bluuebunny/crossref_metadata_2025 huggingface dataset:
The Impact of Immunotherapy on Tumor Evolution
Authors: Thomas-Tikhonenko Andrei
Published: October 2019

We have previously described a mechanism of acquired resistance of B-cell acute lymphoblastic leukemia to CD19-directed chimeric antigen receptor T-cell (CART) immunotherapy. It was based on in-frame insertions in or skipping of CD19 exon 2. To distinguish between epitope loss and defects in surface localization, we used retroviral transduction and genome editing to generate cell lines expressing CD19 exon 2 variants (CD19ex2vs) bearing VSV-G tags. These lines were negative by live-cell flow cytometry with an anti-VSV-G antibody and resistant to killing by VSV-G directed antibody-drug conjugates (ADC), suggestive of a defect in surface localization. Indeed, pulse-chase and α-mannosidase inhibitor assays showed that all CD19ex2vs acquired ER-specific high mannose-type sugars, but not complex-type glycans synthesized in Golgi. When fused with GFP, CD19ex2vs (including a mutant lacking the relevant disulfide bond) showed colocalization with ER markers, implicating protein misfolding. Mass spectrometric profiling of CD19-interacting proteins demonstrated that CD19ex2vs fail to bind to the key tetraspanin CD81 and instead interact with ER-resident chaperones such as calnexin and ER transporters involved in antigen presentation. Thus, even the intact domains of CD19ex2vs cannot be easily targeted with ADCs or current CD19 CARTs but could serve as sources of peptides for MHC-restricted presentation and TCR-mediated killing.

Disclosures

Thomas-Tikhonenko: Children's Hospital of Philadelphia: Patents & Royalties.
Clinical Implications and Biomarkers of Clonal Evolution
Authors: Dawson Sarah-Jane
Published: October 2019

Recent technological advances in the detection and characterization of cell free circulating tumor DNA (ctDNA) are providing new opportunities for treatment tailoring based on real-time monitoring of clonal evolution. Circulating biomarkers can provide a "liquid biopsy" alternative to tissue biopsies allowing noninvasive tumor genotyping and the serial analysis of genomic changes from a simple blood test. The emerging clinical benefits of ctDNA analysis in the management of solid tumours has led to an expansion of opportunities in the use of ctDNA for disease monitoring in haematological malignancies. Research in this area has rapidly expanded exploring the role of ctDNA for risk stratification, monitoring tumour burden and identifying the early emergence of treatment resistance. In the management of lymphoma, comprehensive analysis of somatic genomic alterations in plasma DNA has emerged as an important clinical tool. The use of ctDNA analysis to follow tumour specific genomic alterations identified on an individual basis can be used to help identify minimal residual disease post treatment and guide therapeutic decisions. Moreover, ctDNA analysis can been utilized to identify distinct biological subtypes of lymphoma and provide insights into patterns of genomic evolution following treatment. The role of ctDNA as a minimally invasive strategy for disease monitoring has also recently been demonstrated in chronic lymphocytic leukemia (CLL) showing additional potential of this technique in haematological malignancies with a circulating disease component. In CLL, ctDNA analysis can reveal changes in the disease across tissue compartments, including the bone marrow and lymph nodes, providing additional information to that which can be assessed by monitoring the circulating disease alone. Serial ctDNA analysis in CLL can allow clonal dynamics to be monitored over time and can identify the emergence of genomic changes associated with Richter's syndrome. Here, I will focus on the use of ctDNA technology to follow disease burden and track genomic changes in patients receiving novel therapies for lymphoid malignancies, as well as the promises and challenges in developing these tools for clinical application.

Disclosures

No relevant conflicts of interest to declare.
Blood Donation and Iron Deficiency
Authors: Mast Alan E.
Published: October 2019

In the United States about 40,000 people per day donate blood. Donors are from all racial/ethnic backgrounds and range in age from 16 to over 80 years old. They are allowed to repeatedly donate blood every 56 days, as long as they maintain their hemoglobin above 12.5 g/dL for females or 13.0 g/dL for males. Although each donation removes 200-250 mg of iron from the donor, testing for iron deficiency is not performed. Thus, blood donation can be viewed as a large clinical study to examine the physiological effects of repeated phlebotomy of approximately 10% of an individual's blood volume. In addition, blood donors are altruistic by nature and typically are very willing to participate in research studies. We have utilized this rich clinical population to perform studies on iron metabolism and red blood cell biology in an otherwise healthy population. These include a study of over 750,000 donors to define the demographics of anemia. A study detailing the body's response to blood donation, finding that recovery of iron stores takes over 90 days, if the donor takes daily iron pills following donation, and much longer if they do not. Despite this long time for recovery of iron stores, some donors are capable of repeatedly donating blood every 56 days for several years without experiencing a low hemoglobin deferral. Study of these "superdonors" has identified potential associated genetic variants and behaviors that allow frequent donation without development of anemia. Teenagers are allowed to donate blood in the United States at the same 56 day donation frequency and hemoglobin cut-off values as adults even though they have increased iron requirements for continued growth and neurocognitive development. A study detailing the impact of blood donation on teenager hemoglobin and iron stores has found that they are more susceptible to iron deficiency than adults, and, therefore, should have extra protections to prevent donation-induced iron deficiency.

Disclosures

Mast: Novo Nordisk: Research Funding.
New Advances Along the Common Coagulation Pathway
Authors: Camire Rodney M.
Published: October 2019

Hemostasis is achieved through spatially and temporally regulated thrombin generation following vascular injury. Blood coagulation factor V plays an important role in this process as it has a major impact on thrombin production. FV is a large, heavily glycosylated protein sharing homology and domain organization with FVIII (A1-A2-B-A3-C1-C2). It is an inactive procofactor and must be cleaved by thrombin or other proteases to remove the B-domain to yield activated FV (FVa). FVa is a cofactor for FXa in the prothrombinase complex, the enzyme that activates prothrombin. Given the profound effect FVa has on thrombin formation, FV activation represents an important step in hemostasis. Mechanistic studies have revealed that proteolysis within the FV B-domain serves to remove key autoinhibitory sequences that serve to conceal/block FXa binding site(s). These sequences consist of an evolutionary conserved basic region (BR) in the middle of the B-domain and flanking acidic regions (AR1 and AR2) which define the minimal sequences necessary to maintain FV as a procofactor. Removal of either of these regions results in a molecule that can function in prothrombinase. These sequences are intact and present in plasma FV; however, FV released by activated platelets and FV cleaved by FXa represent active forms FV(a) that lack the BR. Recent work indicates these forms of FV(a) may be regulated by TFPIathrough molecular mimicry. TFPIahas a C-terminal basic tail which shares remarkable homology to the FV BR suggesting TFPIamay be an important regulator at the level of prothrombinase. Remarkably, another form of FV missing the BR, resulting from alternative splicing, has been recently described. The FV-East Texas, FV-Amsterdam, and FV-Atlanta alterations all result in the enhanced production of an alternatively spliced form of FV (FV-short) that has most of the B-domain removed including the BR. While these forms of FV should be constitutively active, studies have shown that FV-short circulates in complex with TFPIa. Further, in each of the clinical cases, TFPIalevels are substantially elevated resulting in a bleeding phenotype. While there are several remaining unanswered questions, the FV(a)-TFPIα interaction as well as the identification of FV-short, has the potential to fundamentally alter our understanding of the regulation of the initiation of coagulation.

Disclosures

Camire: Bayer: Consultancy; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Spark Therapeutics: Membership on an entity's Board of Directors or advisory committees.
A Direct Oral Anticoagulant Edoxaban Enhanced Fibrinolysis Via Inhibition of Thrombin-Activatable Fibrinolysis Inhibitor Activation and Enhancement of Plasmin Generation in Human Plasma
Authors: Morishima Yoshiyuki , Honda Yuko , Furugohri Taketoshi
Published: October 2019

Introduction: Direct oral anticoagulants are as effective as vitamin K antagonists for the treatment of venous thromboembolism and are associated with less bleeding. We have reported that a direct oral factor Xa inhibitor edoxaban exerts a thrombus resolution effect in a rat model of venous thrombosis and enhances tissue plasminogen activator (t-PA)-induced clot lysis in human plasma. However, the mechanism underlying the thrombus resolution effect and fibrinolysis enhancement by edoxaban remains to be determined.

Purposes: To evaluate the effect of edoxaban on plasmin generation during clot lysis in human plasma in vitro. To determine the role of thrombin-activatable fibrinolysis inhibitor (TAFI) in the enhancement of fibrinolysis by edoxaban.

Methods: Pooled human normal plasma or TAFI-deficient plasma (both containing 180 ng/mL t-PA and 0.1 nM thrombomodulin) was mixed with edoxaban, an activated TAFI (TAFIa) inhibitor (a fibrinolysis enhancer) potato tuber carboxypeptidase inhibitor (PCI), or vehicle. Clot was induced by adding 2.5 pM tissue factor and 4 µM phospholipids. To monitor the clot formation and lysis, the absorbance of plasma at 405 nm was measured every 30 sec. Clot lysis time was defined as the interval between the time of the midpoint of the clear to maximum turbidity transition and the midpoint of the maximum turbidity to clear transition. Plasmin-α2 antiplasmin complex (PAP) concentration was measured by ELISA as an indicator of plasmin generation.

Results: In normal plasma, PCI accelerated clot lysis and increased plasma PAP concentration. There was a correlation between plasma PAP concentration and percent of clot lysis, indicating that plasma PAP concentration is an appropriate marker of fibrinolysis. Edoxaban at clinically relevant concentrations (75, 150, and 300 ng/mL) significantly shortened clot lysis time and elevated plasma PAP concentration. The additive combined effect of edoxaban and PCI was observed. In TAFI-deficient plasma, clot lysis time was shortened and plasma PAP concentration increased compared with normal plasma. In these samples, the effects of edoxaban and PCI on clot lysis and plasma PAP concentration were markedly diminished as compared with normal plasma.

Conclusions: Edoxaban at clinically relevant concentrations enhanced t-PA-induced clot lysis with increasing plasma PAP concentration in human plasma. The effects of edoxaban on clot lysis and plasmin generation were diminished in TAFI-deficient plasma. A TAFIa inhibitor PCI exerted similar effects. These data suggest that edoxaban enhanced fibrinolysis via inhibition of TAFI activation and enhancement of plasmin generation.

Disclosures

Morishima: Daiichi Sankyo Co., Ltd.: Employment. Honda:Daiichi Sankyo Co., Ltd.: Employment. Furugohri:Daiichi Sankyo Co., Ltd.: Employment.
Recent Survival Trends in Diffuse Large B-Cell Lymphoma (DLBCL)-Did We Make Any Progress Beyond Rituximab?
Authors: Epperla Narendranath , Hallack Neto Abrahão Elias , Costa Luciano J
Published: October 2019

Background

While rituximab revolutionized outcomes in DLBCL, little is known regarding survival improvements at population level beyond the introduction of rituximab. The advent of novel agents, improvements in supportive care and expansion of autologous hematopoietic cell transplantation (auto-HCT) to older individuals may have improved survival of DLBCL patients. We hypothesized that outcomes for patients diagnosed with DLBCL in the U.S. has continued to improve in recent years and intended to measure improvements and identify possible disparities by comparing survival of DLBCL between two consecutive post-rituximab eras.

Methods

We used the population-based SEER-18 registry to calculate the incidence and relative survival rates (RSR) of DLBCL in the U.S. for two consecutive eras since broad availability of rituximab in upfront DLBCL treatment, 2002-2007 (era-1, early years after adoption of rituximab) and 2008-2013 (era-2, availability of novel agents, broader use of auto-HCT and improvement in supportive care). We included adult patients with microscopically confirmed DLBCL as first malignant neoplasm regardless of histologic subtype and survival follow up to the end of 2015.

Results

There were a total of 56,625 DLBCL patients diagnosed between 2002 and 2013, of which 27,306 patients were diagnosed during era-1 and 29,319 during era-2. Median follow up of survivors was 125 months in era-1 and 53 months in era-2. The median age at diagnosis was 66 years with slight male predominance in both the eras. There were no differences in age, gender, race/ethnicity and stage characteristics. There was a slight decline in incidence of DLBCL (era-1 vs. era-2), 8.11 (95% CI=8.01-8.2) vs. 7.82 (95% CI=7.73-7.91) cases per 100,000 persons (P<0.0001). Overall there was a modest improvement in DLBCL RSR, with 5-year RSR improving from 59.5% to 62.5%. Improvement was mostly evident on first year after diagnosis and affected all subsets of patients (Table), but was more pronounced in patients with advanced stage (Figure).

Conclusions

There has been limited improvement in the survival of adult patients with DLBCL beyond the introduction of rituximab, highlighting the need for experimental therapies and calling for the incorporation of novel therapies earlier in treatment, especially for high-risk cases.

Disclosures

Costa: Sanofi: Honoraria; Celgene: Honoraria, Research Funding; Karyopharm: Research Funding; Janssen: Research Funding; Amgen: Honoraria, Research Funding; BMS: Research Funding; Abbvie: Research Funding.
The Impact of Pain Control on Invasive Clinical Procedure on Children with Leukemia
Authors: WANG Jiayi , He Yingyi , Liang Zhimin , Ye Tiezhen , Zhang Hui
Published: October 2019

Background: Palliative care is becoming more and more important for systemic cancer treatment in developed countries, while this remains infamous topic in developing countries, such as in China. Contemporary treatment strategies have greatly contributed to the improved outcome in childhood cancer patients, survivorship comes at the cost of developing some treatment-related health condition, such as pain-related depression, chronic pain etc. Thus, developing a well-tolerated pain control methods is of great importance within the cancer treatment. Objective: To evaluate the impact and outcome of different pain control applications on invasive procedure in children with leukemia, and record the adverse reactions. Methods: The enrollment of childhood leukemia patients in our hospital from November 2011 to November 2016 were divided into four groups, that is successively midazolam + local anesthesia (group A), midazolam + ketamine +local anesthesia (group B), midazolam + fentanyl + local anesthesia (group C), and fentanyl + propofol + local anesthesia (group D). The efficacy and adverse reactions were systemically recorded. The inter-group diffferences were calculated using x2 test. Results: No significancewas observed in age, gender, and disease distribution in these four groups by ANOVA ONEWAY analysis. The sedation outcome is more pronunced in group D than others. Also, the quality of procedural pain control in group D was the best (P<0.01). In terms of the analgesic effect, group B and D were better than that group A and C. There was significant difference in Hallucination was more easily detected in group B and C. Systemic recovery was delayed in group B other than group A, C, and D. Basing on the survey, we did found that the family members were more willing to accept pain control treatment for their sick kids under the safety assurance. The compliance was significantly improved in group D. Conclusion: Upon adequate auxiliary breathing preparation and rigorous monitor, propofol combined with low-dose fentanyl was the best sedative/analgesic option for pain control within leukemia patients receiving invasive procedure.The outcome of propofol combined with low-dose fentanyl wasvery safe, satisfactory and compliable. Up to now, this study is the first pain control study for invasive procedure in China mainland, it deserves being paid attention.

Disclosures

No relevant conflicts of interest to declare.
Hairy Cell Leukemia Variant
Authors: Ashangari Chandralekha , Cheruku Divya , Tumula Praveen K.
Published: October 2019

Introduction: Hairy cell leukemia-variant (HCL-V) is an uncommon disorder, accounting for approximately 0.4% of chronic lymphoid malignancies and 10% of all HCL cases, without sexual predominance. Here is an attempt to submit a rare case presentation of HCL-V.

Case:A 66 year old female presented to the clinic after she was referred from her primary care for further evaluation of pancytopenia. Past medical history was significant for diabetes mellitus and hypertension. Splenomegaly was noted on examination. Initial bloodwork showed a leukocyte count of 4.9, hemoglobin level of 11.8, platelet count of 78. LDH was 166. CT scan showed significant splenomegaly along with retroperitoneal adenopathy. Peripheral blood flow cytometry demonstrated a CD11c positive monoclonal B cell population suggestive of Hairy Cell Leukemia variant, positive for CD19, CD20, CD22, CD45, CD52, and kappa (moderate-bright), negative for CD5, CD10, CD23, CD38, CD103, CD123, BRAF V600. Left shift myeloid elements were noted but no significant myeloid blasts were noticed. She was diagnosed HCL-V with 17p deletion and 90% marrow involvement. She was treated with 8 doses Rituximab. After 1 year, she relapsed and was started on Cladribine and Rituximab weekly for 8 doses. She relapsed 1 year after Cladribine and Rituximab with splenomegaly and cytopenias, and she was started on Ibrutinib. 4 months after treatment with Ibrutinib, her cytopenias improved with normalization of spleen size. She is tolerating off label medication Ibrutinib well and currently asymptomatic.

Discussion: This case represents an extremely rare hematological malignancy a distinct case of HCL-v due to 17p deletion and refractory to multiple therapies. Patients with multiply relapsed HCL-v are also appropriate candidates for moxetumomab pasudotox because HCL-v retains a high level of expression of CD22. Hairy cell leukemia variant is a chronic lymphoproliferative disorder whose characteristics resemble the classic form and splenic marginal zone lymphoma, making differential diagnosis essential for therapeutic follow-up, as hairy cell leukemia variant has a decreased response to drugs prescribed for the classic form. Immunophenotyping is a key tool for diagnosis, as some markers are different between these neoplasms. Therefore, immunophenotyping, clinical, morphological, and immunohistochemical manifestations must be considered in the differential diagnosis.

Figure. Figure.

Disclosures

No relevant conflicts of interest to declare.
Performance of Diagnostic Tests for Plasma Cell Myeloma
Authors: Zagoya Gilda , Ruiz-Argüelles Alejandro , Ruiz-Arguelles Guillermo J.
Published: October 2019

Introduction. In vitro laboratory tests to diagnose of plasma cell myeloma vary considerably in sensitivity, specificity and positive and negative predictive values. We compared the performance of quantification of free immunoglobulin light chains with other methods used to detect a monoclonal protein in serum and/or urine.

Objective. Compare sensitivity, specificity and positive and negative predictive values of several in vitro laboratory tests to detect monoclonal proteins in serum and urine in persons with plasma cell myeloma.

Methods. 70 subjects with plasma cell myeloma and 50 controls were studied. Diagnostic tests included: (1) quantification of free and total immunoglobulin light-chains by immune assays; (2) immune fixation of heavy-and light-chains in serum and urine after gel electrophoresis; and (3) serum protein capillary electrophoresis. Diagnosis of plasma cell myeloma was based on clinical and radiological criteria, bone marrow examination and flow cytometric immune phenotyping with monoclonal antibodies to CD56, CD19, CD138 (CD38) and CD45. Sensitivity, specificity and positive and negative predictive values for each tests were estimated from contingency tables.

Results. Quantification of free immmunoglobulin light-chains had the highest sensitivity and specificity and best positive and negative predictive values. Immune fixation of serum immunoglobulins was next best. Quantification of total immunoglobulin light-chains was the least sensitive and specific with the worst positive and negative predictive values. Quantitation of free light-chains had the additional advantage of objectivity (independence from observer bias). The immune fixation test was the most subject to observer bias.

Conclusion. Quantification of free immunoglobulin light-chains had the best sensitivity, specificity and positive and negative predictive values for diagnosing plasma cell myeloma. (Table 1)

Disclosures

No relevant conflicts of interest to declare.
Vascular Proteomics Reveals Critical Roles of ADAMTS18 in Stroke Associated with Imbalanced Level of Von Willebrand Factor Versus ADAMTS13
Authors: Zhu Rui , Pan Yi-Hsuan , Lu Tiantian , Dang Suying , Liao Chen-Chung , Zhang Wei
Published: October 2019

Background and Purpose -Stroke is the second leading cause of death worldwide. Acute ischemic stroke (AIS) is frequently induced by internal carotid artery thrombosis. Clinically, most arterial thrombus formation is initiated by platelets adhering to the damaged vessel wall. The adherent platelets become activated and bind with fibrinogen and von Willebrand factor (vWF), allowing formation of a platelet aggregate or mural thrombus. Identifying new mechanism involved in this pathogenesis is critical for understanding the molecular basis of thrombus formation and ultimately developing new therapeutic interventions for stroke. The ADAMTS (A disintegrin and metalloproteinase with thrombospondin motifs) family comprises 19 secreted metalloproteinases that cleave extracellular matrix components and have diverse functions in numerous disease and physiological contexts. Several ADAMTS genes (ADAMTS -7,-8, 12, and 13) have been shown to be involved in cardiovascular disease and pediatric stroke. ADAMTS18 remains "orphan" protease. We have previously shown that ADAMTS18 deficiency causes abnormal remodeling of the common carotid artery (CCA) and an increase in carotid thrombosis and size of cerebral infarct in mice. Thus, the stroke-prone Adamts18 KO mice could be valuable for identification of molecular signatures of accelerated carotid thrombosis related to stroke. One of the major steps yet to be taken by the field is the widespread use of proteomics to uncover the critical molecular events and signaling pathway involved in ADAMTS gene-related stroke.

Methods and Results -Here, we carried out the first proteomics comparison of protein expression patterns in CCA and abdominal artery (AA) between Adamts18 knockout (KO) mice and their wild-type (WT) littermates. A total of 1654 proteins were identified in four separate CCA samples (0.4-1 mg/wet weight each), and 1810 proteins were detected in four separate AA samples (2.3-3.6 mg/wet weight each). The abundances of 154 proteins in CCA and 55 proteins in AA were significantly different between KO and WT mice. Ingenuity® Pathway Analysis revealed that ADAMTS18 deficiency predisposes mice to stroke by altering the abundance of many proteins related to cytoskeleton, prothrombotic activity, coagulation, angiogenesis, oxidative homeostasis, and energy metabolism. Western blotting and qRT-PCR analyses further revealed the imbalanced level of von Willebrand factor and ADAMTS13, the pivotal pathogenic factors of ischemic stroke, in CCA of KO mice.

Conclusion and Significance -Results of this first vascular proteomic study of Adamts18 KO mice establish a substantial new resource for investigation of how ADAMTS gene affects cellular phenotypes of stroke. These new findings contribute to inform novel prevention strategies and direct therapeutic development for stroke.

Disclosures

No relevant conflicts of interest to declare.
A Small Nucleolar RNA Linked to Diamond-Blackfan Anemia
Authors: Mayeux Adeline , Montel-Lehry Nathalie , Saby Manon , Anton Aurore , Karboul Narjesse , Galimand Julie , Bourdeau Helene , Levy Jonathan , Tabet Anne-claude , Narla Mohandas , MacInnes Alyson W. , Dufourcq Pascale , Blader Patrick , Leblanc Thierry , O' Donohue Marie-francoise , Gleizes Pierre-Emmanuel , Da Costa Lydie
Published: October 2019

Diamond-Blackfan anemia (DBA) is a rare congenital erythroblastopenia associated with haploinsufficiency of more than 15 ribosomal protein genes. We identified in the French registry a DBA patient exhibiting a large heterozygous deletion in ribosomal protein gene RPSA, making it a new candidate as a DBA-associated gene. The deletion was confirmed by qPCR using specific intron/exon primers that could discriminate the RPSA gene from pseudo-genes. Consistent loss of function of this allele, RPSA mRNAs were underrepresented in erythroid cultures and in lymphoblastoid cells (LCLs) derived from the patient. Additionally, an important decrease in erythroid proliferation was observed in erythroid progenitor and precursor cells derived from the patient's bone marrow CD34+ cells, together with a delay in erythroid differentiation, the activation of the p53 pathway, and a G0/G1 cell cycle arrest, which are regular features of DBA patient cells. Similar to other RP genes linked to DBA, interruption of RPSA expression in zebrafish embryos strongly affected development of the head and erythroid cell differentiation, supporting its relevance as a candidate DBA gene. Northern blot analysis also revealed impaired pre-ribosomal RNA (pre-rRNA) processing in patient LCLs compared to controls. But unexpectedly, the observed pre-rRNA profile was clearly different from that observed in HeLa cells upon RPSA knockdown and suggested a dysfunction of pre-rRNA processing upstream of that produced by a shortage of RPSA. We noticed that the RPSA gene hosts the intron-encoded H/ACA snoRNAs snoRA6 and snoRA62, the latter being included in the deletion. Q-RT-PCR analysis of patient cells showed decreased levels of snoRA62 when compared to controls and to other RP-mutated DBA patients. Strikingly, knocking out the snoRNA62 coding sequence in human haploid HAP1 cells with Crispr/Cas9 resulted in a pre-rRNA maturation defect similar to that observed in the RPSA+/mut DBA patient, thereby revealing a role for snoRA62 in human pre-rRNA processing. These data strongly suggest that, in addition to RPSA haploinsufficiency, the primum movens of DBA in this particular patient includes the deletion of the intronic snoRA62 hosted in the RPSA gene. To our knowledge, this is the first time that a snoRNA is linked to DBA. We will discuss this ongoing work in light of our most recent results in cells and zebrafish models.

Disclosures

No relevant conflicts of interest to declare.
Occurrence and Severity of DLI Associated Chronic Gvhd Influence the Clinical Outcomes in Relapsed Acute Leukemia after Allogeneic Hematopoietic Stem Cell Transplantation
Authors: Yu Wenjing , Mo Xiao-Dong , Zhang Xiaohui , Xu Lan-Ping , Wang Yu , Yan Chen-Hua , Chen Huan , Chen Yu-Hong , Han Wei , Wang Feng-Rong , Wang Jing-Zhi , Liu Kaiyan , Huang Xiaojun
Published: October 2019

The aim of this study was to investigate the occurrence and severity of chemotherapy plus donor lymphocyte infusion (Chemo-DLI) associated chronic graft-versus-host disease (cGVHD) in a consecutive cohort of patients with acute leukemia who experienced relapse after allogeneic hematopoietic stem cell transplantation (allo-HSCT; n = 104). The 5-year cumulative incidence of complete remission (CR) after Chemo-DLI was 81.0% (95% CI, 73.3-88.7%) and 84.6% (95% CI, 74.5-94.7%) in the moderate and severe cGVHD groups, respectively, which was significantly higher than that of the mild cGVHD 40.9% (95% CI, 29.3-52.5%) and non-cGVHD groups 29.2% (95% CI 23.1-35.3%) (Figure 1). The cumulative incidence of non-relapse mortality was comparable between patients with and without cGVHD . The 5-year probabilities of progression-free survival after Chemo-DLI were 42.9% (95% CI, 26.2-70.2%) and 34.6% (95% CI, 15.3-78.2%) in moderate and severe cGVHD groups, respectively, which were both significantly higher than those of mild cGVHD 9.1% (95% CI, 2.4-34.1%) and non-cGVHD groups 8.3% (95% CI 3.3-21.3%) (Figure 2). The 5-year probabilities of overall survival after Chemo-DLI were 56.7% (95% CI, 38.9-82.7%) and 43.1% (95% CI, 22.1-84.0%), in moderate and severe cGVHD groups, respectively, which were both significantly higher than those of the mild cGVHD 9.1% (95% CI 1.8-47.1%) and non-cGVHD groups 14.9% (95% CI, 7.3-30.2%) (Figure 3). Our observations highlight the close relation between cGVHD and immune-mediated graft-versus-leukemia (GVL) effect in patients with relapse receiving Chemo-DLI; however, mild cGVHD may not be associated with a sufficiently strong GVL effect to induce remission and improve survival.

Disclosures

No relevant conflicts of interest to declare.
Syndecan-2 Surface Expression Identifies Hematopoietic Stem Cells with Increased Repopulating Capacity
Authors: Termini Christina , Li Michelle , Kim Joyce , Zhao Liman , Chute John P
Published: October 2019

Syndecans are transmembrane glycoproteins, which can regulate cell proliferation, growth, and adhesion through interactions with neighboring proteins within the plasma membrane or at the cytoplasmic interface. Although syndecans have been described to regulate aberrant signaling in hematological malignances, the role of syndecans in regulating normal hematopoietic stem cell (HSC) proliferation, differentiation, and self-renewal is largely unknown. We demonstrate that syndecan-1 and syndecan-3 are expressed on the surface of < 10% of murine hematopoietic stem and progenitor cells, whereas, syndecan-4 is expressed on 50% of lineage negative (Lin-) progenitor cells and 98% of c-Kit+Sca-1+Lin- (KSL) hematopoietic stem/progenitor cells, KSL CD34-CD48-CD150+/- short-term HSCs, and 100% of CD34-CD48-CD150+ long-term HSCs. Interestingly, we find that syndecan-2 is expressed by 11% of KSL CD34-CD48-CD150+/- short-term HSCs and 36% of CD34-CD48-CD150+ long-term HSCs. More specifically, our data demonstrate a 15-fold increase in syndecan-2 surface expression on KSL CD34-CD48-CD150+ HSCs compared to Lin- progenitor cells (p<.0001, ****). Collectively, these data suggest that syndecan-2 may be a marker for long-term HSCs. In keeping with this hypothesis, we found that syndecan-2+ CD34- KSL cells produce two-fold more granulocyte, erythrocyte, monocyte, megakaryocyte (GEMM) colonies compared to syndecan-2- CD34- KSL cells (p=.0017, **). Cell cycle analyses revealed a significant increase in BrdU incorporation in syndecan-2+ KSL cells compared to syndecan-2- KSL cells (90% versus 40%, p<.0001, ****). Competitive repopulation assays comparing syndecan-2+ or syndecan-2- CD34- KSL bone marrow cells demonstrated that mice transplanted with syndecan-2+ CD34- KSL cells displayed threefold increased donor multilineage hematopoietic cell repopulation compared to mice transplanted with syndecan-2- CD34- KSL cells. These data suggest that syndecan-2 expression marks a highly proliferative population of HSCs with increased multilineage repopulating capacity and that syndecan-2+ HSCs can be readily isolated to enhance the efficacy of hematopoietic cell transplantation.

Disclosures

No relevant conflicts of interest to declare.
Identification of High Serum Apolipoprotein A1 As a Favorable Prognostic Indicator in Patients with Multiple Myeloma
Authors: Liu Peng , Liang Lifan , Li Jing
Published: October 2019

Purpose

To explore the prognostic significance of serum lipid profiles in patients with multiple myeloma (MM).

Patients and methods

The study retrospectively enrolled 307 MM patients in Zhongshan Hospital, Shanghai, China, from 2007 to 2016. We evaluated the prognostic significance of the prediagnostic serum lipid profile [cholesterol, triglyceride, low-density lipoprotein (LDL), high-density lipoprotein (HDL), Apolipoprotein A1 (Apo A1) and Apolipoprotein B (Apo B)]. Prognostic factors identified through univariate and multivariate analysis were used to construct a new model based on Lasso Cox regression.

Results

In our cohort, lipid levels showed significant difference between ISS stages (P<0.001 in cholesterol, P<0.001 in LDL, P=0.002 in HDL, P<0.001 in Apo A1, P<0.001 in Apo B). However, only Apo A1 showed statistically significance in overall survival (OS), progression free survival (PFS) and cause specific survival (CSS) (P=0.038, P=0.028, P=0.011) in univariate Cox regression. Patients with higher Apo A1 displayed longer OS (median OS, 67 months vs. 30 months; P<0.001). Also, Apo A1 was revealed to be independent prognostic parameters through multivariate analysis. Combining the Apo A1 level, a Zhongshan Score model was constructed with Lasso regression for prognosis prediction. This model exhibited higher accuracy than International Staging System (ISS) and Durie and Salmon (DS) system.

Conclusion

Among all the serum lipid profiles, serum Apo A1 is a powerful prognostic indicator for patients with MM.

Disclosures

No relevant conflicts of interest to declare.
ZEB2 and LMO2 Drive Immature T-Cell Lymphoblastic Leukemia Via Distinct Oncogenic Mechanisms
Authors: Goossens Steven , Wang Jueqiong , Tremblay Cedric , Van Vlierberghe Pieter , Berx Geert , Taghon Tom , Haigh Jody J
Published: October 2019

ZEB2, a E-box binding homeobox transcription factor, is able to induce epithelial-to-mesenchymal transition in the context of solid tumors. As such, its expression is correlated with cancer cell invasion, dissemination and metastasis, but also with the acquisition of cancer stem cell properties and therapy resistance.

Using conditional knockout mice, we previously have shown that ZEB2 is an essential hematopoietic transcription factors, indispensable for normal embryonic(1) and adult(2) hematopoiesis. In addition, using conditional overexpressing mice we have demonstrated that ZEB2 is a bona fide driver of an immature subtype of T-ALL with increased leukemia-initiating potential(3).

Here, we investigated the effects of ZEB2 overexpression on normal T-cell differentiation. Already from young age, ZEB2-overexpressing mice have a smaller thymus, with increased percentages of early T-cell precursors. Detailed flow cytometric analysis indicate a T-cell differentiation delay at the beta-selection checkpoint, with accumulation of DN3 (CD4- CD8- CD44- CD25+) cells. Using in vitro differentiation assays on OP9-DL1 stromal cells, we demonstrate that the perturbed T-cell differentiation is independent of the thymic micro-environment. Lowering the dose of exogenous interleukin-7 levels in the culture media can partially rescue this phenotype, which suggests that ZEB2-overexpressing thymocytes are delayed in T-cell differentiation due to their inability to downregulate the interleukin-7 receptor (IL7R) levels.

Both the perturbed T-cell differentiation, and the latency/penetrance of immature subtype of the T-ALL formed in ZEB2-overexpressing mice is similar to what has been described for LMO2-overexpressing mice. Based on this phenocopy, we hypothesized that ZEB2 and LMO2 induce immature T-ALL via converging oncogenic pathway and/or via regulating similar crucial genes for the T-cell differentiation program. However, in contrast to LMO2, ZEB2-overexpresssing pre-leukemic thymocytes do not display aberrant self-renewal potential as demonstrated by transplantation experiments in sublethal irradiated syngeneic hosts. In addition, by using multiple Cre-transgenic lines, allowing the expression of ZEB2 at different stages of T-cell differentiation, we demonstrate that the oncogenic potential of ZEB2 is independent of the perturbed T-cell differentiation observed during the early stage of the disease.

Based on these results we conclude that ZEB2 and LMO2 both drive the development of an immature subtype of T-ALL, but via distinct oncogenic mechanisms.

(1) Goossens S. et al., Blood 2011 May 26; 117(21):5620-30.

(2) Li J. et al., Blood 2017 Jan 26; 129(4):460-472.

(3) Goossens S. et al., Nat Commun. 2015 Jan 7;6:5794.

Disclosures

No relevant conflicts of interest to declare.
Atomic-Level Dissection of the Polyclonal Immune Response to the Human Platelet Alloantigen, HPA-1a (PlA1)
Authors: Zhi Huiying , Ahlen Maria Therese , Thinn Aye Myat Myat , Weiler Hartmut , Curtis Brian R. , Skogen Bjorn , Zhu Jieqing , Newman Peter
Published: October 2019

Alloantibodies to platelet-specific antigens are responsible for two clinically-important bleeding disorders: Post-transfusion purpura and fetal/neonatal alloimmune thrombocytopenia (FNAIT). The HPA-1a/1b (also known as PlA1/A2) alloantigen system of human platelet membrane glycoprotein (GP)IIIa is controlled by a Leu33Pro polymorphism, and is responsible for ~80% of the cases of FNAIT. Local residues surrounding polymorphic residue 33 are suspected to have a profound effect on alloantibody binding and subsequent downstream effector events. To define the molecular requirements for HPA-1a alloantibody binding, we generated transgenic mice that expressed murine GPIIIa isoforms harboring select humanized residues within the PSI and EGF1 domain, and examined their ability to support the binding of a series of monoclonal and polyclonal HPA-1a-specific antibodies. Humanizing the PSI domain of murine GPIIIa was sufficient to recreate the HPA-1a epitope recognized by some HPA-1a-specific antibodies, however humanizing distinct amino acids within the linearly distant, but conformationally close, EGF1 domain was required to enable binding of others. Using a series of transgenic mice and recombinant constructs, we reveal previously unsuspected, complex heterogeneity of the polyclonal alloimmune response to this clinically important human platelet alloantigen system. High-resolution mapping of this alloimmune response may improve diagnosis of FNAIT by providing cells that distinguish specific alloantibody subpopulations, and should facilitate the rational design and selection of contemplated prophylactic and therapeutic anti-HPA-1a reagents.

Disclosures

Curtis: Ionis Pharmaceuticals: Consultancy.
The Clinical Significance of Tumor Associated Macrophages in Myelodysplastic Syndromes
Authors: Yang Yan , Wu Yu
Published: October 2019

Objective:

The purpose of this study is to investigate the relationship between the infiltration of tumor associated macrophages (TAMs) and the disease risk stratification and survival of patients with myelodysplastic syndrome (MDS) and to explore the role of TAMs in the clinical prognosis.

Methods:

We retrospectively collected and analyzed 115 patients initially diagnosed wih myelodysplastic syndromes from January, 2010 to July, 2017 in West China Hospital of Sichuan University. Both bone marrow biopsy specimens and clinical data of the patients enrolled were collected. All patients were assessed prognosis by international prognostic scoring system (IPSS). We quantified the involvement of macrophage (MΦ), alternatively activated macrophage (M2 MΦ) and classic activated macrophage (M1 MΦ) in bone marrow specimen by staining with anti-CD68 monoclonal antibody, anti-CD163 monoclonal antibody, and anti-iNOS monoclonal antibody respectively. Log-rank test was used to evaluate the difference of overall survival (OS) among different subgroups. Logistic regression was used to evaluate the effect of clinical parameters on patients' OS. Cox proportional-hazards models were used to estimate the independent risk factors influencing the prognosis of patients.

Results:

1 In the high risk group of MDS, the composition of CD163+MΦ was higher than that of the low risk group(p < 0.001). In the low risk group of MDS, the composition of iNOS+ MΦ was higher than that of the group with high risk(p < 0.001).

2 The median survival time and 3-year survival rate in the high CD163+MΦinfiltration group were significantly lower than those in the low infiltration group (13 months vs 43 months, 27% vs 64%, Log-rank test, P<0.001). The median survival time and 3-year survival rate in high iNOS+MΦ infiltration group were significantly higher than those in low infiltration group (42. 5 months vs 11 months, 65% vs 27% Log-rank test, P< 0.001).

3 According to the infiltration degree of iNOS+MΦ and CD163+MΦ, the M1 (iNOShigh/CD 163low)and M2 TA(iNOSlow/CD163high) MΦ were further distinguished (The median OS were 53m and 15m respectively, p< 0.001).

4 In multivariate analysis, high infiltration of CD163+MΦ and CD68+MΦ, low infiltration of iNOS+MΦ and low PLT level were independent risk factors for patients' OS .

Conclusion and Discuss:

The degree of bone marrow TAMs subtype infiltration in MDS patients was associated with disease risk group. CD163+M Φ was the main TAMs in the high risk group of MDS,while iNOS+M Φ was the main TAMs in the low risk group. There was a positive correlation between the infiltration of M1-TAMs and OS. On the contrary, there was a negative correlation between the infiltration of M2-TAMs and OS. Analyzing TAMs subsets in bone marrow of patients with MDS is helpful to evaluate disease risk and prognosis.

Disclosures

No relevant conflicts of interest to declare.
Intravenous Administration of Recombinant Factor VIII Induces Immune Tolerance to Subcutaneous Turoctocog Alfa Pegol (SC N8-GP) in Humanized Hemophilia A Mice
Authors: Reedtz-Runge Stine Louise , Weldingh Karin , Schiøler Schultz Heidi , Holm Millner Anders , Rode Frederik , Wiinberg Bo , Kjelgaard-Hansen Mads
Published: October 2019

Introduction

Turoctocog alfa pegol (N8-GP) is a glycoPEGylated recombinant factor VIII (rFVIII) under development for treatment of hemophilia A (HA). Patients with severe HA receiving intravenous (IV) replacement therapy have 20-40% risk of developing inhibitory FVIII antibodies. It is unclear whether the absence of inhibitors in some patients is due to lack of response or due to peripheral tolerance. Induction of peripheral tolerance may be dependent on regulatory T-cells (Tregs) since they are suggested to be involved in regulation of the anti-FVIII antibody response.

In this study, tolerance induction following IV rFVIII administration was assessed in humanized HA mice, described as a suitable model for tolerance induction as only a fraction produce anti-FVIII antibodies after IV FVIII exposure (Steinitz KN, et al. Blood 2012;119(17):4073-4082).

Aims

To explore whether IV exposure to rFVIII induces peripheral tolerance to subcutaneous N8-GP (SC N8-GP) exposure in humanized HA mice, and whether Tregs play a role in tolerance induction.

Methods

HLA-DR transgenic FVIII-deficient mice lacking murine MHC class II were treated with rFVIII IV once per week for 8 weeks, then challenged with SC N8-GP once a week for 8 weeks. The role of CD25+ Tregs was studied by treating one group of mice with depleting anti-CD25 antibodies during the IV exposure period.

The presence of anti-FVIII antibodies was measured prior to IV and SC dosing, and at study end. Mice with anti-FVIII antibodies after IV rFVIII exposure were excluded from the analysis.

Results

No anti-FVIII antibodies were detected after challenge with SC N8-GP in mice pretreated with IV rFVIII. Depletion of CD25+ Tregs (confirmed by flow cytometry) during the IV exposure did not significantly affect the anti-FVIII antibody response.

Conclusion

It was demonstrated that peripheral tolerance to SC N8-GP was induced in humanized HA mice not responding to IV rFVIII exposure. Depletion of CD25+ Tregs during IV exposure was insufficient to abolish the induction of tolerance.

Disclosures

Reedtz-Runge: Novo Nordisk A/S: Employment. Weldingh:Novo Nordisk A/S: Employment. Schiøler Schultz:Novo Nordisk A/S: Employment. Holm Millner:Novo Nordisk A/S: Employment. Rode:Novo Nordisk A/S: Employment. Wiinberg:Novo Nordisk A/S: Employment. Kjelgaard-Hansen:Novo Nordisk A/S: Employment.
Daratumumab for Delayed Red Cell Engraftment after Hematopoietic Stem Cell Transplant
Authors: Chapuy Claudia I , Kaufman Richard M , Alyea Edwin P. , Connors Jean M.
Published: October 2019

Allogeneic stem cell transplantation represents a curative treatment option for patients with malignant hematologic diseases, including myelodysplastic syndrome (MDS). In 25-50% of transplants, HLA-matched allogeneic stem cell donors have some degree of ABO incompatibility with the recipient. Major ABO incompatibility is caused by recipient antibodies directed against donor red blood cells (RBCs) and may lead to delayed RBC recovery and transient pure red cell aplasia (PRCA). We describe a case of treatment-refractory pure red cell aplasia following a major ABO-mismatchedallogeneic stem cell transplant successfully treated with daratumumab.

The 72-year-old patient with MDS received a matched unrelated donor transplant with a major ABO incompatibility, recipient blood group O from blood group A donor. Despite prompt engraftment with WBC and platelet recovery he remained anemic and RBC transfusion dependent. Bone marrow biopsy showed <1% erythroid elements concerning for post-transplant PRCA. 200 days post-transplant he had persistent circulating anti-A antibodies and no signs of red cell recovery. Standard treatments including tapering of immunosuppression, steroids, rituximab and erythropoiesis stimulating agents had no effect. The patient remained transfusion dependent with 1-2 units per week. Following treatment with daratumumab 390 days post transplant, he achieved transfusion-independence promptly within one week of treatment with daratumumab and continues to have normal erythropoiesis 10 months after daratumumab treatment. Anti-A antibodies became undetectable. The patient tolerated daratumumab well and no signs of GVHD or opportunistic infections were observed.

We describe the use of daratumumab in patients with PRCA after major ABO mismatch allogeneic stem cell transplant. We hypothesized that a selective treatment targeting the CD38 positive plasma cells would decrease anti-A antibody production and allow for RBC recovery. Our findings suggest that direct targeting of residual host plasma cells with an anti-CD38 targeted agent such as daratumumab might be a valid treatment option that needs to be considered for patients refractory to standard treatments.

Figure. Figure.

Disclosures

No relevant conflicts of interest to declare.