bluuebunny/crossref_metadata_2025_split huggingface dataset:
Specific Immune Responses for Leukemia-Associated Antigens Against Myeloid Leukemic Cells Are Increased By Immune Checkpoint Inhibition
Authors: Greiner Jochen , Götz Marlies , Schneider Vanessa , Schrezenmeier Hubert , Wiesneth Markus , Bullinger Lars , Döhner Hartmut , Hofmann Susanne 
Published: October 2019

Abstract
Immunotherapy in cancer treatment has gained importance in the last few years. The efficacy of immunotherapeutic approaches such as immune-checkpoint inhibitors, chimeric antigen receptor T cells (CARs) or bi-specific T cell activating antibodies becomes more and more obvious. However, mechanisms of these immune responses and responsible antigen structures have to be further elucidated. Leukemia-associated antigens (LAA) represent immunogenic antigens that are candidates for specific immunotherapy since they are target structures relevant for elimination of malignant cells by cytotoxic T cells (CTL).
In this work, we investigated the influence of PD-1 antibody Nivolumab and CTLA-4 antibody Ipilimumab on the antigen-specific immune responses by specific T cells against leukemic myeloid blasts in functional T cell assays using ELISpot Assays, tetramer-analysis and colony-forming immunoassays. We investigated T cell responses stimulated against known LAAs like RHAMM, PRAME, WT-1, SSXIP2, Proteinase 3, Survivin, Aurorakinase A and NPM1 against several AML cell lines and samples of AML patients. Expression of different LAAs were measured and correlated to functional T cell assays.
Colony forming unit immunoassays displayed a significant inhibition of CFU (colony forming units) in AML patient samples when adding T cells stimulated against various LAA. In all patient samples, effectors activated against at least one LAA were successful to decrease the colony number significantly. The LAAs PRAME, RHAMM and WT1 showed highest frequency and intensity of immunogenic reactions: PRAME stimulated CTL induced an immune response in 83% of tested samples in CFU, stimulated with WT1 in 75% and with RHAMM in 58% of colonies from AML samples. Specific immune responses of cytotoxic T cells were additionally detected by ELISpot assays and correlated to results detected in CFU. Immune effects increased adding nivolumab to the CTL for several days before starting CFU immunoassays whereas no effect was measured when CTL were incubated with Ipilimumab. The combination of Nivolumab and Ipilimumab showed no additional effect of immune responses compared to Nivolumab alone.
Taken together, the immune checkpoint inhibitor Nivolumab increases specific T cell responses of LAA-stimulated cytotoxic T cells and the cytotoxic effect of T cells against blasts of AML patients. No additional effect was detected with Ipilimumab. These data suggest that PD-1 antibodies could be an immunotherapeutic approach in AML and combination with LAA-directed vaccination strategies might open interesting application possibilities.

Disclosures
Greiner: BMS: Research Funding.
Bone Marrow Macrophage Galectin-3 Regulates Platelet Production through Recognition of O-Glycans on Megakaryocytes
Authors: Lee-Sundlov Melissa M , Grozovsky Renata , Giannini Silvia , McGrath Martina , Ramsey Haley E , Sola-Visner Martha , Hoffmeister Karin M. 
Published: October 2019

Abstract
Bone marrow (BM) macrophages maintain both survival and retention of hematopoietic stem cells and regulate erythropoiesis. The role of macrophage lectins and glycans in thrombopoiesis remains unclear. We report a novel role for bone marrow macrophage galectin-3 in maintaining platelet counts, by phagocytosing megakaryocytes (MKs) expressing the Thomsen-Friedenreich (TF) antigen, which is often exposed under pathological conditions, such as cancer and malignancies.
The TF antigen is a disaccharide presented in cryptic form on O-glycans and covered by a sialic acid moiety. The sialyltransferase ST3Gal1 transfers sialic acid onto the TF antigen. To investigate the role of O-glycans in thrombopoiesis, we generated mice with increased TF antigen in MKs by generating St3gal1loxP/PF4+ mice specifically lacking ST3Gal1 in the MK lineage. As expected, St3gal1loxP/PF4+ circulating platelets and BM MKs had increased TF antigen expression, compared to controls, as evidenced by peanut agglutinin (PNA) binding. Other blood cell lineages had no increase in TF antigen expression. St3gal1loxP/PF4+ mice developed mild thrombocytopenia, but surprisingly had virtually normal platelet clearance. BM MK colony forming units and in vitro proplatelet production were normal in St3gal1loxP/PF4+ mice, suggesting that extrinsic factors in the St3gal1loxP/PF4+BM environment affected platelet production. St3gal1loxP/PF4+ BM smears revealed increased hemophagocytosis, indicative of an increase in phagocytic macrophages. In vivo macrophage ablation by injection of clodronate-encapsulated liposomes significantly reduced the numbers of activated macrophages, thereby normalizing blood platelet counts and size. Flow cytometric phenotypic analysis of BM-derived macrophages showed an increased population of activated macrophages in St3gal1loxP/PF4+ mice, compared to controls, specifically macrophages with increased galectin-3 expression, a ligand for the TF antigen. Immunofluorescence staining of BM sections using a specific antibody towards the TF antigen showed that MK progenitors and pro-platelet-like structures expressed TF antigen in control BMs, which is significantly increased in St3gal1loxP/PF4+ mice and co-localized with galectin-3 expressing macrophages, supporting the notion that MK O-glycans and macrophage galectin-3 play a role in thrombopoiesis under steady state and pathological conditions. Consistent with this notion, galectin-3 deficient mice have slightly, but significantly increased blood platelet counts. We conclude that galactin-3 plays a minor role in normal thrombopoiesis. Activation of galectin-3 expressing macrophages by the MK TF antigen leads to MK phagocytosis, inhibition of platelet formation and thrombocytopenia.

Disclosures
No relevant conflicts of interest to declare.
The G-Protein Coupled Receptors and G-Proteins Alpha Expression in Mantle Cell Lymphoma
Authors: Wasik Agata M , Mundt Filip , Christensson Birger , Sander Birgitta 
Published: October 2019

Abstract
Mantle cell lymphoma (MCL) is a B cell Non-Hodgkin lymphoma comprising approximately 8% of malignant lymphomas. MCL is considered to be an incurable disease even though survival has improved with novel biological therapies that interrupt survival signaling from the lymphoma microenvironment. The signaling pathways that are deregulated and the factors leading to systemic dissemination of the disease are still poorly understood. There is also a clear need to identify additional druggable targets for therapy (Saba and Wiestner, Curr Opin Hematol, 2014). Druggable targets may be G-protein coupled receptors (GPCRs) and G-proteins. GPCRs belong to the family of seven transmembrane receptors, which upon activation by extracellular ligands transfer the signals inside the cell for activation of the specific pathway. The signaling involves G-proteins. G-proteins consist of the subunits: alpha, beta and gamma and their subtypes. During GPCR activation the G alpha subunit is released and the subtype of G-protein subunit determines which downstream signaling pathways (including Akt, PI3K, ERK, JNK, MAPK, NFkappaB and others) is then activated. In the immune cells different subtypes of G-proteins alpha are instrumental for leukocyte migration, architecture of B cells compartments in spleen, thymus, lymph nodes, and gastrointestinal tract (Hwang et al., PlosOne, 2013) and BCR-mediated cell death (Misra et al., J Exp Med, 2010). G alpha13 signaling is involved in development of germinal centre-derived lymphomas (Muppidi et al., Nature, 2014).
In order to map the aberrant expression of GPCR and G-proteins in MCL, the expression of genes encoding for GPCRs and G-protein alpha subunits was analyzed by Affymetrix U133A Array of seventeen MCL biopsies and eight non-malignant lymph nodes. Among investigated GPCR and G-protein alpha subunit genes 25/298 were differently expressed in MCL compared to reactive lymph nodes (moderated t-test, adj. p=or< than 0.05). The four most significantly different genes expressed were: LPAR6, GPRC5C, OPN3 and GNAZ (all adj.p<0.003). The latter, GNAZ, encodes for G-protein alpha z. The analysis of GNAZ by qPCR in 77 additional MCL biopsies from a population-based Stockholm MCL cohort showed varied expression of GNAZ. Interestingly, the MCL cases, which expressed the highest levels of GNAZ, had significantly worse survival (Kaplan-Meier, log rank, p<0.003) suggesting that GNAZ has, a yet unknown, role in MCL biology. Further analysis showed that GNAZ expression was correlated to lymphocytosis (Mann-Whitney test, p=0.023) and leukocytosis (Mann-Whitney test, p=0.011) in MCL patients but not to: high tumor cell proliferation, blastoid morphology nor to p53 immunoreactivity. Our results suggest that GNAZ-signaling is of importance for cell-cell interaction in the tumor microenvironment and/or for egress of lymphoma cells to the blood.

Disclosures
No relevant conflicts of interest to declare.
Expression and Function of Tigit in B-Cell Non-Hodgkin Lymphoma
Authors: Yang Zhi-Zhang 
Published: October 2019

Background:T cell Ig and ITIM domain (TIGIT) is an immune checkpoint molecule and a novel member of CD28 family. TIGIT is expressed on NK cells, effector and memory T cells, and Treg cells. Upon ligation with CD155, TIGIT delivers an inhibitory signal and negatively regulates anti-tumor responses. While important in normal T-cell biology, the expression and function of TIGIT in the blood and tumor microenvironment of patients with B-cell non-Hodgkin lymphoma (NHL) is completely unknown.
Goal: To phenotypically and functionally characterize TIGIT+T cell subsets in B-cell NHL and compare expression of TIGIT on intratumoral T cells to normal controls.
Results: In peripheral blood, TIGIT expression was not detected on resting T cells. In contrast, when we analyzed biopsy specimens from B-cell NHL patients, we observed that TIGIT is variably but highly expressed on a subset of intratumoral T cells. A median of 41.1% (range: 24.9-56.1, n=5) of CD4+ or 22.2% (range: 13.4-33.3, n=5) of CD8+ T cells from tumor specimens expressed TIGIT on the cell surface. In normal tonsil tissue, expression level of TIGIT was modest or negligible. A median of 12.0% (range: 9.1-15.8, n=5) of CD4+ or 3.9% (range: 1.3-5.7, n=5) of CD8+ T cells express TIGIT on the cell surface, which is significantly lower than that in B-cell NHL (p=0.005 and p=0.003 for CD4+ and CD8+, respectively). We observed that both CD45RA+ and CD45RA- T cells express TIGIT with the greatest expression on the CD45RA- population in B-cell NHL specimens. Furthermore, we found that TIGIT+ T cells coexpressed other immune checkpoint molecules including PD-1 and TIM-3. Functionally, TIGIT+ T cells displayed reduced cytokine production, as the number of IFN-γ- and IL-2-producing cells was lower in the TIGIT+ population than in TIGIT-T cells. Unlike TIM-3 that is coexpressed with PD-1 and whose expression is upregulated by IL-12, TIGIT expression is not upregulated by IL-12, suggesting that a different mechanism is involved in TIGIT upregulation in B-cell NHL.
Conclusion: Taken together, these results indicated that TIGIT is highly expressed on intratumoral T cells in B-cell NHL and is expressed on a population of T-cells with suppressed immune function. TIGIT, while co-expressed with PD-1 and TIM-3, is not upregulated by cytokines such as IL-12, suggesting that it is regulated by an alternate pathway. Inhibition of TIGIT signaling may be an additional mechanism to prevent T-cell suppression and exhaustion in B-cell NHL.

Disclosures
No relevant conflicts of interest to declare.
Ocular MALT Lymphoma Free from the Chlamydia Psittaci Infection ; Analysis of 150 Cases
Authors: Park Gyeongsin , Lee Hye Won , Cho Uiju , Yang Suk Woo , Cho Seok-Goo , Lee Youn Soo , Kang Chang Suk 
Published: October 2019

Abstract
Background: Clamydophila psittaci (C. psittaci) has been proposed as an etiologic factor for extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma) in the ocular region. However, previous studies showed varied association rates ranging 0% to 87%, not constant even in the series of same geographical areas, which suggest that there could be some technical variance in detecting methods for C. psittaci. The authors validated five sets of primers for detecting C. psittaci DNA and investigated its association with ocular MALT lymphomagenesis.
Material and Methods: Five sets of PCR primers including 4 previously reported and one newly designed were evaluated with positive control C. psittaci DNA (acquired from Korea Centers for Disease Control and Prevention). One hundred fifty cases of confirmed ocular MALT lymphomas were collected from pathology archives of our institutes from 2008 to 2014, and entered into this study. DNA was extracted from archival paraffin block sections with Qiagen DNA extraction kits. Quality of DNA was assessed with NanoDrop and beta-globin PCR. Standard PCR was performed together with negative (H2O and tonsil DNA) and positive control C. psittaci DNA.
Results: In all five primer sets for C. psittaci DNA, each PCR product showed clear positive band and negative with appropriate positive and negative controls, respectively. All 150 ocular MALT lymphoma cases showed positive for Beta-globin DNA control, but negative for C. psittaci DNA in any of PCR product with five primer sets.
Conclusion: These results suggest that possibility of the pathogenetic role of C. psittaci in ocular MALT lymphoma would have been overestimated so far, at least in Korean people.

Disclosures
No relevant conflicts of interest to declare.
Outcome of Bone Marrow Biopsy in Patients with Diffuse Large B-Cell Lymphoma Patients Staged by Combined 18 FGD-PET/CT Scan
Authors: Hamed Rasha Abdel Tawab , Kotp Rami , Turecotte Eric , Sawan Baseem , Morine Annie 
Published: October 2019

Abstract
DLBCL represents 1/3 of non-Hodgkin lymphoma, in 60% of the cases the disease presented in advanced stage (III-IV). Extranodal organs are involved in 40% of cases; BM involvement in 11% to 27% of cases. BMB is an invasive procedure, & it also could represent false negative result in patients with patchy pattern of involvement or if involvement else where the routine biopsy site. Study included 102 patients, above the age of 18 years, confirmed to have newly diagnosed DLBCL with no any other malignancy. Every patient had a baseline PET-Ct, bone marrow biopsy. PET-CT detected BM infiltration in 23 patients, i.e 22.5%. BMB were positive in 20 patients, while PET-CT showed BM involvement in 23 patients.- 94 patients had concordant negative ( 75 patients ) or positive ( 19 patients), PET-CT & BMB results. One patient had positive BMB and negative PET-CT. All patients with stage I & II had concordant PET-CT & BMB results One patient graded stage III by PET-CT showed 1-2% BM infiltration & upstaged to stage IV. Of the 49 patients graded stage IV by PET-CT, 3 had positive BM involvement PET-CT and negative BMB. the sensitivity of PET-CT was 95%, the specificity of PET-CT was 96.2%. the PPV was 86.4% & the NPV was 98.7%. PET-CT showed 95.9% accuracy. Our results suggest PET-CT as a powerful tool to evaluate BM infiltration in patients with DLBCL, with overall concordance exceeding 94% (100%for stage I & II ). For patients with advanced stage IV disease, PET-CT was able to retrieve patients with BM involvement & false negative BMB.

Disclosures
No relevant conflicts of interest to declare.
HSC Hierarchy in Primary Myelofibrosis
Authors: Triviai Ioanna N , Zeschke Silke , Panagiota Victoria , Heuser Michael , Stocking Carol , Kroeger Nicolaus 
Published: October 2019

Abstract
Primary Myelofibrosis (PMF) is a Myeloproliferating Neoplasm (MPN) of hematopoietic stem cell origin, characterized by expansion of aberrant myeloid progenitors in the chronic phase that can lead to bone marrow fibrosis development and/or osteosclerosis. In 20-25% of PMF cases transformation to acute myeloid leukemia (AML) is observed. Identification of multiple molecular lesions suggests complex clone dynamics that indicates exceeding sub-clone dominance as PMF progresses. Our aim is to determine the HSC hierarchy orchestrating initiation and development of PMF.
In our previous studies we reported a CD133+ HSC population in PMF peripheral blood that represents the aberrant long-term stem cell fraction responsible for PMF reproduction of chronic and acute phases in vivo. Molecular analysis of PMF xenografts indicates sustenance of genetically different HSC clones exhibiting variation in both their engraftment potential and reproduction of PMF parameters in the first mouse model of the disease.
To further characterize the succession of molecular lesions determining HSC clone propagation we performed targeted exon sequencing of PMF HSC from 100 PMF patients for 54 genes. Mutations in the epigenetic regulators ASXL1 and EZH2 were detected in 38% and 15% of PMF patients respectively. HSC clonal evolution was determined by single cell molecular and phenotypic analysis in vitro and graft analysis in vivo.
Mutations detected in the epigenetic regulators ASXL1 and EZH2 represent founding molecular lesions at the top of PMF HSC hierarchy. ASXL1 mutations precede and are connected with sustenance of clonal hematopoiesis without any significant influence on the HSC differentiation potential. EZH2 mutations are connected with impaired erythropoiesis in vitro and anemia, high engraftment and expansion of pre-leukemic clones in vivo. Occurrence of JAK2 and CALR mutations in the background of mutated epigenetic regulator genes is connected with expansion of HSC subclones and reproduction of chronic phase PMF as atypical myelopoiesis, splenomegaly and induction of fibrosis.
Our results indicate genetic heterogeneity of PMF neoplastic HSC is comprised of three different mutational clusters. Mutations in epigenetic regulator genes (Group 1) precede and shape the epigenetic landscape conferring genetic instability to sustain the expansion of pre-leukemic clones (Group 3 mutations). JAK2 and CALR mutations (Group 2) occur later on and are connected with aberrant myelopoiesis of chronic phase disease. HSC clonal dynamics reflect genotype related phenotypes as determinants of chronic and acute phases of PMF.

Disclosures
No relevant conflicts of interest to declare.
KIR Gene Haplotype: An Independent Predictor of Clinical Outcome in MDS Patients
Authors: Daher May , Marin David , Sobieski Catherine , Shaim Hila , Basar Rafet , Mendt Vilchez Mayela Carolina , Gokdemir Elif , Li Li , Imahashi Nobuhiko , Muftuoglu Muharrem , Champlin Richard E. , Shpall Elizabeth J. , Kantarjian Hagop M. , Garcia-Manero Guillermo , Rezvani Katayoun 
Published: October 2019

Abstract

Myelodysplastic syndromes (MDS) are a group of hematopoietic disorders affecting the myeloid lineage, characterized by cytopenias and clonal evolution to AML. Immune responses against MDS, partly mediated by NK cells, have the potential to affect disease progression. However, immune evasion remains an important barrier. NK cells express both activating and inhibitory killer immunoglobulin-like receptors (KIRs) that interact to regulate NK effector function. Based on the number and distribution of inherited KIR genes, individuals can be classified in two broad haplotypes. Haplotype A comprises a single activating KIR (aKIR) gene (KIR2DS4), while haplotype B incorporates various combinations of aKIR genes (up to 6). We hypothesized that the aKIR gene repertoire may be useful in refining predictions of clinical outcomes in MDS. Thus, we studied the variations in aKIR gene content and haplotype in MDS and their relationship to the risk of AML transformation and patient outcomes.
We first compared the number of aKIR genes in 108 MDS patients treated at MDACC with that in 139 HSC donors. The median number of aKIR genes was significantly lower in MDS than controls: 2 (range 0-6) vs 3 (range 0-6), p=0.001. Functional studies revealed that compared to healthy controls, NK cells from MDS patients demonstrate less Interferon gamma production (p<0.0001) and less degranulation (p=0.0028) in response to K562 cell line, and have lower killing ability evidenced by chromium release assay (p=0.04).
We next examined the influence of KIR haplotype on the risk of AML transformation and outcomes in the cohort of 108 MDS patients (28 KIR haplotype A and 80 KIR haplotype B patients). On multivariate analysis, cytogenetic risk group and KIR haplotype were identified as independent predictors of MDS progression to AML. The relative risk of MDS-AML transformation in patients with haplotype A vs. haplotype B was 2.67 (1.13-6.31) (p=0.02). Similarly, cytogenetic risk group, IPSS and KIR haplotype independently predicted survival. MDS patients with KIR haplotype A had worse adjusted PFS (RR with 95%CI 2.96 [1.59-5.52], p=0.001) and OS (2.25 [1.17-4.31], p=0.02), compared to patients with haplotype B.
These novel findings may help identify a subgroup of MDS patients with a high risk of disease progression and poor outcomes, who would likely benefit from adoptive NK cell therapy.

Disclosures
Kantarjian: ARIAD: Research Funding; Bristol-Myers Squibb: Research Funding; Amgen: Research Funding; Pfizer Inc: Research Funding; Delta-Fly Pharma: Research Funding; Novartis: Research Funding.