of rapamycin (mTOR). mTOR plays an important role in T cell differentiation and mTOR deficient T cells differentiate into regulatory T cells. In this manner, metformin enhances immunoregulatory response in an individual. mTOR is responsible for B cell proliferation and germinal center (GC) differentiation. Thus, reduction of B cell differentiation into antibody-producing plasma cells occurs via downregulation of mTOR. Due to the lack of suggested treatment for SS, metformin has been considered as a treatment strategy and is expected to ameliorate salivary gland function.

PD-1 Expression in Chronic Lymphocytic Leukemia and Large B-Cell Richter Transformation (DLBCL-RT): A Distinguishing Feature for DLBCL-RT and Promising Surrogate Marker for Clonal Relatedness

Purpose: The programmed death-1 (PD-1) pathway is crucial in tumor immunity evasion and its blockade has emerged as an effective anti-cancer immunotherapy. PD-1 and PD-L1 expression has not been thoroughly examined in chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) and their associated large B-cell Richter transformation (DLBCL-RT). This study investigates PD-1 and PD-L1 expression and explores its clinicopathological significance in CLL/SLL and DLBCL-RT.Methods: PD-1 and PD-L1 expression was assessed by immunohistochemistry in 39 CLL/SLL, 15 DLBCL-RT with prior history of CLL/SLL, and 26 DLBCL without prior history of CLL/SLL (22 de novo DLBCL and 4 with prior history of other low-grade B-cell lymphoma). The clonal relationship of DLBCL and the corresponding preceding CLL/SLL was molecularly determined in 10/15 DLBCL-RT. Clinical outcomes were evaluated by Kaplan-Meier analysis.Results: PD-1 expression in CLL/SLL neoplastic B-cells was weak and restricted to the larger prolymphocytes/paraimmunoblasts within the proliferation centers (PCs) and accentuated PCs of all sizes. Expression was not observed on the background monotonous-appearing small B-cells. Neoplastic B-cells PD-1 expression was highly prevalent in DLBCL-RT and demonstrated increased intensity in comparison to the CLL/SLL prolymphocytes/paraimmunoblasts. In contrast, neoplastic B-cell PD-1 expression was only rarely seen in DLBCL without prior history of CLL/SLL (12/15 vs. 1/26, P<0.0001). In DLBCL-RT, neoplastic B-cell PD-1 expression demonstrated excellent concordance (90%) with the molecularly-defined CLL/SLL clonal relatedness. Strong PD-1 expression was observed in the scattered background small tumor infiltrating T-cells (TIL) in the entire study cohort, and served as an internal control for the PD-1 immunostain. PD-L1 expression was observed on the background immune cells histiocytes and dendritic cells, absent on the CLL/SLL neoplastic B-cells, and rarely seen on the large neoplastic B-cells in DLBCL-RT or other DLBCL (1/15 vs. 1/26, P>0.05). DLBCL-RT prognosis was significantly inferior to the other DLBCL (median overall survival 18.3 vs. 167.3 months, P=0.001) and PD-1 expression was associated with adverse outcome in the combined DLBCL cohort (median overall survival 18.3 vs. 167.3 months, P=0.02).Conclusions: PD-1 expression is weak and restricted to the PC prolymphocytes/paraimmunoblasts in CLL/SLL and increased on the large neoplastic B-cells in DLBCL-RT, suggesting a biological continuum. It is highly prevalent in DLBCL-RT but rare in other DLBCL. Neoplastic B-cell PD-1 expression is a distinguishing feature for DLBCL-RT and a promising surrogate marker for CLL/SLL clonal relatedness in DLBCL-RT, which have important prognostic and therapeutic implications. The restricted PD-1 expression in PCs may also aid in precise PC evaluation in CLL/SLL. DisclosuresDing:Merck: Research Funding. Parikh:AstraZeneca: Honoraria; Pharmacyclics: Honoraria; Pharmacyclics: Research Funding. Shanafelt:Genentech, Celgene, Pharamacyclics, Jansen, GlaxoSmithKline, AbbVie, Hospira, and Cephalon: Research Funding. Ansell:Seattle Genetics: Research Funding; Merck: Research Funding; Celldex: Research Funding; Affimed: Research Funding; Bristol-Myers Squibb: Research Funding.

Regulation and Impact of BCR Induced Autophagy in Chronic Lymphocytic Leukemia

Introduction: Chronic lymphocytic leukaemia (CLL) is characterised by the accumulation of malignant B-cells within the peripheral blood and secondary lymphoid organs. CLL patients can be broadly divided into two groups depending on the mutational status of the immunoglobulin variable region genes (IGHV), with unmutated and mutated V- genes associated with progressive and indolent disease respectively. B cell receptor (BCR) expression and signalling capacity is pivotal to CLL pathogenesis and whilst drug which targeting this pathway are revolutionising the treatment of this disease. Resistance is already occurring and therefore a better understanding of this pathway is urgently required if we are to identify novel therapeutic strategies to treat this largely incurable disease. Autophagy primarily acts as a protective mechanism against cellular stress induced by starvation, cytokine removal, hypoxia, reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress and is important for normal B-cell development. However autophagy can also promote tumorigenesis and resistance to therapy. The role of autophagy in CLL cases in response to therapy has previously been investigated, however its role in CLL biology and how it is regulated has not previously been evaluated.Results: We investigated basal expression of a number of autophagy markers between CLL cases and normal donor B cells (NDB). We demonstrated that basal expression of LC3B-II p=0.0005, GABARAPL2 p=0.0113, ATG3 p=0.0005 and ATG7 p=0.034 was greater in CLL samples compared to NDB. Moreover even greater LC3B-II expression was observed in U-CLL cases compared to M-CLL (p=0.02). Using a pre-determined cut-off, LC3B-II expression identified a subset that had a shorter time to first treatment from first lymphocytosis (p=.017) and identified samples particularly within the M-CLL subset which were more likely to progress. Next using tissue extracted from CLL lymph nodes we showed greater LC3B expression in areas with higher levels of Ki67 by immunohistochemistry, indicating an association between proliferation centers and autophagy. This suggested that autophagy might be induced following BCR signalling. Indeed basal expression of LC3B-II was greater in samples with increased BCR signalling capacity. Therefore we investigated the role of the BCR in regulating autophagy. Activation of the CLL BCR with bead immobilised (BI) anti-IgM significantly induced expression of LC3B-II (p<.0001) at 2, 4 and 24 hours, with greater levels induced in U-CLL compared to M-CLL cases. Similar changes were observed with anti-IgD but at reduced levels compared to anti-IgM treatment. Interestingly using confocal microscopy and transmission electron microscopy we demonstrated that the beads coated with anti-IgM were being endocytosed by the CLL cells and the autophagosomes formed around the beads. To confirm that the BI anti-IgM induced autophagy was not solely a result of bead endocytosis we also treated CLL and NDB with soluble anti-IgM and anti-IgD. Soluble anti-IgM significantly induced LC3B-II (p=0.006) and GABARAPL2 (p=0.04) expression compared to untreated controls in CLL cases, but at slightly reduced levels compared to BI anti-IgM. In contrast soluble anti-IgD induced minimal changes or no change at all. Similar changes were also observed in NDB. Next we examined autophagic flux to determine whether BI anti-IgM increased RNA and protein expression or simply inhibited autophagosome breakdown. CLL samples treated with BI anti-IgM increased LC3B and GABARAP RNA expression within 2h (p=.0017 & p=.022 respectively) whilst treatment with BI anti-IgM plus HCQ significantly increased LC3B-II protein expression up to 24 hours compared to HCQ or anti-IgM alone (p<.0001). Confirming that activation of the BCR increased autophagic flux. To confirm the role of the BCR in the regulation of autophagy we treated CLL cells with ibrutinib or fostamatinib to inhibit BCR signalling capacity or treated cells with IL-4 to increase sIgM expression and signalling as we have previously demonstrated. As expected BCR kinase inhibitors prevented anti-IgM induced autophagy. In contrast IL-4 increased sIgM and signalling and consequently induced autophagy highlighting the importance of the BCR in regulating autophagy in CLL. Together these data suggest that the autophagy pathway might be therapeutically targeted for the treatment of CLL. DisclosuresStrefford:Roche: Research Funding. Forconi:AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Infinity: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Research Funding; Janssen-Cilag: Speakers Bureau. Steele:Portola Pharmaceuticals: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria.

Hyper-Activation of the Adenosinergic Axis in the Eμ-TCL1 Chronic Lymphocytic Leukemia Mouse Model Offers Therapeutic Opportunities

Background. Tumor immunosuppression is a major cause for treatment failure and disease relapse, both in solid tumors and leukemia. Several indications suggest that increased extracellular adenosine levels, which potently suppress T cell responses and skew macrophages towards an M2 phenotype, characterize the tumor microenvironment. Hence, there is intense investigation to identify drugs that target this axis.We previously showed that CLL cells, particularly those endowed in the proliferation centers, express CD39 and CD73, the enzymes that lead to extracellular adenosine production. We also showed that the A2A adenosine receptor is expressed at high levels in CLL lymph nodes, likely due to the effect of hypoxia in positively modulating adenosine production.Aim of the work. The main aim of this work is to study the expression and the role of the adenosinergic axis in vivo, determining the impact of this signaling pathway in CLL progression and modulation of immune responses. To this purpose, we exploited the Eμ-TCL1 (TCL1) mouse model based on the adoptive transfer of spleen-purified leukemic cells in naïve C57BL/6 immunocompetent recipients (WT).Results. Firstly, expression of the adenosinergic axis (CD39, CD73, CD26 and A2A) both on the leukemic side and on bystander immune cells was assessed by comparing cells obtained from the spleen, the bone marrow (BM) and peripheral blood (PB) of TCL1 and WT mice. Flow cytometry analysis of B220+/CD5+ leukemic cells showed that CD39 was constantly expressed at high levels, in contrast to normal circulating B cells from naïve mice. CD73 expression was more heterogeneous, with both CD73+ and CD73- leukemic clones. These data recapitulate the phenotype observed in CLL patients and sustain the hypothesis of variable levels of adenosine in tissues colonized by leukemic cells. In contrast, CD26, the adenosine deaminase-anchoring protein was poorly detected, as opposed to WT B cells. Real-time PCR data obtained from purified cell populations confirmed this expression pattern and also indicated dramatic up-regulation in the expression of the A2A receptor, suggesting that leukemia development is accompanied by increased ability to respond to extracellular adenosine. The development of CLL is usually associated with specific T cell and myeloid subset changes. Multiparametric flow cytometry analyses, with selective markers, were adopted to identify naïve, memory, central memory, effector memory and effector T cells, as well as inflammatory, intermediate or patrolling monocytes. Data obtained confirm that in TCL1 mice the naïve T cell subset was dramatically reduced compared to WT mice, with the simultaneous increase in memory, exhausted and regulatory T cell sub-populations. Both CD4+ and CD8+ lymphocyte subsets in TCL1 mice were highly CD39+ compared to the normal counterpart, as opposed to CD73 that was expressed in distinct subpopulation of cells, including regulatory T cells. No significant differences were highlighted for CD26, in all the tissues considered, while the A2A receptor became expressed at very high levels in all cellular components of the leukemic niche. In the monocyte compartment, we observed a reduction of the inflammatory subset, with a parallel increase of patrolling population, functionally similar to M2 tumor-supportive macrophages.Given the role of low oxygen tension as a critical modulator of the adenosinergic system, hypoxia levels were determined, comparing tumor-bearing and WT mice. Mice were treated with pimonidazole hydrochloride, a hypoxia marker, 2 hours prior euthanasia. Flow cytometry and immuno-histochemistry analyses indicated that leukemic engraftment is paralleled by increased hypoxia levels in the lymphoid tissues, such as spleen or BM.Conclusions.Taken together, these results show that the adenosinergic axis is over-expressed in the TCL1 mouse model and confirm that the presence of leukemic cells polarizes the host immune cells, favoring the establishment of a tumor supportive environment. This could be due, at least partly, to the presence of a highly hypoxic leukemic niche. These observations confirm what described for CLL patients, indicating that this animal model can be used to test the impact of adenosine-targeting therapies, in combination with drugs that directly target the clone. DisclosuresNo relevant conflicts of interest to declare.

Reactive Oxygen Species and Metabolic Gene Expression Profiling Further Differentiate CXCR4/CD5 Fractions in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) clones are made up of slow growing, long-lived mature B cells that accumulate in the blood and secondary lymphoid organs. Studies using deuterium exchange into CLL cell DNA of patients drinking heavy water led to the phenotypic classification of intraclonal subfractions that differ in time since last cell division. Cells that have most recently divided, termed the proliferative fraction (PF), are CXCR4DimCD5Bright and older cells that are in a resting state, termed the resting fraction (RF), are CXCR4BrightCD5Dim. These studies suggest a model of the life cycle of a CLL cell that involves exiting of the PF from the tissue into the blood and subsequent homing of the RF from the blood to the tissue to survive and/or recapitulate the division cycle.The CXCL12/CXCR4 axis is central to this trafficking of CLL cells out of and into proliferative niches in the tissue. Moreover, activation of this axis is associated with an increase in the transcriptional activity of sterol response element binding protein-1 (SREBP1) in cancer. SREBP1 regulates genes involved in lipid synthesis and uptake. Since CLL cells preferentially rely on fatty acid (FA) metabolism for energy demands, this concurs with the proposed life cycle of CLL subfractions. CXCL12thereby exerts two functions on the CXCR4BrightCD5DimRF: directional trafficking to the tissue and altering the transcriptome to adapt to the lipid rich environment in the lymph node [LN] that the cells in this fraction will soon encounter. Indeed, our microarray analysis has confirmed that SREBP1a and c are more highly expressed in the RF [1.012x] compared to the PF that express higher levels of the transcriptional targets such as lipoprotein lipase [LPL: 1.201x] fatty acid synthase [FASN: 1.17x] glucose-6-phosphate dehydrogenase [G6PD: 1.505x] and pyruvate kinase [PKM2: 1.433x]. Cancer cells are known to have a concomitance to FA synthesis and oxidation during proliferation. This appears to be true in CLL if we infer that the higher expression of genes involved within the most recently divided cells of the PF are a reflection of how actively dividing cells behave in the LN.Since the main source of reactive oxygen species (ROS) is a by-product of oxidative phosphorylation, we examined the extent that the CLL CXCR4/CD5 subfractions differed in ROS levels as a result of their individual metabolic demands during proliferation. We found that PF has significantly higher levels of ROS compared to the RF, regardless of patient IGHV gene mutation status. Additionally, microarray analysis has revealed that the PF express significantly higher levels of genes involved in response to oxidative stress compared to the RF; these include superoxide dismutase [SOD1: 1.149x], catalase [CAT: 1.462] , hemeoxygenase-1 [HMOX1: 1.695], and glutathione peroxidase [GPX1:1.898] . This suggests that this population is uniquely suited to survive under harsh conditions that result from their distinctive demand for fatty acid oxidation to fuel growth in contrast to normal B-cells. This makes targeting these protective enzymes an attractive therapeutic strategy, as an accumulation of damaging levels of ROS will induce cell death.Finally, we have noted a rare population of cells that are CD5BrightCXCR4Negative that contain the highest levels of ROS in CLL patient PBMCs. Initial studies suggest that these CD5BrightCXCR4Negative cells are more enriched in patient LNs and might represent the cells actively dividing in proliferation centers. If so, those few in the peripheral circulation bearing this phenotype may be the closest link to the tissue resident dividing cells. Consistent with this, concurrent quantitation of ROS and cell cycle analysis by flow cytometry confirmed that higher levels of ROS are present in MEC1cells in S, M, and G2 phases. Current studies involve comparing gene expression profiles of various CXCR4/CD5 fractions, including the CD5Bright CXCR4Negative subset, from the blood and LN based on high ROS and reciprocal densities of CXCR4 and CD5. By sorting this rare population from PB, we may conveniently study those actively dividing CLL cells in the LN in lieu of obtaining rare biopsy samples. DisclosuresBarrientos:AbbVie: Consultancy, Research Funding; Gilead: Consultancy, Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy, Research Funding; Janssen: Consultancy. Kolitz:Boehringer Ingelheim, Cantex, Erytech, and Millennium: Research Funding; Gilead, Magellan, Novartis, Pharmacyclics, and Seattle Genetics: Consultancy; Celgene, Jazz: Equity Ownership; Gilead, Magellan, and Novartis: Honoraria; Gilead, Novartis, and Seattle Genetics: Other: Travel Support.

Tumor Microenvironment (TME) Regulation of Programmed Death-1 (PD1) Receptor and Its Ligands PDL1 and PDL2 in Chronic Lymphocytic Leukemia (CLL)

The PD1 pathway is involved in the inactivation of immune effectors and is potentially reactive against neoplastic cells in the TME. T cells from CLL patients exhibit defective immunity, decreased proliferative capacity, and impaired effector function leading to T cell exhaustion. These functional defects coincide with higher CD244, CD160 and PD1 expression on T cells. In the Eμ-TCL1 transgenic CLL model, PDL1 checkpoint blockade restores immune dysfunction and impairs leukemia growth. PD1/PDL1 ligation also affects BCR signaling, and because PD1 is also expressed on CLL cells, PD1 interference might directly influence tumor growth and proliferation directly.We studied the 1) expression the PD1 receptor and its ligands and their correlation with TFTT; 2) role of the TME in controlling PD1 axis and miRNA expression; 3) role of the BCRi ibrutinib (IB) in the expression of the receptor and ligands.Early-stage CLL patients were prospectively enrolled at diagnosis (n=211, O-CLL1: clinicaltrial.govID:NCT00917540). Gene expression (GE) profiling was performed using the GeneChipVR Gene1.0 STArray (Affymetrix) and validated by qPCR (Thermofisher) and flow-cytometry (FC, BD Biosciences) in an independent subset of patients. CLL lymph node samples underwent in situ immunolocalization analyses. Autologous T-cells (AAT) were obtained by in vitro exposure of patient T-cells with anti CD3/CD28 Dynabeads (Thermofisher) and IL2 in co-culture with CLL cells. Cultures were monitored until substantial clumping was observed and tested for PD1 axis expression. HS5 stromal cells were used in co-culture experiments with B-CLL (ratio 1:100). MiR-424 expression was evaluated by qPCR using validated TaqMan® MicroRNA Assays (Thermofisher). In selected experiments IB was added to cell culture. PD1 GEP of circulating B-CLL cells was significantly higher than of ligands. No differences in GEP of PD1 or its ligands were observed stratifying cases based on CD38, ZAP-70, or FISH markers. Significantly higher PDL1 GE was detected in IGHV-M cases. GEP results were validated by qPCR on 27 independent CLL samples. The impact of GE of the PD1 axis on clinical outcomes in CLL cases (median follow-up 39 months, range 6−82 months) indicated TTFT was significantly shorter in cases with higher levels of PDL2, while no significant differences were detected based on higher or lower PD1 or PDL1 levels. Multivariate analysis indicated higher PDL2 gene expression retained an independent prognostic power (HR=1.9, 95%CI 1.1-3.4, P=.022) in predicting TTFT together with IGHV-UM status, B-lymphocytosis≥5000/mm3 (P=.020), and CD38 expression (P=.021). In situ immunolocalization of CLL tissue infiltrates showed variable expression of PD1 that characterized small lymphoid elements, while PDL1 and PDL2 (PDL2>PDL1) characterized larger medium-sized elements within proliferation centers. Co-localization studies revealed PD1 co-expression of CD20+CLL cells and in scattered CD3+ cells. Both PDL1 and PDL2 were variably expressed in CD20+CLL cells; few T-cells and macrophages showed expression of both ligands within CLL infiltrates. FC analysis of CLL cells after AAT co-culture showed a higher percentage expression of circulating PD1 axis members (n=27, PDL1>>PDL2>PD1) compared to baseline indicating that the changes in PD1 axis expression may be due to events triggered by activation of the TME. In contrast, T cell populations had higher expression of CD3+, CD4+ and CD8+ cells bearing both PD1 and PDL1, while no substantial increment for either CD3+, CD4+ or CD8+/PDL2+ cells following AAT co-culture. GE analysis also showed a similar increase in PDLs (n=15, PDL2>>PDL1>PD1) expression with respect to non-activated B-CLL cells. Interestingly, a 48h co-culture of B-CLL with HS5 stromal cells (n=6) showed a similar increase in PD1 axis GE pattern as AAT cultures. Expression of miR-424 also decreased significantly (<80%) after AAT co-cultures (n=6), indicating its potential involvement in PDL1 regulation. IB reduced PD1/PDL1 gene and protein expression on CLL B-cells; the IB inhibitory effect was also observed only on CD8+ cells.We indicate that PDL2 expression characterizes a subset of high-risk early stage CLL patients. PD1 axis expression is characteristic of the CLL TME whereby modulatory co-stimulatory signals may be counteracted by IB. Further studies will examine the role of miR-424 regulation with this cellular context. DisclosuresNo relevant conflicts of interest to declare.

Co-Stimulatory versus Cell Death Aspects of Agonistic CD40 Monoclonal Antibody Selicrelumab in Chronic Lymphocytic Leukemia.

Simple SummaryPrevious observations have shown that CD40 activation of CLL cells via coculture with CD40L-expressing fibroblasts increases sensitivity to cell death by CD20 mAbs rituximab and obinutuzumab. We studied the activity of the fully human-agonistic CD40 mAb selicrelumab in primary CLL cells in relation to cell activation, induced pro-survival profile and sensitization for cell death by aCD20 mAbs. We found that the pro-survival effect of selicrelumab is minimal, while cell death by combined selicrelumab plus anti-CD20 antibody treatment is maintained. Thus, further investigation of applying selicrelumab combined with anti-CD20 mAbs in a therapeutic setting might be considered.Objectives: Chronic lymphocytic leukemia (CLL) is a common form of leukemia with a heterogeneous clinical course that remains incurable due to the development of therapy resistance. In lymph node proliferation centers, signals from the microenvironment such as CD40 ligation through interaction with follicular T helper cells shield CLL cells from apoptosis. Previous observations have shown that, despite CD40-induced changes in apoptotic mediators resulting in cell survival, CD40 activation also increases sensitivity to cell death by CD20 mAbs rituximab and obinutuzumab. To further investigate these observations, we here studied the activity of the fully human agonistic CD40 mAb selicrelumab in primary CLL cells in relation to cell activation, induced pro-survival profile, and sensitization for cell death by aCD20 mAbs, in vitro. Methods: CLL cells from peripheral blood were isolated by the Ficoll density method. The expression of activation markers and cytokine production following CD40 stimulation was quantified by flow cytometry and ELISA. The anti-apoptotic profile of CLL induced by stimulation was evaluated by the expression of BCL-2 proteins with Western blot, and resistance to venetoclax with flow cytometry. Cell death induced by the combination of selicrelumab and aCD20 mAbs was quantified by flow cytometry. Results: CLL cells treated with selicrelumab upregulated co-stimulatory molecules such as CD86, TNF-α and death receptor CD95/Fas. In contrast to the CD40 ligand-transfected NIH3T3 cells, induction of resistance to venetoclax by selicrelumab was very moderate. Importantly, selicrelumab stimulation positively sensitized CLL cells to CD20-induced cell death, comparable to CD40 ligand-transfected NIH3T3 cells. Conclusions: Taken together, these novel insights into selicrelumab-stimulatory effects in CLL may be considered for developing new therapeutic strategies, particularly in combination with obinutuzumab.

Biological method of plant protection is one of the green economy development directions

The importance of the biological method of plant protection as one of the prospects for the green economic development in Russia and in the world is discussed in the article. The modern directions of the Russian agricultural science development for the transition to environmentally friendly agricultural production are analyzed. The experience of biological preparations and biological agent’s application in Russia and abroad is considered in the historical aspect. A differentiated approach has been proposed for chemical treatments prescribing, carrying them out only in pest proliferation centers, on border land and catching band, which makes it possible to almost halve the pesticides use. The field research results on the study of the spatial distribution of useful entomofauna in irrigated agricultural landscapes are presented, ways of increasing the species diversity and number of beneficial insects (entomophages and parasites) in agrocoenosis due to the redistribution of useful entomocomplex species in agrocoenosis and natural agricultural landscape are shown.

Ligand Bound Fatty Acid Binding Protein 7 (FABP7) Drives Melanoma Cell Proliferation Via Modulation of Wnt/β-Catenin Signaling.

Fatty acid-binding protein 7 (FABP7) involved in intracellular lipid dynamics, is highly expressed in melanomas and associated with decreased patient survival. Several studies put FABP7 at the center of melanoma cell proliferation. However, the underlying mechanisms are not well deciphered. This study examines the effects of FABP7 on Wnt/β-catenin signaling that enhances proliferation in melanoma cells. Skmel23 cells with FABP7 silencing and Mel2 cells overexpressed with wild-type FABP7 (FABP7wt) and mutated FABP7 (FABP7mut) were used. Cell proliferation and migration were analyzed by proliferation and wound-healing assay, respectively. Transcriptional activation of the Wnt/β-catenin signaling was measured by luciferase reporter assay. The effects of a specific FABP7 inhibitor, MF6, on proliferation, migration, and modulation of the Wnt/β-catenin signaling were examined. FABP7 siRNA knockdown in Skmel23 decreased proliferation and migration, cyclin D1 expression, as well as Wnt/β-catenin activity. Similarly, FABP7wt overexpression in Mel2 cells increased these effects, but FABP7mut abrogated these effects. Pharmacological inhibition of FABP7 function with MF6 suppressed FABP7-regulated proliferation of melanoma cells. These results suggest the importance of the interaction between FABP7 and its ligands in melanoma proliferation modulation, and the beneficial implications of therapeutic targeting of FABP7 for melanoma treatment.

Mushroom Bodies of Stick and Leaf Insects (Phasmatodea: Insecta): Structure and Sources of Formation

It has been established that mushroom bodies of stick insects are formed by Kenyon cells collected in numerous groups of cells the processes of which originally form individual bundles going into the calyx. The majority of such bundles lose their identity already in the cellular layer, and therefore, complex bundles usually enter the calyx. The merging of bundles progresses in the calyx, and even the peduncle keeps a fascicular structure. Only lobes have a classical concentric structure. In the embryonic period, mushroom bodies are formed by two proliferative centers with one neuroblast per center. It is supposed that the neuroblasts of mushroom bodies of stick insects are neuroblasts of type II and that groups of Kenyon cells are generated by intermediate neural progenitors.

Microenvironment Remodeling and Subsequent Clinical Implications in Diffuse Large B-Cell Histologic Variant of Richter Syndrome.

IntroductionRichter Syndrome (RS) is defined as the development of an aggressive lymphoma in the context of Chronic Lymphocytic Leukemia (CLL), with a Diffuse Large B-Cell Lymphoma (DLBCL) histology in 95% cases. RS genomic landscape shares only a few features with de novo DLBCLs and is marked by a wide spectrum of cytogenetic abnormalities. Little is known about RS microenvironment. Therapeutic options and efficacy are limited, leading to a 12 months median overall survival. The new targeted treatments usually effective in CLL fail to obtain long-term remissions in RS.MethodsWe reviewed available PubMed literature about RS genomics, PD-1/PD-L1 (Programmed Death 1/Programmed Death Ligand 1) pathway triggering and subsequent new therapeutic options.ResultsData from about 207 patients from four landmark papers were compiled to build an overview of RS genomic lesions and point mutations. A number of these abnormalities may be involved in tumor microenvironment reshaping. T lymphocyte exhaustion through PD-L1 overexpression by tumor cells and subsequent PD-1/PD-L1 pathway triggering is frequently reported in solid cancers. This immune checkpoint inhibitor is also described in B lymphoid malignancies, particularly CLL: PD-1 expression is reported in a subset of prolymphocytes from the CLL lymph node proliferation centers. However, there is only few data about PD-1/PD-L1 pathway in RS. In RS, PD-1 expression is a hallmark of recently described « Regulatory B-cells », which interact with tumor microenvironment by producing inhibiting cytokines such as TGF-β and IL-10, impairing T lymphocytes anti-tumoral function. Based upon the discovery of high PD-1 expression on tumoral B lymphocyte from RS, immune checkpoint blockade therapies such as anti-PD-1 antibodies have been tested on small RS cohorts and provided heterogeneous but encouraging results.ConclusionRS genetic landscape and immune evasion mechanisms are being progressively unraveled. New protocols using targeted treatments such as checkpoint inhibitors as single agents or in combination with immunochemotherapy are currently being evaluated.

Abstract IA23: The role of EZH2 gain-of-function mutations in lymphoma

Abstract Follicular lymphomas (FLs) are slow-growing, indolent tumors containing extensive follicular dendritic cell (FDC) networks and recurrent EZH2 gain-of-function mutations. Paradoxically, FLs originate from highly proliferative germinal center (GC) B cells with proliferation strictly dependent on interactions with T follicular helper cells. Herein, we show that EZH2 mutations initiate FL by attenuating GC B-cell requirement for T-cell help and driving slow expansion of GC centrocytes that become enmeshed with and dependent on FDCs. By impairing T-cell help, mutant EZH2 prevents induction of proliferative MYC programs. Thus, EZH2 mutation fosters malignant transformation by epigenetically reprograming B cells to form an aberrant immunologic niche that reflects characteristic features of human FLs, explaining how indolent tumors arise from GC B cells. Citation Format: Wendy Béguelin. The role of EZH2 gain-of-function mutations in lymphoma [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr IA23.

Identification of enhancers that drive the spatially restricted expression of Vsx1 and Rx in the outer proliferation center of the developing Drosophila optic lobe.

Combinatorial spatial and temporal patterning of stem cells is a powerful mechanism for the generation of neural diversity in insect and vertebrate nervous systems. In the developing Drosophila medulla, the neural stem cells of the outer proliferation center (OPC) are spatially patterned by the mutually exclusive expression of three homeobox transcription factors: Vsx1 in the center of the OPC crescent (cOPC), Optix in the main arms (mOPC), and Rx in the posterior tips (pOPC). These spatial factors act together with a temporal cascade of transcription factors in OPC neuroblasts to specify the greater than 80 medulla cell types. Here, we identify the enhancers that are sufficient to drive the spatially restricted expression of the Vsx1 and Rx genes in the OPC. We show that removal of the cOPC enhancer in the Muddled inversion mutant leads to the loss of Vsx1 expression in the cOPC. Analysis of the evolutionarily conserved sequences within these enhancers suggests that direct repression by Optix may restrict the expression of Vsx1 and Rx to the cOPC and pOPC, respectively.

The N-terminal peptide generated after activation of prophenoloxidase affects crayfish hematopoiesis

The circulating hemocytes of invertebrates are important mediators of immunity, and hemocyte homeostasis is of high importance for survival and health of crustaceans. The prophenoloxidase (proPO)-activating system is one of the most essential immune reactions, which can be activated by pattern recognition proteins from microorganisms. Activation of proPO by the proPO activating enzyme generates an N-terminal peptide, with cleavage site after Arg176, as well as the active enzyme phenoloxidase, which is the key enzyme for melanization. In the present study we demonstrate a role for the N-terminal proPO-peptide in hematopoiesis. Injection of this proPO-peptide increased the number of circulating hemocytes and especially granular hemocytes. We also show that the reactive oxygen species (ROS) production in the anterior proliferative center was enhanced after proPO peptide injection, which is a prerequisite for rapid hemocyte release from the hematopoietic tissue. Moreover, this peptide had an effect on ROS production in in vitro cultured hematopoietic cells and induced spreading of these cells within 72 h. Taken together, our findings show a role of the N-terminal proPO peptide in stimulation of hematopoiesis in crayfish, Pacifastacus leniusculus.

Integrative analysis of spontaneous CLL regression highlights genetic and microenvironmental interdependency in CLL

AbstractSpontaneous regression is a recognized phenomenon in chronic lymphocytic leukemia (CLL) but its biological basis remains unknown. We undertook a detailed investigation of the biological and clinical features of 20 spontaneous CLL regression cases incorporating phenotypic, functional, transcriptomic, and genomic studies at sequential time points. All spontaneously regressed tumors were IGHV-mutated with no restricted IGHV usage or B-cell receptor (BCR) stereotypy. They exhibited shortened telomeres similar to nonregressing CLL, indicating prior proliferation. They also displayed low Ki-67, CD49d, cell-surface immunoglobulin M (IgM) expression and IgM-signaling response but high CXCR4 expression, indicating low proliferative activity associated with poor migration to proliferation centers, with these features becoming increasingly marked during regression. Spontaneously regressed CLL displayed a transcriptome profile characterized by downregulation of metabolic processes as well as MYC and its downstream targets compared with nonregressing CLL. Moreover, spontaneous regression was associated with reversal of T-cell exhaustion features including reduced programmed cell death 1 expression and increased T-cell proliferation. Interestingly, archetypal CLL genomic aberrations including HIST1H1B and TP53 mutations and del(13q14) were found in some spontaneously regressing tumors, but genetic composition remained stable during regression. Conversely, a single case of CLL relapse following spontaneous regression was associated with increased BCR signaling, CLL proliferation, and clonal evolution. These observations indicate that spontaneously regressing CLL appear to undergo a period of proliferation before entering a more quiescent state, and that a complex interaction between genomic alterations and the microenvironment determines disease course. Together, the findings provide novel insight into the biological processes underpinning spontaneous CLL regression, with implications for CLL treatment.