bluuebunny/crossref_metadata_2025 huggingface dataset:
High Dimensional Immune Profiling in Smoldering Multiple Myeloma Identifies Novel Organizing Features of the Tumor Microenvironment
Authors: Cho Hearn Jay , Perumal Deepak , Rahman Adeeb H , Kim-Schultze Seunghee , Yesil Jennifer , Auclair Daniel , Casneuf Tineke , Adams Homer , Parekh Samir , Gnjatic Sacha , Fernandez Nicolas
Published: November 2019

Multiple myeloma (MM) is a malignancy of plasma cells that arises from premalignant Monoclonal Gammopathy of Undetermined Significance (MGUS) and often progresses through an asymptomatic Smoldering (SMM) phase lasting months or years before manifesting clinical symptoms warranting therapy. Current research indicates that the tumor microenvironment (TME) in the bone marrow may play a significant role in governing progression to symptomatic disease. Therefore, understanding of the interactions between malignant plasma cells and the TME in early disease states is critical in the pursuit of therapies that will prevent progression to symptomatic disease. We performed high dimensional genomic and immunologic analysis of bone marrow specimens from 73 subjects with SMM. We performed RNA-seq on the malignant plasma cells isolated by anti-CD138 magnetic bead positive selection, mass cytometry (CyTOF) and T cell receptor sequencing (TCR Seq) of CD138-depleted bone marrow mononuclear cells, and proteomics, seromic, and grand serology analysis of bone marrow plasma. These samples and assays provided a broad view of the tumor cells and the cellular and soluble components of the TME. Each of these assays identified self-organizing clusters of subjects, indicating that subgroups of SMM patients shared common characteristics in the tumor or TME populations. We then applied novel bioinformatic methods to compare data from pairs, trios, quartets, and quintets of assays to identify communities of subjects with similar immunologic and genomic characteristics. Integrated analysis of CyTOF, proteomic, and TCR Seq resolved three distinct communities with a high degree of significance. These communities shared distinct cellular and proteomic features that suggested early adaptive, activated adaptive, or innate immune characteristics. These results suggest that the continuum from MGUS to MM does not consist of a single pathway in either the tumor cells or the TME, and that complex interactions ultimately determine progression. This suite of assays (CyTOF, proteomics, and TCR Seq) may be applicable in translational and clinical studies to understand key tumor and immune determinants of SMM and lead to rationally designed therapy to replicate these conditions to prevent progression to symptomatic disease.

Disclosures

Cho: Genentech: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; The Multiple Myeloma Research Foundation: Employment; Takeda: Research Funding; BMS: Consultancy; Agenus: Research Funding; GSK: Consultancy. Adams:Janssen Pharmaceuticals R&D: Employment, Other: Own Stock. Parekh:Foundation Medicine Inc.: Consultancy; Celgene Corporation: Research Funding; Karyopharm Inc.: Research Funding.
Patients with Bone and Bone Marrow Involvement Had Better OS and PFS in Patients with Aggressive Non-Hodgkin Lymphoma Treated with CD19 CAR T Cells
Authors: Zhou Lili , Aibin Liang , Li Shaoguang , Ye Shiguang , Li Ping
Published: November 2019

Factors associated with complete remission and durable remission after CD19 chimeric antigen receptor (CAR)-modified T-cell immunotherapy for aggressive B-cell non-Hodgkin lymphoma (NHL) have not been identified. We report multivariable analyses of factors affecting response and progression-free survival (PFS) in patients with aggressive NHL treated with CD19 CAR T cells. The best overall response rate was 71%, with 32% of patients achieving complete remission. The median PFS and OS of patients with aggressive NHL who achieved complete remission were 11 months and 12 months. The median PFS and OS of patients with bone and bone marrow involvement without soft tissue involvement were 14 months and 18 months. The median PFS and OS of patients with soft tissue involvement were 1 months and 4 months. We report complete remission rates and long-term survival rates in patients with bone and bone marrow involvement were significantly higher than those in patients with soft tissue involvement.

Figure

Disclosures

No relevant conflicts of interest to declare.
The Impact of Aminoacid Substitution at Position 116 Class I HLA, in Unrelated Donor Transplants
Authors: Chiusolo Patrizia , Lamparelli Teresa , Sapienza Giuseppe , Dominietto Alida , Raiola Anna Maria , Di Grazia Carmen , Giammarco Sabrina , Gualandi Francesca , Metafuni Elisabetta , Rossi Monica , Minnella Gessica , Sica Simona , Bacigalupo Andrea , Angelucci Emanuele
Published: November 2019

Background : The role of aminoacid substitution at position 116 of class I HLA antigens, has been the subject of several contributions, suggesting the possibility of permissive or non permissive mismatches.

Hypothesis. Permissive mismatched at position AA116, yield outcomes comparable to 10/10 matched grafts and superior to AA116 non permissive mismatches.

Patients: We have analyzed 358 unrelated donor transplants (UD) to test this hypothesis. All donors were matched at class II for DRB1 and DQ alleles; 226 were also matched at high resolution for A,B and C alleles; 84 had 1 permissive AA116 mismatch (Pmm), and 48 had 1 non permissive mismatch (NPmm). The 3 groups were comparable for patients age (p=0.5), donors age (p=0.9), diagnosis (p=0.2) and intensity of the conditioning regimen (p=0.4); both NPmm and Pmm had more patients with advanced disease, as compared to matched patients. The stem cell source was peripjheral blood for the large majority of patients.

Results.

The cumulative incidence (CI) of acute GvHD grade II-IV (p=0.01) and III-IV (p=0.001) was greater in patients with 1 allele mismatch, compared to matched patients, irrespective of AA116 permissive substitution. The CI of non relapse mortality (NRM) at 5 years 29%, 35%, 50% respectively in patients grafted from 10/10 matched donors, 1 allele Pmm donors and 1 allele NPmm donors (p=0.005). GvHD with or without infections, as a cause of death was recorded in 19%, 23% and 33% of the three groups respectively (p=0.1). The CI of relapse was respectively 18%, 30%, 20% (p=0.1). The actuarial survival at 5 years was 68% for 10/10 matched patients, 63% for 1 allele Pmm patients and 47% for 1 allele NPmm patients.

Conclusions. We confirm that aminoacid substitution at position 116 of class I HLA antigens, is a risk factor for non relapse mortality and survival. Patients with permissive mismatched AA116 donors have outcome comparable to patients grafted from matched donors.

Figure

Disclosures

Angelucci: Novatis: Honoraria, Other: Chair Steering Committee TELESTO protocol; Celgene: Honoraria, Other: Participation in DMC; BlueBirdBio: Other: Local advisory board; Jazz Pharmaceuticals: Other: Local advisory board; Roche: Other: Local advisory board; Vertex Pharmaceuticals Incorp., and CRISPR Therapeutics: Other: Participation in DMC.
The Neurotransmitter Receptor Gabbr1 Regulates Proliferation and Function of Hematopoietic Stem and Progenitor Cells
Authors: Elujoba-Bridenstine Adedamola , Shao Lijian , Zink Katherine , Sanchez Laura , Cox Brian , Pajcini Kostandin , Tamplin Owen J
Published: November 2019

Hematopoietic stem and progenitor cells (HSPCs) have multi-lineage potential and can be used in transplants as a curative treatment for various hematopoietic diseases. HSPC function and behavior is tightly regulated by various cell types and factors in the bone marrow niche. One level of regulation comes from the sympathetic nervous system that innervates the niche and releases neurotransmitters to stromal cells. However, the direct regulation of HSPCs via cell surface expression of neural receptors has not been functionally explored. Using imaging mass spectrometry, we detected strong and regionally specific gamma-aminobutyric acid (GABA) neurotransmitter signal in the endosteal region of mouse bone marrow. GABBR1 is known to be expressed on human HSPCs (Steidl et al., Blood 2004), however its function in their regulation has not been investigated. Based on published mouse HSPC single cell RNA-seq data (Nestorowa et al., Blood 2016), we found that a subset of HSPCs expressed the GABA type B receptor subunit 1 (Gabbr1). We confirmed by surface receptor expression that a subset of mouse bone marrow HSPCs express Gabbr1 protein. Using the same single cell RNA-seq data as above, our own gene set enrichment analysis (GSEA) revealed positive correlation of Gabbr1 expression with genes involved in immune system processes, such as response to type I interferons. We generated a CRISPR-Cas9 Gabbr1 mutant mouse model on a C57/BL6 background suitable for hematopoietic studies. Analysis of Gabbr1 mutant bone marrow cells revealed a reduction in the absolute number of Lin-Sca1+cKit+ (LSK) HSPCs, but no change in the number of long-term hematopoietic stem cells (LT-HSCs). With further hematopoietic profiling, we discovered reduced numbers of white blood cells in peripheral blood that was primarily due to fewer B220+ cells. We show that Gabbr1 null HSPCs display reduced proliferative capacity, as well as diminished reconstitution ability when transplanted in a competitive setting. An in vitro differentiation assay revealed the impaired ability of Gabbr1 null HSPCs to produce B cell lineages. We tested our predicted association with type I interferon response by administration of poly(I:C) and found reduced HSPC proliferation in Gabbr1 null mice. Our results may translate well to humans, as a rare human SNP within the GABBR1 locus was found that correlates with altered leukocyte counts (Astle et al., Cell 2016). Our results indicate an important role for Gabbr1 in the regulation of HSPC proliferation and differentiation, highlighting Gabbr1 as an emerging factor in the modulation of HSPC function and behavior.

Disclosures

No relevant conflicts of interest to declare.
Long Range Sequencing Shows Improved Resolution in the Detection of Beta Globin Cluster Variants
Authors: Rangan Aruna , Hein Molly S. , Koganti Tejaswi , Jenkinson William G. , Hilker Christopher A. , Blommel Joseph H. , Porter Tavanna R. , Jen Jin , Hoppman Nicole , He Rong , Viswanatha David S. , Aleff Ross , Hoyer James D. , Wieben Eric , Klee Eric W. , Kipp Benjamin R. , Oliveira Jennifer L.
Published: November 2019

Introduction: Globin gene sequencing has long been impeded by repetitive regions, homology and structural variation. Currently, long-read sequencing technology has been limited by high cost and error rate. These challenges can be mitigated by sample multiplexing of targeted capture sequencing and the use of consensus reads, respectively. This ongoing study analyzes the utility of long range sequencing technology in identifying historically problematic beta globin cluster mutations.

Materials and methods: Long range sequencing was performed on complex beta globin gene cluster cases utilizing the Sequel platform (Pacific Biosciences, Menlo Park, California, United States). Sample input was 1 microgram of DNA extracted from whole blood. Circular Consensus Sequence (CCS) FASTQ sequences files were mapped to reference genome (GRCh37/hg19) using NGMLR, followed by annotation of variants using a custom bioinformatics workflow. Comparison to multiplex ligation-dependent probe amplification (MLPA), Sanger sequencing, array CGH, short-range NGS (300 bp), hemoglobin protein, and clinical data was performed.

Results: CCS read length varied from 1 to 10 kb, average 3.5. Analysis of the beta globin cluster region showed impressive superiority and comprehensiveness to Sanger, MLPA, array CGH and short range sequencing technology: 1) single nucleotide variants (SNVs) were identified to a sensitivity and specificity of 99.5%; 2) large structural variants (SVs) such as large deletions, duplications, insertions, crossovers and fusions spanning many kilobases were characterized in the heterozygous, homozygous and compound heterozygous state to a precise genomic coordinate (table 1); 3) phasing SNVs identified Hb S haplotypes (Central African region (CAR), Benin, Senegal, Arab-Indian and Cameroon).

Conclusion: 1) Long range sequencing holds significant promise in genotyping hemoglobin disorders. This method improves efficiency with potential for a single test offering with increased resolution, particularly in large deletion analysis. Complex cases greatly benefited from improved range and resolution. 2) The decreased requirement of blood volume for extensive testing is of particular interest for the pediatric population. 3) Genotyping of Hb S patients detected by positive newborn screening will help predict need for prophylaxis or therapy by assessing variant zygosity, phasing and haplotypes. In conclusion, we believe that long range sequencing has immense potential to contribute extensively to thalassemia and hemoglobinopathy diagnostics and is far superior to the currently available technologies for beta globin cluster analysis.

Disclosures

Jen: Celgene: Employment.
Hematopoietic Stem Cells Increase Quiescence during Aging
Authors: Kovtonyuk Larisa V. , Ashcroft Peter , Spaltro Gianluca , Tata Nageswara Rao , Skoda Radek C. , Bonhoeffer Sebastian , Manz Markus G.
Published: November 2019

Introduction: Definitive hematopoietic stem cells (HSCs) sustain blood production from fetal development throughout life. In mice, most of steady state, young adult HSCs are in the G0 phase of cell cycle (quiescence), and are estimated to divide roughly once a month. Daily hematopoietic production is thus mainly sustained by highly proliferative downstream hematopoietic progenitor cells (HPCs). Aged haematopoiesis was demonstrated to be distinct from young haematopoiesis in various aspects such as i) a shift from lymphopoiesis to myelopoiesis, ii) functional decline of HSCs (self-renewal, homing), and iii) HSCs pool expansion. While several studies attempted to address whether changes in HSCs turnover during aging can account for the distinct aging associated phenotype and function, it remained to be determined whether aged HSCs overall cycle more or less frequently than young HSCs.

Methods: To construct data-based, quantitative models, we measured turnover rates and compartment sizes of populations of HSCs, HSPCs and granulopoiesis/granulocytes, i.e. a post-mitotic mature hematopoietic linage with a short half-life. We examined four age groups: 3 week, 2 month, 1 year and 2 year old mice. Mice in each group were i.p. injected every 4 hours with 1 mcg EdU up to a maximum time of 48 hours. HSC, HSPC and granulopoiesis/granoulocyte compartment sizes and snapshot cell-cycle analysis was performed by FACS at multiple sampling points in BM and peripheral blood (PB), respectively. Based on this data, we built a mathematical model of HSC turn-over and HSPC differentiation during ageing. Moreover, we evaluated HSC cycling by CFSE dilution in steady-state transplantation experiments (as described before; Takizawa et al., J Exp Med 2011).

Results: In line with previous reports, the HSCs compartment size gradually increased with age from 3wk old mice to 2 year old mice. In sharp contrast, cycling activity of HSCs as determined by EdU incorporation decreased gradually and significantly with increasing age. This was driven by decreased activation from the quiescent state, while the time that actively cycling HSCs require to progress through cell-division remains constant with age. Multipotent Progenitor (MPP) cycling showed a non-significant trend towards slower turn-over. These results were confirmed by complementary CFSE-dilution experiments. Mathematical modeling of HSC proliferation and differentiation revealed a higher probability of self-renewing divisions in 3 week old mice as compared to 2 month, 1 and 2 year old mice, with the latter both having nearly equal chances of self-renewing versus differentiating divisions.

Conclusions: Our data clarifies the long-standing question, how the HSC pool increases with age. Instead of an increase in active cycling, an increase in HSC quiescence is responsible for the increased size of the HSCs pool in aging.

Disclosures

No relevant conflicts of interest to declare.
Fetal Hemoglobin Promotes P. Falciparum Growth in Sickle Cell Disease Erythrocytes
Authors: Archer Natasha M. , Duraisingh Manoj
Published: November 2019

Introduction: We have demonstrated that sickle hemoglobin (HbS) polymerization in low oxygen (O2) is the main driver of sickle cell trait (AS) resistance to P.falciparum malaria. This suggests that homozygous sickle cell disease (SS) individuals should have even greater resistance to malaria. Instead SS individuals infected by P. falciparum often have increased malaria morbidity and mortality compared to individuals with normal hemoglobin (AA) or AS. The reasons for this paradox are poorly understood. We propose that fetal hemoglobin (HbF) inhibits polymerization thereby allowing parasite proliferation. To test this hypothesis the following experiments were performed.

Methods: AA and SS erythrocytes were collected within two weeks of the assay date. P. falciparum 3D7 parasites were used for growth assays. Growth assays were performed at 1, 3, 5, 7.5, 10 and 16% O2 using synchronized schizonts obtained by magnet purification via the MACS system. Staging was performed via light microscopy analysis of May-Grünwald-Giemsa-stained cytospins. Parasite Multiplication Rate (PMR) was determined via flow cytometry using parasitemia at 64 and 16 hours post infection (hpi) as previously described.

Results: In contrast to AS erythrocytes, SS RBCs contain varying amounts of HbF that differ by cell and by individual. In hypoxic SS RBCs with low (3.8%) HbF, P. falciparum parasites remain immature (early trophozoites) in 1, 3, and 5% O2 (Fig. 1) at 36 hpi, when they should have matured into schizonts. However, in SS RBCs with high (22.6%) HbF, 12, 16 and 27% of parasites matured into schizonts in 1, 3 and 5% O2 respectively compared to 0, 0, and 1% in the SS RBCs with 3.8% HbF. Preliminary data also demonstrate that the parasite multiplication rate (PMR), a surrogate of proliferation, in SS RBCs improves with increasing O2 and HbF. Using SS/Hereditary Persistence of Fetal Hemoglobin RBCs with 42% HbF, proliferation in 5% O2 or higher is exponential with a PMR of 2.6 or greater (Fig. 2). In RBCs isolated from SS patients treated with hydroxyurea with HbF of 26.4 or 21%, PMR is 2 or more in 7.5% O2 or greater.

Conclusion: These data demonstrate that HbF promotes P. falciparum growth in SS erythrocytes. In addition, the data initiates a resolution of the malaria paradox. HbS in both hypoxic AS and SS surely inhibits P. falciparum growth due to HbS polymerization at low O2. But high levels of anti-sickling HbF observed in erythrocytes from many individuals with SS reverses this inhibition despite the increased content of HbS in SS individuals. Even small increases in P. falciparum proliferation within the broadly compromised SS host may enhance the malaria associated morbidity and mortality seen in this population. These data further suggest the importance of anti-malarial prophylaxis in patients with SS especially those treated with hydroxyurea or anti-sickling agents.

Disclosures

No relevant conflicts of interest to declare.
Outcome of Patients with Newly-Diagnosed Therapy-Related AML (t-AML) and AML with Myelodysplasia-Related Changes (AML-MRC) Treated with Liposomal Daunorubicin and Cytarabine (CPX-351) at an Academic Community-Based Health System
Authors: Khoury John , Anusim Nwabundo , Macari David , Duenn Angela , Jaiyesimi Ishmael
Published: November 2019

Method: A query was generated to retrieve all cases treated with CPX-351 at Beaumont Health System. SPSS software was used to perform descriptive statistics.

Results: We found 11 cases treated with CPX-351 from 2017 to 2019. Six patients were male and 5 females. The median age at diagnosis was 67. Eight patients were Caucasian, two African American and one Asian. The median body mass index (BMI) was 27.1 and median body surface area (BSA) was ! treated with CPX-351 after a diagnosis of AML-MRC and 4 patients (36.3%) with t-AML. The median hospital stay was 35 days. The median units of red blood cells transfusion during hospital stay were 9 units and the median number of transfused platelet units was 6. Only 2 patients (18.1%) achieved complete response (CR) and 2 patients (18.1%) achieved complete response with incomplete blood count recovery (CRi). Three patients developed allergic reactions presented as a rash, 2 patients had significant bleeding and all 11 patients developed neutropenic fever. Three patients died during induction therapy and 2 patients underwent allogeneic hematopoietic stem cell transplant. Six patients (54.5%) died within 90 days of starting induction therapy.

Conclusion: t-AML and AML-MRC remain to have dismal prognosis. Our study highlights the high early mortality rate associated with induction therapy with CPX-351. The most common cause of death was refractory or relapsed leukemia. The high incidence of neutropenic fever was also more prevalent to what has been reported.

Disclosures

No relevant conflicts of interest to declare.
Imatinib (IMA) Low Dose Reduction Program (LDR) in Chronic Myeloid Leukemia Ph+ (CML Ph+) in a Single Reference Institution in Mexico City. Ongoing Preliminary Results
Authors: Hurtado Monroy Rafael , Vargas-Viveros Pablo , Silvia Carrillo Munoz
Published: November 2019

Imatinib (IMA) total stop therapy has interesting worldwide results in selected patients (pts) with CML Ph+ who have achieved 4 to 5 log reduction of BCR/ABL copies, however there is no information of LDR.

The observation that IMA intolerance induces a dose reduction in our cases and, the fact that during the follow-up of their RT-PCR for BCR/ABL remains in > 4 log reduction after two years, was the rationale for an open prospective LDR program with the main objective to optimizing CML Ph+ treatment due to economical limitations and drug availability in Mexico. Herein we report our ongoing preliminary results:

A total of 73 of 152 (48%) pts was eligible for LDR after achieving Major Molecular Response (MMR) who reduced of > 4 log in at least two RT-PCR determinations in one or two years. The molecular response was assessed by peripheral blood real-time polymerase chain reaction at selected laboratory, able to express the results according to the International Scale (Hughes T, et al. Blood 2006;108:28-37). LDR included reduction of IMA to 25% of the 400mg for the chronic phase and 33% for the accelerated phase or those who reduced from 800 to 600 and even 400mg.

Among 73 pts, 11 (15%, historic group) were intolerant to IMA and their median dose reduction was 50% (Min-Max; 25-100%) with a median follow-up of 28 months (12-48) with only one relapse after 48 months of dose reduction. The LDR included 62 of 152 (40%) pts with a median age of 37 years (18-74), the gender was, female/male (n-30/32), the median time (MT) duration of CML was 8 years (4-18 years), the MT of reduction was 16 months (12-30). In 52 of 62 pts (83%) the proportion of LDR was 25% and the others in the range of 800 to 400mg. Molecular relapse was seen in 4 cases (6.4%) of the 25% reduction group and in 2 cases of the 50% (600-to 800mg) that represented 1.2%.

The LDR program is an interesting alternative for optimizing CML Ph+ IMA treatment with range from one to two and a half years in MMR with a 7.2% global relapse rate which is lower than the total interruption of TKIs in previous literature reports; however, the inclusion of more pts with follow-up as well, is mandatory to validate our program and the economic impact.

Disclosures

No relevant conflicts of interest to declare.
Novel Thermostable Inhibitor of Contact Activation Tica Effectively Blocks Contact Activation in Low Tissue Factor Thrombin Generation
Authors: Van De Berg Tom , Suylen Dennis P.L. , Thomassen M.G.L. Christella D. , van Oerle Rene , Spronk Henri M.H. , Apitz Rafael , Hemker H. Coenraad , Hackeng Tilman M.
Published: November 2019

Background: Thrombin generation and other clotting assays suffer from a wide variation of pre-analytical variables. One of those pre-analytical variables is contact activation through blood withdrawal methods, different syringes, differences in blood coagulation tubes, blood transport and sample handling. It has been shown that the addition of contact activation inhibitors in low tissue factor activated thrombin generation leads to a correction of the, in these circumstances significant, increase in thrombin generation due to contact activation. We compare the novel 'thermostable inhibitor of contact activation' (TICA) to the current standard 'corn trypsin inhibitor' (CTI).

Aim: Comparing the effectiveness of novel contact activation inhibitor TICA to the current standard CTI in low tissue factor-induced thrombin generation and recalcification in sodium citrate anticoagulated platelet poor plasma (PPP) and platelet rich plasma (PRP).

Methods: We compared TICA, Corn trypsin inhibitor and plasma without contact activation inhibitors in low tissue factor PPP thrombin generation and in PRP recalcification thrombin generation, the latter the most sensitive condition for contact activation. In addition, we compared low tissue factor activated thrombin generation in plasma from severe hemophilia A patients with and without TICA during and after blood drawing. Thermostability - as a measure of shelf life - was measured and compared to CTI.

Results: TICA is able to fully block contact activation in PRP recalcification experiments and is comparable to CTI in doing so. TICA significantly lowers low tissue factor induced thrombin generation by blocking contact activation. Pre-loading vacuum blood collection tubes with contact activation inhibitors is superior in inhibiting contact activation compared to addition of the inhibitor during the thrombin generation assay itself. TICA did not alter coagulation activity when added to FXIIa deficient plasma in thrombin generation. In contrast to CTI TICA is heat stable which will be of benefit to shelf life of pre-loaded blood drawing tubes.

Conclusion: TICA is able to fully block contact activation and has several advantages over CTI.

Disclosures

No relevant conflicts of interest to declare.
CD39 As a Master Regulator of Pulmonary Thrombosis in Sickle Cell Disease
Authors: Brzoska Tomasz , Tutuncuoglu Egemen , Tofovic Stevan P. , Jackson Edwin K. , Gladwin Mark T , Sundd Prithu
Published: November 2019

Acute systemic painful vaso-occlusive crisis (VOC) often serves as an antecedent to acute chest syndrome (ACS), which is a type of acute lung injury and the leading cause of mortality among sickle cell disease (SCD) patients. Thrombocytopenia secondary to pulmonary thrombosis is major risk factor for ACS, however, only 20% of ACS patients are diagnosed with pulmonary thrombosis as an underlying cause of ACS. Although clinical evidence supports the presence of prothrombotic state in subset of SCD patients, the molecular, cellular and genetic mechanisms that selectively render subset of SCD patients at either higher or lower risk of developing pulmonary thrombosis remains elusive. Adenosine diphosphate (ADP) released from lysed erythrocytes can activate platelets by stimulating their purinergic P2Y1 and P2Y12 receptors, however, P2Y12 receptor antagonists have not shown any benefit in clinical trials, justifying the need for better understanding of purinergic signaling in SCD.

Here, we use intravital lung microscopy in transgenic humanized SCD mice to show that intravenous administration of ADP triggered pulmonary thrombosis in control mice but failed to trigger pulmonary thrombosis in SCD mice. In contrast, collagen evoked pulmonary thrombosis identically in both control and SCD mice. Identical to intravital findings, IV ADP administration also evoked transient thrombocytopenia in control but not SCD mice, while, IV collagen led to comparable drop in platelet count in both SCD and control mice. ADP is metabolized by the ecto-nucleoside-tri-phosphate-diphosphohydrolase-1 (E-NTPDase1) CD39. IV administration of fluorescent analogue of ADP, N⁶- ethenoadenosine- 5'- O- diphosphate (ε-ADP) followed by invivo microdialysis and HPLC analysis revealed impaired ε-ADP degradation in SCD mice, suggestive of decreased CD39 activity.

Our current findings suggest that loss of CD39 activity in SCD possibly prevents ADP-mediated pulmonary thrombosis. Currently, experiments are underway to identify pathways contributing to loss of CD39 activity in SCD, how that affects purinergic signaling and whether selective activation vs deactivation of this pathway is responsible for risk of pulmonary thrombosis in only 20% of ACS patients.

Disclosures

Gladwin: Globin Solutions, Inc: Patents & Royalties: Provisional patents for the use of recombinant neuroglobin and heme-based molecules as antidotes for CO poisoning; United Therapeutics: Patents & Royalties: Co-inventor on an NIH government patent for the use of nitrite salts in cardiovascular diseases ; Bayer Pharmaceuticals: Other: Co-investigator.
Characterization of Leukemic Stem Cells Heterogeneity in Chronic Myeloid Leukemia
Authors: Warfvinge Rebecca , Sommarin Mikael , Dhapola Parashar , Pfisterer Ulrich , Geironson Ulfsson Linda , Safi Fatemeh , Krishna Thakur Ram , Richter Johan , Karlsson Göran
Published: November 2019

In chronic myeloid leukemia (CML), a rare subset of leukemic stem cells (LSC) persists in patients responding to conventional tyrosine kinase inhibitor (TKI) therapy. The failure to eradicate these LSCs results in indefinite therapy dependence and a risk of leukemic relapse. However, the conventional LSC compartment (Lin-CD34+CD38-) is highly heterogeneous where only a subpopulation is believed to be functional, TKI-insensitive LSCs.

Previously, using single-cell gene expression analysis we characterized the heterogeneity within the LSC population (Lin-CD34+CD38-) in CML patients using a selected panel of 96 primers. Interestingly, by comparing LSC heterogeneity at diagnosis with the heterogeneity following 3 months of TKI therapy we uncovered a therapy-insensitive, quiescent subpopulation, which could be isolated at high-purity using a combination of the surface markers: Lin-CD34+CD38-CD45RA-cKIT-CD26+ (Warfvinge, Geironson, Sommarin et al., 2017).

Here, we expand the single-cell analysis of CML LSC populations to include combined immunophenotype-/RNA sequencing analysis (CITE-seq). CITE-seq allows for unbiased, further in-depth transcriptome analysis as wells as immunophenotypic characterization by pre-staining cells with a panel of DNA-barcoded antibodies prior to sequencing. DNA-barcoded antibodies convert the protein expression into readable sequences through unique oligo-conjugates as identifiers.

Using CITE-seq with a panel of 44 distinct surface markers designed to immunophenotypically differentiate between stem/progenitors cells and leukemic clones we simultaneously characterize the molecular and immunophenotypic heterogeneity within Lin-CD34+/Lin-CD34+CD38- CML stem/progenitor compartment at diagnosis. Additionally by comparing the LSCs transcriptome from patients with different therapeutic outcome after 12 months of therapy we describe how differences in heterogeneity and the presence of immunophenotypic therapy-insensitive LSCs at diagnosis (Lin-CD34+CD38-CD45RA-cKIT-CD26+) contribute to therapy response.

Disclosures

Richter: Novartis: Consultancy; Pfizer: Consultancy, Research Funding.
Impact of Histologic Type on Survival in Primary Parotid Lymphomas
Authors: Al-Juhaishi Taha , Khurana Arushi , Shafer Danielle , Yazbeck Victor
Published: November 2019

Introduction:

Primary extranodal lymphomas of the salivary glands constitute 12% of head and neck lymphomas, and less than 5% of salivary gland neoplasms. Parotid gland is the most commonly involved structure. Mucosa-associated lymphoid tissue (MALT) lymphoma is considered the most common histology in parotid lymphomas and carry a favorable prognosis. In this study, we sought to explore the impact of histologic subtype on survival.

Methods:

The Surveillance, Epidemiology and End Results (SEER) database was used to identify patients (≥18 years) diagnosed with primary parotid lymphomas between 1975 and 2016. B-cell, T-cell, and Hodgkin's lymphomas were included. Treatment effect (surgery, radiation, chemotherapy) was analyzed when available. Only stage I lymphomas by Ann-Arbor staging system were included to ensure parotid gland as the primary site. Overall survival was estimated using the Kaplan-Meier method, and compared using Log-Rank test. Cox proportional hazards models were used for adjusted survival analyses.

Results:

A total of 1482 patients were included in the analysis. Median age was 66 years (18-102), and the majority were female (n=904, 61%) and Caucasian (n=1074, 72.5%). MALT was the most common histology (N=681, 46%), followed by follicular lymphoma (FL; N=407, 27.5%), and diffuse large B-cell lymphoma (DLBCL; N=310, 21%). Small lymphocytic lymphoma was the least common histology (CLL/SLL; N=3, 0.8%). A total of 1084 patients (73%) had surgery, 433 (29%) had systemic therapies and 691 (47%) had radiation. Median overall survival was 13 years [12-14.5; 95% CI]. Survival was different by histological type (Figure 1, P<0.001). Compared with MALT, DLBCL was associated with higher mortality (HR 2.05 [1.60-2.63], P <0.001), while FL was not significantly different (HR 1.11 [0.89-1.40], P=0.32); independent of other variables. In a multivariate model; surgery (HR 0.81 [0.66-0.99], P=0.042) and radiation (HR 0.67 [0.56-0.82], P <0.001), but not systemic therapies (HR 0.93 [0.76-1.15], P=0.55) were associated with lower mortality (Figures 2-4). Adjusted mortality decreased by a relative 3% annually (HR 0.97 [0.95-0.99], P=0.03).

Conclusion:

Primary parotid lymphomas are rare extranodal lymphomas affecting mostly elderly Caucasian women. Using a large population database, MALT was still the most common histology and carried a favorable prognosis with a median overall survival of greater than 10 years despite the high median age at diagnosis. DLBCL histology did significantly worse which highlights the need for potentially more aggressive interventions to improve outcomes. Surgery and radiation were associated with improved outcomes.

Disclosures

Yazbeck: Gilead Sciences: Research Funding; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.
Caudal Type Homeobox 2 Is Required for Leukemogenesis but Not for Normal Hematopoieisis
Authors: Jones Morgan A , Zhao Meiling , Liu Lu , Feng Ying , Ross Adam , Fearon Eric R , Li Qing
Published: November 2019

Caudal Type Homeobox 2 (CDX2) is a caudal-related homeobox family transcription factor known to be developmental regulator involved in murine axial elongation and anteroposterior vertebral patterning and cell fate determination in the intestine. In adults, CDX2 expression is restricted to the small intestine and the colon. Interestingly, CDX2 is overexpressed in ~90% of human acute myeloid leukemias and the majority of acute lymphoblastic leukemias. Hence, aberrant expression of CDX2 may play a contributing role in leukemogenesis. The role of CDX2 in normal hematopoietic development is unknown. To assess the role of CDX2 in normal hematopoiesis we characterized hematopoietic development in Cdx2 fl/fl Mx1-Cre+ mice after poly (I:C)-mediated gene deletion compared to wildtype littermates. Under steady state conditions, no significant defects were identified in long-term hematopoietic stem cells (LT-HSCs), multipotent progenitors, myeloid progenitors, or mature cell lineages. To test if long-term hematopoietic stem cells were functionally impaired following Cdx2 deletion, competitive transplantation assays were performed. We found that CDX2 was not required for long-term hematopoietic reconstitution in the competitive transplant setting. Together, these data establish that CDX2 is not required for adult steady state hematopoiesis or LT-HSC function. We then asked whether or not CDX2 was essential for leukemogenesis. To interrogate this, Cdx2 fl/fl Mx1-Cre+, Cdx2 fl/+ Mx1-Cre+, and wildtype littermates were treated with poly (I:C) to facilitate gene deletion or for control purposes. Bone marrow cells from the mice were then transformed using well-established HOXA9/MEIS1 or MLL-AF10 leukemogenesis models. In both systems, Cdx2-deficiency prevented leukemia development, whereas wildtype controls succumbed to leukemia within 200 days. Cdx2 heterozygous knockout animals had a delayed onset of leukemia compared to wildtype animals. Taken together, these data indicate that CDX2 is not required for steady state adult hematopoiesis or LT-HSC function, but is necessary for the development of leukemia. Our findings suggest CDX2 has potential as a novel therapeutic target that could be exploited in the majority of AML and ALL patients across a broad mutational spectrum. The nomination of CDX2 as a key target for the majority of leukemias is significant, as current targeted treatment strategies are limited to only particular patient subsets.

Disclosures

No relevant conflicts of interest to declare.
Recurrent JAK3 Mutations in Post-Transplant Lymphoproliferative Disorder
Authors: Butzmann Alexandra , Jangam Diwash , Kumar Jyoti , Sridhar Kaushik , Pinsky Benjamin A , Ohgami Robert S.
Published: November 2019

Post-transplant lymphoproliferative disorders (PTLD) are diverse in morphology, immunophenotype and clinical outcomes, occurring in immune compromised patients after hematopoietic stem cell transplantation (HCT) or single organ transplantation (SOT). Predicting the outcome of PTLDs is complex. In most of cases PTLD is driven by Epstein-Barr virus (EBV) infection, though the etiology of EBV negative cases is unknown. There have been only few studies investigating the mutational landscape and gene expression of PTLD.

In our study we performed targeted DNA deep sequencing and RNA expression profiling (RNA-Seq) on the hematopoietic lymphoid tissues of 15 post-transplant patients with PTLD and 16 post-transplant patients with follicular hyperplasia (FH) after HCT or SOT.

Recurrent mutations in JAK3 were identified in five of 16 PTLD cases and one of 16 follicular hyperplasia cases. We additionally identified mutations in the PIK3CD gene in four PTLD cases and NOTCH1 mutations in 2 PTLD and four FH cases. 17 other genes were commonly mutated in both groups. Gene expression profiles differed between both groups. Genes involved in regulating the immune system were unregulated in PTLD. We also saw a stronger expression of T-cells and a weaker expression of B-cells within the PTLD group when compared with FH.

Our findings suggest an important role for immune regulatory pathways in the pathogenesis of PTLD, with the JAK/STAT pathway being a central recurrent key metabolic pathway.

Figure

Disclosures

Ohgami: Agilent technologies: Other: received support/funding.
RNA-Sequencing Based Assessment of Targets, Risk and Long Term Survival for Personalized Treatment of Multiple Myeloma
Authors: Emde Martina , Beck Susanne , Benes Vladimir , Moreaux Jerome , Seckinger Anja , Hose Dirk
Published: November 2019

Aim of our study is to implement RNA-sequencing as basis for risk adapted and targeted treatment-strategies in multiple myeloma. We establish assessment of malignant plasma cell proliferation and risk-stratification de novo (HDHRS) by RNA-sequencing, transfer microarray-based risk scores to RNA-sequencing, and report expression of actionable including immune-oncological targets.

Methods. Transcriptome profiling was performed on CD138 purified malignant plasma cell samples from previously untreated symptomatic multiple myeloma patients undergoing high-dose melphalan treatment and autologous stem cell transplantation. RNA-sequencing was performed on an Illumina NextSeq500-platform using 5ng of total RNA (n=535), and, in the same cohort, by gene expression profiling using Affymetrix U133 2.0 DNA-microarrays (n=534). iFISH was performed using a ten-probe panel (n=535). Clinical data and survival of patients treated with high-dose therapy and autologous stem cell transplantation were assessed (n=534/533/534 for RNA-seq/GEP/iFISH) with median follow up of 64 months. Data were validated using the independent COMPASS cohort (n=767 pts., median follow up of 31 months).

Results. RNA-sequencing is feasible in more than 90% of myeloma patients. It allows assessment of proliferation (RPI) and risk, both de novo (HDHRS) and regarding microarray-based scores (UAMS GEP70-, EMC92-, Rs-score). All scores are significantly predictive for overall and event-free survival in GEP are likewise in RNA-seq with hazard ratio for high risk to low risk group of 3.06, 4.3, 2.84, 3.93 and 3.30 in overall survival of test group. (See as example gene expression based proliferation index / RNA-seq based proliferation index, Fig. 1). The de novo RNA-seq risk stratification (HDHRS) superiorly delineates three significantly different groups of 40%, 38%, and 22% of patients with 5-year survival rates of 84%, 67%, and 32%, respectively (Fig.2). It likewise discerns a group of long term surviving patients with a survival rate of 57% and 49%, at 10 and 12 years, respectively. Targets expressed in normal plasma cells with varying expression height in malignant include BCMA and CD38, those aberrantly expressed CSF1, CD20 and HGF. Mutated actionable transcripts include BRAF V600E/K (target), the proteasome and cereblon (resistance).

In conclusion, RNA-seq allows assessment of risk, long term survival and targets in multiple myeloma both in phase III multicenter trials as well as in clinical Routine.

Disclosures

Moreaux: Diag2Tec: Other: Co-founder of Diag2Tec company.
VISTA Is a Novel Regulator of Macrophage Biology
Authors: Shah Parth , Eltanbouly Mohamed , Smits Nicole , Sarde Aurelien , Noelle Randolph , Mabaera Rodwell
Published: November 2019

Background:V-domain Ig suppressor of T cell activation (VISTA) is an immune checkpoint receptor with an established role as an inhibitory receptor on T cells. However, its role in myeloid cell biology remains unclear. Owing to the much higher expression of VISTA on monocytes and macrophages, we investigated its role in regulating myeloid biology.

Methods: Mouse bone marrow-derived macrophages (BMDMs) were differentiated and treated with agonistic anti-VISTA mAb under steady-state and conditions of M1 inflammatory polarization (LPS + IFN-𝛾). Human CD14+ monocytes were isolated from peripheral blood and treated with anti-VISTA in vitro. Phosphoarrays were used to assess signaling downstream of VISTA engagement and Luminex analysis was used to evaluate cytokine production. Time-course total RNA-seq was performed on BMDMs (at 1, 2, 4, 8 and 16 hours of anti-VISTA treatment). To evaluate the effect of anti-VISTA in vivo, single-cell RNA-seq (scRNA-seq) was performed on FACS sorted CD11b+ myeloid populations 12 hours after mice were treated with intravenous anti-VISTA.

Results:

Anti-VISTA treatment of BMDMs and human monocytes in vitro induced rapid phosphorylation events including robust phosphorylation of Raf-1 kinase at the suppressive serine residue 43.Next, we generated a comprehensive timestamp of VISTA activation in BMDMs in vitro by time-course deep RNA-seq analysis of agonistic anti-VISTA treated cells under rigorous M1 inflammatory polarization. This revealed a unique activation state marked by the upregulation of inhibitory cytokines IL-10 and IL1RA as well as ablation of IL-12 family cytokines, suggestive of an M2-like signature. The presence of an M2-like immunoregulatory signature defined by upregulation of the IL-10 was also seen in macrophages identified post clustering of in vivo scRNA-seq data on myeloid cells.

Conclusion:

Using novel agonistic anti-VISTA antibodies, we reveal that VISTA triggering on myeloid cells can induce an alternative anti-inflammatory cell-state, even under conditions of strong inflammatory polarization. This introduces VISTA as an intrinsic checkpoint of macrophage tolerance.

Disclosures

Noelle: Immunext: Employment, Equity Ownership, Patents & Royalties, Research Funding.
Acquired Dysfibrinogenemia Krakow III after Everolimus Therapy
Authors: Merkulova Alona A. , Mitchell Steven C. , Merkulov Sergei , Wolberg Alisa S. , Marguerite Neerman-Arbez , Schmaier Alvin H.
Published: November 2019

A 45 yo woman with a medical history of common variable immunodifficiency (CVID) was referred for a bleeding disorder. The bleeding was manifested daily with ecchymosis and epistaxis, but prior to the visit, she developed a lower pelvic mass that was a massive hematoma requiring transfusion. In 2011 she had a splenectomy for thrombocytopenia. After splenectomy, her liver enlarged and one year prior to being seen, she was started on everolimus as part of a protocol to reduce her liver size.

Bleeding started 3-6 months after starting everolimus treatment, first manifesting with recurrent lower gastrointestinal hemorrhage. In 2011 she had normal PT and aPTT. At the present time her PT remains normal at 11.43+0.67 (Mean+SD) (normal 9.7-12.7 sec); but her aPTT is abnormal at 43+6.1 (normal 28-38 sec); clottable fibrinogen (Fb) 305+72 (normal 200-400 mg/dl); Fb antigen, 428+115 mg/dl (normal 196-441); ratio of clottable Fb/Fb antigen = 0.71+ 0.06; reptilase time 20+2.2 (normal 14-23 sec); and her thrombin time of 19+1.6 is prolonged (normal 10-16 sec). Blood coagulation factors XI, IX, VIII, VII, X, V, II, and XIII were normal. Further, she did not have excess alpha-1-antitrypin, antithrombin, or protein C to suppress thrombin generation. On a 1:1 mixing study of normal plasma:patient plasma, her thrombin time corrected to normal. Since the patient had an abnormal thrombin time with serious, spontaneous, clinical bleeding with a normal reptilase time, we postulated a fibrin polymerization defect. On a fibrin polymerization assay, the patient required 4-fold greater amounts (32 nM vs 8 nM) of human alpha thrombin to achieve complete fibrin polymerization (See Figure 1 below). On complete fibrinogen sequencing, the patient was found to have a pathologic mutation [Fb gamma chain exon 3, 124G>A, Gly42Ser (Gly16Ser in the mature gamma chain without the signal peptide)]. This defect had been previously described as Fibrinogen Krakow III (Pietrys D et al. Thromb Haemost. 2011;106:558-60). However, neither parent of the patient has a bleeding disorder. Sequencings of her parents' Fb gamma chains show no mutation.

Since first being seen, the patient had a serious bleed into her right sacral plexus leaving her with a permanent foot drop. We managed her bleeding with cryoprecipitate replacement acutely and oral tranexamic acid as an outpatient. Everolimus is an mTor inhibitor which is a regulator of protein synthesis. We postulated that everolimus therapy produced the acquired abnormal Fb gamma synthesis, producing the fibrin polymerization defect associated with bleeding. Upon urging, the patient stopped everolimus therapy and has not had a major bleeding incident in 8 months although the abnormal thrombin time persists.

Figure 1

Disclosures

Wolberg: Novo Nordisk: Research Funding.