Low CD20 Expression Research Articles

P11: Proteoglycans expression correlates with the phenotype of malignant and non-malignant EBV-positive B-cell lines

In vitro Epstein-Barr virus (EBV) infects B-cells resulting in immortalization, In vivo the virus resides latent in resting B-cells. Rarely the EBV-host cell interaction may contribute to development of malignant lymphoma. It is well known that both c-myc translocation and the viral infection are observed in patients with EBV+ Burkitt’s lymphoma (BL). Proteoglycans (PGs) are complex glycosylated proteins. They are key components of ECM and play a critical role in cell–cell and cell–matrix interactions. Disruptions of such interactions will affect B cell interaction with surrounding stroma and may thus perturb the cell phenotypes. The purpose of this study was to investigate expressinon of proteoglycans in EBV+ cell lines with different origins and phenotypes. We analysed the expression of 12 of the main proteoglycans in primary B cells, lymphoblastoid cell lines (LCLs) generated by EBV infection of normal human B cells in vitro and EBV-positive BL cell lines. An EBV-negative BL cell line was used for reference. According to RT-PCR analysis, primary B-lymphocytes expressed different PGs, mainly serglycin, CD44, perlecan and syndecan-1. The high expression of PGs in normal B cells probably reflects interactions of these cells with the neighbouring cells and microenvironment. B cell lines which carry EBV, in general, showed lower levels of PGs. The PGs expression pattern was similar in LCLs and in primary B cells, however, distinguished by high levels of perlecan and serglycin and low expression of CD44 in LCLs. BL cells showed the most significant down-regulation of PGs compared to primary B cells. There was a correlation between the type of EBV latency program, and PGs expression. Serglycin was expressed at a low levels in BL-cells with EBV latency III-program compared to LCLs, while in EBV latency I BL cells both serglycin and perlecan were down-regulated. Cells with latency I-program show general hypermethylation of the cellular genome in contrast to cells with latency III-program. Thus we explored the possibility of epigenetic regulation of the PG-coding genes by treating cells with 5′-deoxyazacytidine (5-AzaC, a demethylating agent) and Trichostatin A (TSA, a chromatin structure modulator). There was no significant change in PGs expression upon this treatment in LCLs or in EBV latency III BL cells, while EBV latency I BL cells showed up-regulation of several PGs. This suggests that PGs expression is at least partly regulated by epigenetic mechanisms. Interestingly EBV latency is also partly regulated at the epigenetic level. Similar trends were observed for the key ECM components (collagen 1A1, fibronectin and elastin). Normal B lymphocytes expressed collagen, fibronectin and elastin, whereas LCLs and BL cells showed no expression of these. Treatment of these cells with 5-AzaC or TSA resulted in similar changes in PGs expression patterns. Up-regulation of ECM components was detected only in EBV latency I BL cells. Taken together, our data show that proteoglycans are expressed in primary B lymphocytes whereas they are not or only partly expressed in EBV-carrying cell lines, depending on their latency program. Expression of PGs in latency I BL cells is silenced due to hypermethylation, but by another mechanim in latency III BL cells. These results show that PGs expression patterns follow the EBV latency programs . It will be highly interesting to further investigate if EBV and its transformation associated genes are directly involved in control of PGs, as well as how PGs may contribute to major phenotypic properties of EBV-carrying cell lines, such as adhesion, migration and growth in soft agarose – a property associated with the malignant phenotype of BLs.

P148: CD68 expression in inflammatory cell infiltration of nonspecific invasive breast cancer

Background Tumor-associated macrophages play a main role in tumor progression and dissemination. Taking into account the high heterogeneicity of tumor the different clinical impact of macrophages, infiltrating different sites of tumor, could be expected. The aim was to detect the level of CD68+ cells (macrophages) in the different site of stroma in breast tumor in comparison to clinical course. Materials and methods One thirty-six women with nonspecific invasive breast cancer T1-4N0-3M0, who were treated in General Oncology Department of Tomsk Cancer Research Institute (Tomsk, Russia), were included in the present study. Patients did not receive preoperative treatment. The material was fixed in 10–12% neutral formalin. Preparation of the histological material was carried out according to standard procedures. Morphological examination of the surgical specimens was performed by the standard method using a light microscope “Carl Zeiss Axio Lab.A1” (Germany) and slidescanner “MiraxMidiZeiss” (Germany). Metastatic lesion was detected in regional lymph nodes. Immunohistochemical study was performed according to standard procedures. Cytoplasmic expression of these markers was determined in the inflammatory cell infiltrate of different tumor segments: (1) in areas with soft fibrous stroma; (2) in areas with coarse fibrous stroma; (3) in the areas of the so-called “maximum stromal-and-parenchymal relationship” where the individual tumor cells, short strands and groups of tumor cells arranged in soft fibrous stroma; (4) among parenchymal elements; (5) in gaps of ductal tumor structures. Double-stained immunofluorescence was performed according to standard procedures using Leica TCS SP2 laser-scanning spectral confocal microscope (Germany). The following primary antibodies were used: mouse monoclonal anti-human CD68 (BD Biosciences) and rabbit polyclonal anti-stabilin-1 or RS1 (marker of M2 macrophages). Results The highest expression of CD68 in the inflammatory cell infiltrate was detected more frequently in areas with soft fibrous stroma (54%) or the so-called “maximum stromal-and-parenchymal relationship” (79%) in patients with breast cancer. The lowest expression of CD68 was observed in areas with coarse fiber stroma (23%). The CD68-positive cells of the inflammatory infiltrate were located between parenchymal elements of tumor (88%). Inverse correlation ( R = −0.67; p = 0.02) observed between tumor size and the expression of CD68 in the cells of the inflammatory infiltrate in gaps of tubular tumor structures. The CD68 expression in cells of the inflammatory tumor infiltrate was correlated with the presence of metastatic regional lymph nodes. It was found that in the case of the lymph node metastases the average score of CD68 expression in cells of ductal gaps tumor structures was lower (1.4 ± 0.5) in comparison with the negative lymph nodes case (3.1 ± 1.0; F = 10.9; p = 0.007). Same time no correlation between the CD68 expression in the inflammatory cell tumor infiltrate and the rate of tumor malignancy was found. Using confocal microscopy domination of CD68 + /RS1 + cells were found. Conclusion So, low CD68 expression level in ductal gaps tumor structures is associated with the presence of metastatic regional lymph nodes.

Abstract A12: Combination of the anti-CD38 monoclonal antibody daratumumab and all-trans retinoic acid.

Abstract Background: Daratumumab (DARA) is an anti-CD38 monoclonal antibody (mAb) with lytic activity on multiple myeloma (MM) cells, including ADCC (antibody-dependent cellular cytotoxicity) and CDC (complement-dependent cytotoxicity). In current clinical phase I/II trials, DARA induced anti-MM activity; however, the depth of the response varied between patients. Further improvement of DARA-treatment can be achieved by modulating the mechanisms hampering DARA responsiveness. Results: In a sub-analysis of 16 MM patients treated with DARA monotherapy (GEN501 trial), we observed that response to DARA was associated with baseline CD38 expression levels on the MM cells (R2= 0.40; P= 0.009). We also observed a significant decrease of CD38 expression on MM cells during DARA-treatment (median MFI decreased from 900 to 83). Consistent with this idea, in vitro experiments with isogenic MM cell lines expressing different levels of CD38 have revealed that the level of CD38 expression correlates with DARA-mediated ADCC and CDC. Similarly, in bone marrow samples from 125 and 56 MM patients, we observed a significant correlation between CD38 expression and DARA-mediated ADCC (Pearson R= 0.34; P= 0.<0001) as well as CDC (Pearson R= 0.35; P= 0.0038) respectively. Altogether, this suggested that upregulation of CD38 expression, before and during DARA-treatment, may improve the anti-MM activity of DARA. Indeed, all-trans retinoic acid (ATRA) induced a ~5-fold increase in CD38 expression both in 4 MM cell lines and primary MM cells from 5 DARA-naive patients, which resulted in synergistic improvement of DARA-mediated ADCC and CDC. The synergy between ATRA and DARA was observed not only in DARA-naive patients, but also in 2 DARA-treated patients, whose residual MM cells had lower CD38 expression following therapy. Conclusion: Our results provide evidence that CD38 expression levels may predict response to DARA. Furthermore, we show that ATRA increases CD38 expression on MM cells, resulting in enhanced DARA-mediated lysis of MM cells. Our results provide the preclinical rationale for further evaluation of DARA combined with ATRA in MM patients. Citation Format: Inger S. Nijhof, Henk M. Lokhorst, Berris van Kessel, Parul Doshi, Kate Sasser, Tuna Mutis, Niels WCJ van de Donk. Combination of the anti-CD38 monoclonal antibody daratumumab and all-trans retinoic acid. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr A12.

Abstract 1254: Mitochondrial SOD2 regulates EMT and cancer stem cell-like cell populations

Abstract Epithelial-mesenchymal transition (EMT) is involved in cancer cell invasion, metastasis and treatment failure. EMT may promote conversion of a subset of cancer cells defined by low expression of CD44 (CD44L) to those with high expression of CD44 (CD44H), the latter associated with increased malignant properties of cancer cells. Triggered by a variety of stimuli such as transforming growth factor (TGF)-β, hypoxia and chemoradiation, EMT may be activated in cancer cells via stress-induced reactive oxygen species (ROS) in the tumor microenvironment. Excessive ROS may cause cell death or senescence; however, cells undergoing EMT may require a physiological level of ROS. It remains elusive as to how cellular antioxidant capabilities may influence EMT. Mitochondrial superoxide dismutase 2 (SOD2) is frequently overexpressed in oral and esophageal cancers. Here, we investigated mechanisms of SOD2 transcriptional regulation in EMT as well as the functional role of this antioxidant in EMT. Using well-characterized genetically engineered oral and esophageal human epithelial cell lines coupled with RNA interference (RNAi) and flow cytometric approaches, we find that TGF-β stimulates EMT, resulting in conversion of CD44L to CD44H cells, the latter of which display SOD2 upregulation. SOD2 induction in transformed cells was concurrent with suppression of TGF-β-mediated induction of both ROS and senescence. SOD2 gene expression appeared to be transcriptionally regulated by NF-κB and ZEB2, but not ZEB1. Moreover, SOD2-mediated antioxidant activity may restrict conversion of CD44L cells to CD44H cells at the early stages of EMT. In aggregate, this study provides novel mechanistic insights into the dynamic expression of SOD2 during EMT and delineates a functional role for SOD2 in EMT, influencing distinct CD44L and CD44H subsets of cancer cells that have been implicated in oral and esophageal tumor biology. Note: This abstract was not presented at the meeting. Citation Format: Kelly A. Whelan, Hideaki Kinugasa, Koji Tanaka, Satish Srinivasan, Manti Guha, Daret St. Clair, Andres Klein-Szanto, Narayan Avadhani, Alan Diehl, Anil Rustgi, Hiroshi Nakagawa. Mitochondrial SOD2 regulates EMT and cancer stem cell-like cell populations. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1254. doi:10.1158/1538-7445.AM2015-1254

NOTCH1 mutations associate with low CD20 level in chronic lymphocytic leukemia: evidence for a NOTCH1 mutation-driven epigenetic dysregulation.

In chronic lymphocytic leukemia (CLL), NOTCH1 mutations have been associated with clinical resistance to the anti-CD20 rituximab, although the mechanisms behind this peculiar behavior remain to be clarified. In a wide CLL series (n=692), we demonstrated that CLL cells from NOTCH1-mutated cases (87/692) were characterized by lower CD20 expression and lower relative lysis induced by anti-CD20 exposure in vitro. Consistently, CD20 expression by CLL cells was upregulated in vitro by γ-secretase inhibitors or NOTCH1-specific small interfering RNA and the stable transfection of a mutated (c.7541-7542delCT) NOTCH1 intracellular domain (NICD-mut) into CLL-like cells resulted in a strong downregulation of both CD20 protein and transcript. By using these NICD-mut transfectants, we investigated protein interactions of RBPJ, a transcription factor acting either as activator or repressor of NOTCH1 pathway when respectively bound to NICD or histone deacetylases (HDACs). Compared with controls, NICD-mut transfectants had RBPJ preferentially complexed to NICD and showed higher levels of HDACs interacting with the promoter of the CD20 gene. Finally, treatment with the HDAC inhibitor valproic acid upregulated CD20 in both NICD-mut transfectants and primary CLL cells. In conclusion, NOTCH1 mutations are associated with low CD20 levels in CLL and are responsible for a dysregulation of HDAC-mediated epigenetic repression of CD20 expression.

Co-delivery of paclitaxel and gemcitabine via CD44-targeting nanocarriers as a prodrug with synergistic antitumor activity against human biliary cancer

Multi-drug delivery focuses on different signaling pathways in cancer cells that have synergistic anti-proliferative effects. In this study, we developed multi-prodrug nanocarriers (MPDNCs) consisting of poly (l-lysine)–carboxylate PTX (PLL-PTX) and hyaluronic acid-conjugated GEM (HA-GEM) for CD44-targeted synergistic biliary cancer therapy. An in vitro study of cell viability and mRNA expression levels and an in vivo study showed that MPDNCs more effectively inhibit proliferation in CD44-overexpressing cancer cells (HuCCT1) than in cells with lower CD44 expression (SCK) by synergistically inducing apoptosis. Consequently, these results demonstrate that MPDNCs are prodrugs with synergistic cancer therapeutic efficacy and effective cellular uptake at target cells compared to free drugs, indicating their strong potential as efficient multi-drug-carrying nano-platforms for cancer treatment.

Clinical significance of monocyte heterogeneity.

Monocytes are primitive hematopoietic cells that primarily arise from the bone marrow, circulate in the peripheral blood and give rise to differentiated macrophages. Over the past two decades, considerable attention to monocyte diversity and macrophage polarization has provided contextual clues into the role of myelomonocytic derivatives in human disease. Until recently, human monocytes were subdivided based on expression of the surface marker CD16. “Classical” monocytes express surface markers denoted as CD14++CD16− and account for greater than 70% of total monocyte count, while “non-classical” monocytes express the CD16 antigen with low CD14 expression (CD14+CD16++). However, recognition of an intermediate population identified as CD14++CD16+ supports the new paradigm that monocytes are a true heterogeneous population and careful identification of specific subpopulations is necessary for understanding monocyte function in human disease. Comparative studies of monocytes in mice have yielded more dichotomous results based on expression of the Ly6C antigen. In this review, we will discuss the use of monocyte subpopulations as biomarkers of human disease and summarize correlative studies in mice that may yield significant insight into the contribution of each subset to disease pathogenesis.

Expression of the Cancer Stem Cell Markers CD44 and CD133 in Colorectal Cancer: An Immunohistochemical Staining Analysis.

PurposeThe aim of this study was to assess the expressions of CD44 and CD133 in colorectal cancer tissue by using immunohistochemical staining and to analyze the clinical significance of the expressions related to other clinicopathological data and survival results.MethodsOne hundred sixty-two patients with a biopsy-proven colorectal adenocarcinoma who were operated on between January 1998 and August 2004 were enrolled in this study. Immunohistochemical staining for CD44 and CD133 was performed on primary colorectal cancer tissue, metastatic lymph nodes, and synchronous and metachronous metastatic tumor tissues if available.ResultsCD44 expression was stronger in the primary tumor than in metastatic lymph nodes (P < 0.001), and CD133 expression tended to be stronger in primary tumor than in metastatic lymph nodes (P = 0.057). No significant correlation was found between the CD44 and the CD133 expressions. The cases with recurrence showed low expression of CD44 (P = 0.017). CD133 expression was lower in cases with elevated CA 19-9 serum levels (P = 0.028) and advanced T stage (P = 0.038). Multivariate analysis proved that low expression of CD44 was an independent prognosis factor for short disease-free survival (P = 0.028).ConclusionLow CD44 expression was correlated with increased tumor recurrence and short disease-free survival, and low CD133 expression was associated with advanced tumor stage. We suggest that further studies be performed to evaluate whether the immunohistochemical method for determining the CD44 and the CD133 expressions is appropriate for exploring cancer stem-cell biology in patients with colorectal cancer.

Expression of Stem Cell Markers in Preinvasive Tubal Lesions of Ovarian Carcinoma.

In order to better understand the ovarian serous carcinogenic process with tubal origin, we investigated the expression of stem cell markers in premalignant tubal lesions (serous tubal intraepithelial carcinoma or STIC). We found an increased stem cell marker density in the normal fallopian tube followed by a high CD117 and a low ALDH and CD44 expression in STICs raising the question of the role of the stem cell markers in the serous carcinogenic process.

Isolation and Characterization of Human Mesenchymal Stem Cells Derived From Synovial Fluid in Patients With Osteochondral Lesion of the Talus

Background: Recently, mesenchymal stem cells (MSCs) have been suggested as a source for cell-based treatment of cartilage lesions based on the ability of these cells to differentiate into chondrocytes. Purpose: To characterize MSCs derived from the synovial fluid in ankle joints with osteochondral lesion of the talus (OLT). Study Design: Controlled laboratory study. Methods: Synovial fluid was collected from the ankle joints of 28 patients with OLT who underwent arthroscopic marrow stimulation between September 2011 and April 2012. Epitope profiles and multilineage differentiation were assessed to characterize the synovial fluid MSCs. To clarify the origin of synovial fluid MSCs, we assessed gene profiles of MSCs derived from various mesenchymal tissues by reverse transcription–polymerase chain reaction (RT-PCR) analysis. Results: Synovial fluid MSCs expressed CD90 and CD105, showed low expression of CD14 and CD34, and underwent multilineage differentiation in vitro. The RT-PCR revealed strong expression of CD90, CD44, and CD73, whereas CD45 and CD133 were not detected. The colony number of synovial fluid MSCs from OLT significantly increased in stages C and D, as defined by arthroscopic classification. Gene expression profiles indicated that synovial fluid MSCs derived from the patients with OLT were more similar to MSCs from synovium than to MSCs from bone marrow and adipose tissue. Conclusion: This study confirmed that human synovial fluid is a good source of MSCs, with the capacity to differentiate toward several cell lineages. Further study with matched controls of synovial fluid MSCs derived from ankle joints without OLT is required for a more accurate evaluation of synovial fluid MSCs. Clinical Relevance: The findings of this study provide a platform for exploring the potential role of synovial fluid MSCs in OLT and their therapeutic potential in novel joint regeneration strategies.

NOTCH1 Mutations Are Associated with Low CD20 Expression in Chronic Lymphocytic Leukemia: Evidences for a NOTCH1-Mediated Epigenetic Regulatory Mechanism

Background.NOTCH1 mutations are found in about 10% of chronic lymphocytic leukemia (CLL) cases at diagnosis, and with higher frequencies in chemorefractory CLL, CLL in advanced disease phases and in Richter Syndrome transformations (Rossi et al. Blood, 2013). In CLL, NOTCH1 mutations have been recently associated with clinical resistance to anti-CD20 immunotherapy (Stilgenbauer et al. Blood, 2014, Dal Bo et al. AHO, 2014). In lymphoproliferative disorders, susceptibility to rituximab is determined by CD20 levels (Golay et al. Blood, 2001), which are in turn epigenetically modulated via HDAC (Shimizu et al., Leukemia, 2010).Aim. To investigate whether NOTCH1 mutations could affect CD20 expression in CLL.Methods.NOTCH1 mutations and NOTCH1 mutational burden were evaluated by ARMS PCR, QRT-PCR, conventional and next generation sequencing. NOTCH1 protein levels were evaluated by western blot. CD20 expression was evaluated by flow cytometry. Transcript levels of MS4A1, encoding for CD20 protein, were evaluated by QRT-PCR. CLL cells and CLL-like cell line MEC-1 cells were treated with the HDAC inhibitor valproic acid (VPA) at a concentration of 3mM. Susceptibility to rituximab was evaluated by complement dependent cytotoxicity (CDC) assay. MEC-1 cells were transfected with a vector containing a NOTCH1 intracellular domain (NICD) carrying either the 7544-7545delCT or a stop codon at the beginning of NICD sequence (c.5304G>A), as control.Results.i) In a cohort of 452 CLL, 54 cases carried NOTCH1 mutations. NOTCH1-mutated cases (NOTCH1-mut) with high mutational burden showed 3.0- fold higher transmembrane NOTCH1 and 2.1- fold higher NICD protein expression compared to NOTCH1 wild-type cases (NOTCH1-wt). NOTCH1-mut showed a lower Mean Fluorescent Intensity (MFI) of CD20 expression than NOTCH1-wt both in trisomy 12 (tris12, 18 NOTCH1-mut vs. 44 NOTCH1-wt, p<0.001) and in non-tris12 (36 NOTCH1-mut vs. 354 NOTCH1-wt, p=0.005) cases. MS4A1 transcript levels were lower in NOTCH1-mut than in NOTCH1-wt (tris12 CLL, p=0.024; non-tris12 CLL, p=0.002). By performing cell sorting to isolate CD20low and CD20high subpopulations in 4 cases with subclonal NOTCH1 mutations (range 26%-45%), the CD20low subpopulation always showed an enrichment in NOTCH1 mutational burden when compared to the CD20high counterpart, along with lower MS4A1 expression levels. NICD mutated MEC-1 cells, expressing higher NICD transcript (2.2 over the control) and NICD (1.3 over the control) protein, were characterized by lower CD20 (0.1 over the control) and MS4A1 transcript (0.77 over the control) expression. ii) In keeping with CD20 levels, primary NOTCH1-mut CLL cells showed lower relative lysis induced by rituximab than NOTCH1-wt CLL cells (mean relative lysis: NOTCH1-mut, 5% vs. NOTCH1-wt, 30%, p=0.008). This phenomenon was confirmed by using the MEC-1 cell model (mean relative lysis: NICD mutated, 3% vs. control, 30%, p=0.006). iii) NICD mutated MEC-1 cells expressed higher levels of both HDAC1 (2.0 over the control) and HDAC2 (1.4 over the control) transcripts. Moreover, in co-immunoprecipitation experiments, NICD mutated MEC-1 cells were characterized by lower levels of HDAC1/HDAC2 bound to the transcriptional factor CBF-1, due to a competition for the binding to CBF1 with the high NICD levels present in these cells, and suggesting a nuclear interplay between NOTCH1 and HDAC where CBF-1 is the connecting element. The dependency of low CD20 expression by HDAC activity was demonstrated by the capability of VPA to increase CD20 expression in primary NOTCH1-mut CLL cells (1.3 over the control) and NICD mutated MEC-1 transfectants (1.4 over the control).Conclusions. CLL cases carrying NOTCH1 mutations are characterized by low CD20 expression levels that may confer resistance to anti-CD20 immunotherapy. The low CD20 expression, at least in part due to HDAC-dependent repression mechanism(s), can be reverted by HDAC inhibitor therapy. DisclosuresNo relevant conflicts of interest to declare.

The Broad Activity of the CD33/CD3 Bispecific BiTE® Antibody AMG 330 in Primary Human AML Is Impacted By Disease Stage and Cytogenetic/Molecular Risk

Background: Recent studies have demonstrated that AMG 330, a novel CD33/CD3-directed bispecific T-cell engaging (BiTE¨) antibody, is highly active against CD33+ AML cell lines and can potently lyse leukemic blasts from AML patients. Here, we have investigated the preclinical determinants for AMG 330 activity in primary newly diagnosed and relapsed/refractory human AML samples to prospectively understand factors that might contribute to clinical response or resistance.Patients and Methods: Frozen aliquots of Ficoll-isolated mononuclear cells from peripheral blood or bone marrow specimens were obtained from adults with AML who consented to their use for research purposes. CD33 expression on myeloblasts (identified by appropriate CD45 and side-scatter properties) and the percentage of endogenous CD3+ T-cells were quantified by flow cytometry. To determine drug-induced cytotoxicity, AML cells were incubated in culture medium containing various concentrations of AMG 330; in some experiments, exogenous T-cells (isolated via magnetic cell sorting from a healthy adult volunteer who underwent leukapheresis) were labeled with CellVue Burgundy and added at different effector:target (E:T) cell ratios. After 48 hours, cell numbers and drug-induced cytotoxicity, using DAPI to detect non-viable cells, were determined by flow cytometry; AML cells were identified by forward/side scatter properties and negativity for CellVue Burgundy dye. Specific drug-induced cytotoxicity was calculated as: 100 x (1 – live target cellstreated/live target cellscontrol), and presented as mean±SEM.Results: Forty-one of the 49 studied specimens were included in our analyses as they had >40% myeloblasts upon thaw and were >50% and >30% viable at baseline and after 48 hours, respectively. Median age of the patients was 65.3 (range: 23.9-80.0) years. The median percentage of myeloid blasts and CD3+ T-cells in the studied specimens was 87.1% (range: 55.1-97.0%) and 2.0% (range: 0-27.3%), respectively, and median sample viability after 48 hours in culture was 76.7% (range: 31.1-93.5%). These characteristics were similar between the newly diagnosed (n=21) and relapsed/refractory (n=20) AML specimens. In the absence of healthy donor T-cells, AMG 330 resulted in modest cytotoxicity (at 500 pg/mL: 11.8±2.5%) that was correlated with the amount of autologous T-cells (at 500 pg/mL; r=0.566; p=0.0001; Spearman’s rank correlation test). On the other hand, AMG 330 exerted marked cytotoxic activity in several specimens with very low CD33 expression, and there was no correlation between AMG 330 cytotoxicity and CD33 expression (at 500 pg/mL; r=-0.048; p=0.77). In the presence of healthy donor T-cells, AMG 330 cytotoxicity was strictly dependent on the drug dose (e.g. p<0.0001 at E:T=1:3) and E:T cell ratio (e.g. p<0.0001 at 500 pg/mL; Fig. 1). High cytotoxic activity of AMG 330 was observed in specimens with very low CD33 expression, and a correlation between AMG 330 cytotoxicity and CD33 expression was only observed at high E:T cell ratio (3:1) and higher AMG 330 doses (at 500 pg/mL: r=0.465, p=0.0022). In analyses of patient subsets, AMG 330 resulted in significantly higher cytotoxicity in specimens from patients with newly diagnosed AML (n=21) than those with relapsed/refractory disease (n=20) despite similar levels of CD33 on leukemic blasts (median of 837 [30-5,356] vs. 1,018 [7-2,567] arbitrary fluorescence units; Fig. 2A and B). Furthermore, AMG 330-induced cytotoxicity was higher in specimens from patients with cytogenetically/molecularly favorable-risk disease (n=5) as compared to those with intermediate- (n=26) or adverse risk disease (n=10; Fig. 2C and D).Conclusion: AMG 330 causes potent cytolysis of primary human AML cells in vitro in a dose- and E:T cell ratio-dependent manner across the entire spectrum of cytogenetic/molecular risk and disease stage, even in specimens with very low expression of CD33. Lower activity of AMG 330 was observed in relapsed/refractory AML specimens (relative to newly diagnosed AML specimens) and intermediate- or adverse-risk disease specimens (relative to favorable-risk specimens) suggesting the potential presence of yet undefined, CD33-independent, relative resistance mechanisms in defined patient subsets.Figure 1 [Display omitted] Figure 2 [Display omitted] DisclosuresOff Label Use: Use of AMG 330 in AML (AMG 330 is investigational drug). Newhall:Amgen, Inc: Employment. Sinclair:Amgen, Inc: Employment. Frankel:Amgen, Inc: Employment. Kischel:Amgen, Inc: Employment. Chen:Amgen, Inc: Employment. Walter:Amgen, Inc: Research Funding; Seattle Genetics, Inc.: Research Funding; Amphivena Therapeutics, Inc.: Consultancy; Covagen AG: Consultancy.

Key Immune Cell Subsets Are Dysregulated in Patients with Myelofibrosis

Introduction: Myelofibrosis (MF) is a clonal disorder associated mainly with JAK2V617F and MPL mutations. Recently, a new mutation in the gene encoding calreticulin (CALR) was discovered in the majority of JAK2/MPL negative patients. MF is burdened by a high rate of potentially life-threatening infections. The issue of recurrent and opportunistic infections is increased after the introduction in clinical practice of JAK inhibitors with immunosuppressive activity. However, the role of crucial immune cell subsets is still poorly characterized. Here, we investigated the phenotype/function of selected immune cells in MF. Specifically, we focused on circulating regulatory (Tregs) and IL-17-producing T cells (Th17 cells), monocytes and dendritic cells (DCs). Monocyte-derived DCs were also characterized.Methods: We characterized circulating Th17 cells, Tregs, monocytes and DCs of 17 untreated MF patients and 8 healthy controls (HC) by flow cytometry. Th17 cells were identified as CD4+ CD161+ CD196+ cells while Tregs were enumerated as CD4+ CD25high CD127low T cells. We also tested the in vitro suppressive activity of circulating CD4+ CD25+ Tregs with a mixed leukocyte reaction assay. Two subpopulations of circulating DCs, myeloid CD11c+ and plasmacytoid CD123+cells, were enumerated as well. In addition, after immunomagnetic selection, we tested both phenotype of circulating monocytes and their capacity to differentiate into CD14-derived immature and mature DCs, using a specific cytokines cocktail. JAK2V617F and MPL mutations were detected with RT-PCR while the presence of CALR mutations were tested with Exon 9 Next Generation Sequencing assay.Results: JAK2V617F (11 cases), MPL (3 cases), and CARL (3 cases) mutations were detected. We found that circulating CD4+CD25highCD127low Tregs were reduced in MF patients as compared with healthy controls (p=0.043), although their suppressive ability was maintained. We also found a lower number of circulating Th17 cells (p=0.0026) in MF patients. This finding was particularly evident in JAK2V617F+(p=0.008) and CARL+(p=0.03) patients. Despite their number was in the normal range, circulating monocytes from MF patients showed reduced expression of the CD86 co-stimulatory molecule. Moreover, as compared with the normal counterparts, immature monocytes-derived DCs from patients maintained low CD14 expression without upregulating the CD80 co-stimulatory molecule expression (p=0.0063). Interestingly, at variance with plasmacytoid DCs, a reduced number of circulating myeloid DCs was observed in MF patients as compared with that of HC (p=0.01).Conclusions: Here we demonstrated that specific crucial subsets of immune cells show quantitative and/or qualitative abnormalities in MF patients. These findings may be useful to better understand the increased susceptibility of these patients to infections, since Th17 cells play a role in bacterial and fungal infections while myeloid DCs regulate Th1 activity. Of note, DCs inhibition might result in increased propensity to infections and compromised immune response to cancer.In addition, since monocytes are DC precursors, alterations in their differentiation pathway may contribute to develop defective immune responses. DisclosuresMartinelli:NOVARTIS: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.

Construction and evaluation of rats' tolerogenic dendritic cells (DC) induced by NF-κB Decoy method.

To construct and evaluate rats' tolerogenic dendritic cells (DC) through induction by NF-κB Decoy method. GM-CSF and IL-4 were used to transform rats's monocytes into DC, and DC were stimulated with LPS, NF-κB Decoy ODN, and loaded with Bovine Type II Collagen. The following methods were employed to phenotype DC: 1) Observation of cell morphology; 2) Evaluation of cell viability using trypan blue staining; 3) Purity determination of DC through detection of specific markers OX-62; 4) Evaluation of mature state of DC via the determination of the expression of CD80 and CD86; 5) Determination of stimulation capability towards the proliferation of lymphocyte and the secretion of INF-r and IL-10. The activity of DC was more than 92%, and the expression of OX-62 was more than 70%. Most of DC exhibited the phenotype of CD80(+)/CD86(-). Compared with control group and LPS-stimulation group, the less mature adhered cells and hairlike DC were observed in NF-κB decoy group. Significant reduction (p < 0.05) was observed for the positive expression and extension of CD80 and CD86 in cell surface. After loaded with calf type II collagen, the low expression of CD80 and CD86 remains to be existed. The stimulation capability of DC towards lymphocyte in NF-κB decoy group was lower than that in control group (p<0.05) and LPS stimulation group (p < 0.05). NF-κB Decoy ODN method can be successfully applied for construct rats' tolerogenic dendritic cells (DC) with stable morphology and phenotype. The tolerogenic DC exhibited immature immune phenotype, and low capability to stimulate lymphocytes.

IL-7 receptor recovery on CD8 T-cells isolated from HIV+ patients is inhibited by the HIV Tat protein.

Expression of the IL-7 receptor α-chain (CD127) is decreased on CD8 T-cells in HIV infected patients and partially recovers in those receiving antiretroviral therapy with sustained viral suppression. We have shown that soluble HIV Tat protein down regulates CD127 expression on CD8 T-cells isolated from healthy HIV-negative individuals. Tat is taken up by CD8 T-cells via endocytosis, exits the endosome and then translocates to the inner leaflet of the cell membrane where it binds to the cytoplasmic tail of CD127 inducing receptor internalization and degradation by the proteasome. This down regulation of CD127 by Tat results in impaired CD8 T-cell function. Interestingly, suppression of CD127 by Tat is reversible and requires the continual presence of Tat in the culture media. We thus questioned whether the low IL-7 receptor expression evident on CD8 T-cells in HIV+ patients was similarly reversible and if suppression of the receptor could be maintained ex vivo by Tat protein alone. We show here that when CD8 T-cells isolated from HIV+ patients are incubated alone in fresh medium, low CD127 expression on the cell surface recovers to normal levels. This recovery of CD127, however, is completely inhibited by the addition of HIV Tat protein to the culture media. This study then provides evidence that soluble factor(s) are responsible for low CD127 expression on circulating CD8 T-cells in HIV+ individuals and further implicates Tat in suppressing this receptor essential to CD8 T-cell proliferation and function.

Combination of ATG and Rapamycin Prolongs the Persistence of Adoptively-Transferred, Ex Vivo-Expanded Third Party Treg in Cynomolgus Monkeys.

Background: It has been shown that ex vivo-expanded regulatory T cells (Treg) can promote transplant tolerance in small animal models as well as in phase I clinical trials of hematopoietic stem cell transplantation. The issue has been raised of establishing Treg banks for adoptive cell therapy of organ transplant rejection and autoimmune disease. However, little is known about the persistence, stability and fate of infused Treg, especially third-party allogeneic Treg, and the influence of T cell-depleting and other immunosuppressive agents on their survival and therapeutic efficacy. Methods and Results: In this study, we flow-sorted CD4+CD25+CD127- Treg from normal juvenile cynomolgus monkey PBMC and expanded these cells using a protocol with artificial antigen-presenting cells. Three rounds of stimulation expanded the cells over 1000-fold and consistently generated over 108 Treg from 30 ml peripheral blood, while maintaining expression of Foxp3, CD25, CTLA-4, Helios; potent capacity to suppress T cell proliferation in vitro; demethylation status at the Treg-Specific Demethylation Region (TSDR), and down-regulated expression of CD45RA, CCR7, CD62L and upregulation of CXCR3 suggesting that these cells are likely to migrate to inflamed sites immediately after infusion in vivo. More importantly, when CFSE-labeled autologous (auto) and VPD450-labeled allogeneic (allo) ex vivo expanded Tregs were transferred simultaneously into monkeys, the number of auto-Treg was up to 100 per ml peripheral blood for at least 6 days (ie. for the duration of the experiment) in both control and ATG/rapamycin-treated (IS) monkeys. In contrast, by day 3 after their infusion, allo-Treg could not be detected in control monkeys, while they were maintained up to 100 cells per ml peripheral blood for at least 15 days in IS monkeys. These Tregs maintain high expression of Foxp3, CD25, and CXCR3 expression, and low expression of CD127, CCR7, and CD62L. Conclusion: The finding that allo-Treg can survive in IS-treated monkeys and maintain Treg phenotype supports the concept and potential utility of Treg banks for clinical application in transplantation and autoimmune disease and provides valuable information required to take allo-Treg therapy towards phase II/III clinical trials for efficacy assessment.

Mutations in TLR/MYD88pathway identify a subset of young chronic lymphocytic leukemia patients with favorable outcome

AbstractMutations in Toll-like receptor (TLR) and myeloid differentiation primary response 88 (MYD88) genes have been found in chronic lymphocytic leukemia (CLL) at low frequency. We analyzed the incidence, clinicobiological characteristics, and outcome of patients with TLR/MYD88mutations in 587 CLL patients. Twenty-three patients (3.9%) had mutations, 19 in MYD88(one with concurrent IRAK1mutation), 2 TLR2(one with concomitant TLR6mutation), 1 IRAK1, and 1 TLR5. No mutations were found in IRAK2and IRAK4. TLR/MYD88-mutated CLL overexpressed genes of the nuclear factor κB pathway. Patients with TLR/MYD88mutations were significantly younger (83% age ≤50 years) than those with no mutations. TLR/MYD88mutations were the most frequent in young patients. Patients with mutated TLR/MYD88CLL had a higher frequency of mutated IGHVand low expression of CD38 and ZAP-70. Overall survival (OS) was better in TLR/MYD88-mutated than unmutated patients in the whole series (10-year OS, 100% vs 62%; P= .002), and in the subset of patients age ≤50 years (100% vs 70%; P= .02). In addition, relative OS of TLR/MYD88-mutated patients was similar to that in the age- and gender-matched population. In summary, TLR/MYD88mutations identify a population of young CLL patients with favorable outcome.

Comparison of the Proportion of Non‐Classic (CD14+CD16+) Monocytes/Macrophages in Peripheral Blood and Gingiva of Healthy Individuals and Patients With Chronic Periodontitis

Monocyte subsets with low CD14 expression that coexpress CD16 (CD14+CD16+) are called non-classic or hyperinflammatory monocytes. Previous studies have reported an increase in the percentage of CD14+CD16+ monocytes in the peripheral blood of patients with chronic periodontitis (CP). To our knowledge, there are no reports demonstrating the presence of CD14+CD16+ monocyte-derived macrophages (MDMs) in the gingival tissue. The objective of this study is to identify the proportion of non-classic (CD14+CD16+) monocytes/macrophages in peripheral blood and gingiva of healthy individuals and patients with CP. A total of 60 individuals (n = 30 per group) were recruited for the study. Group 1 included 30 individuals with healthy gingiva, and group 2 included 30 patients with CP. Direct immunofluorescent staining was done in 200 μL whole-blood and single-cell suspensions obtained from gingival tissue, with fluorochrome-conjugated monoclonal antibodies against CD14, CD16, and human leukocyte antigen-DR (HLA-DR), and subjected to flow cytometric analysis. The mean percentage of CD14+CD16+ monocytes in the peripheral blood of healthy individuals was 9.10% ± 1.39%, and for patients with CP it was 14.18% ± 2.69% (P <0.05). The mean percentage of CD14+CD16+ MDMs in the gingival tissue of healthy individuals was found to be 0.93% ± 0.33%, whereas in patients with CP, it was 1.92% ± 0.78% (P <0.01). Non-classic monocytes/macrophages showed a high median fluorescent intensity for HLA-DR (DR++). This study demonstrates an increased proportion of CD14+CD16+HLA-DR++ monocytes/macrophages in the peripheral blood and gingiva of patients with CP.

Ceramide-enriched LDL induces cytokine release through TLR4 and CD14 in monocytes. Similarities with electronegative LDL

IntroductionIn vitro ceramide-enriched LDL (CER-LDL) reproduces most of the properties of electronegative LDL (LDL(−)), a heterogeneous subfraction of LDL found in plasma. LDL(−) comprises several modifications of LDL and has an increased content in ceramide (CER). It promotes cytokine release in monocytes through CD14 and TLR4. CER-LDL also induces cytokine release in these cells but the mechanism is unknown. AimTo evaluate TLR4 andCD14 as the putative receptors involved in cytokine release induced by CER-LDL. MethodsCER-LDL was obtained by incubating native LDL with CER-enriched liposomes. CER content in CER-LDL was assessed by thin layer chromatography of lipid extracts. CER-LDL and LDL(−) were incubated for 20h with human monocytes in the presence or absence of a TLR4 signaling inhibitor. Both LDLs were also incubated with two human monocytic cell lines, normal and THP1 overexpressing CD14 (THP1-CD14) cells. The release of IL-6, IL-10 and MCP-1 was evaluated by ELISA in culture medium. ResultsThe release of IL-6, IL-10 and MCP-1 induced by CER-LDL in monocytes was inhibited by VIPER (90% inhibition), a specific TLR4 inhibitor. The cytokine release induced by CER-LDL was negligible in THP1, cells presenting a very low CD14 expression. In contrast, the induction of cytokine release in THP1-CD14 was high and dependent on the CER content in LDL. ConclusionCER-LDL induces IL-6, IL-10 and MCP-1 release through the activation of CD14 and TLR4 in monocytes, reproducing the behavior of LDL(−). The increased content of CER in LDL(−) is then related to the inflammatory action of LDL(−).

De novo diffuse large B‐cell lymphoma with a CD20 immunohistochemistry‐positive and flow cytometry‐negative phenotype: Molecular mechanisms and correlation with rituximab sensitivity

CD20 is expressed in most B-cell lymphomas and is a critical molecular target of rituximab. Some B-cell lymphomas show aberrant CD20 expression, and rituximab use in these patients is controversial. Here we show both the molecular mechanisms and the clinical significance of de novo diffuse large B-cell lymphomas (DLBCL) that show a CD20 immunohistochemistry (IHC)-positive and flow cytometry (FCM)- negative (IHC[+]/FCM[−]) phenotype. Both IHC and FCM using anti-CD20 antibodies L26 and B1, respectively, were analyzed in 37 of the 106 cases of de novo DLBCL; 8 (22%) of these cases were CD79a(+)/CD20(+) with IHC and CD19(+)/CD20(−) with FCM. CD20 (MS4A1) mRNA expression was significantly lower in IHC(+)/FCM(−) cells than in IHC(+)/FCM(+) cells (P = 0.0005). No genetic mutations were detected in MS4A1 promoter and coding regions. Rituximab-mediated cytotoxicity in the CDC assay using IHC(+)/FCM(−) primary cells was significantly lower than in IHC(+)/FCM(+) cells (P < 0.05); however, partial effectiveness was confirmed. FCM using rituximab detected CD20 more efficiently than B1. No significant difference was observed between IHC(+)/FCM(−) and IHC(+)/FCM(+) patients in overall survival (P = 0.664). Thus, lower expression of CD20 mRNA is critical for the CD20 IHC(+)/FCM(−) phenotype. Lower CD20 expression with FCM does not rule out rituximab use in these patients if expression is confirmed with IHC. FCM using rituximab may be more informative than B1 for predicting rituximab effectiveness in IHC(+)/FCM(−) cases.