bluuebunny/crossref_metadata_2025 huggingface dataset:
Combination of Cord Blood T Regulatory Cells with Ruxolitinib Decreases Side Effects and Improves Survival in Xenogenic Graft Vs. Host Disease
Authors: Zeng Ke , Huang Meixian , Li Li , Lyu Mi-Ae , Ma Hongbing , Nishimoto Mitsutaka , Sadeghi Tara , Ahmed Sairah , Andersson Borje S. , Parmar Simrit
Published: November 2020

Background: Recent approval of ruxolitinib (rux) for steroid refractory graft versus host disease (GvHD) has revolutionized the field and provided tremendous choice for the patients. However, the side effects including thrombocytopenia leads to dug discontinuation and intolerance. We have previously shown that adoptive therapy with cord blood (CB) derived T regulatory (Treg) cells can prevent and treat GvHD. We hypothesized that the addition of CB Treg therapy to rux based therapy can augment overall efficacy.

Methods: CellTrace Violet suppression assay was performed to evaluate the suppressor function of CB Treg cells in the presence or absence of rux. Xenogenic GvHD mouse model was utilized where the NSG mice underwent sublethal irradiation on day -1 followed by injection of 1x107 donor peripheral blood (PB) mononuclear cells (MNCs) on day 0. Oral rux at 1 mg daily was fed continuously to the mice in the presence or absence of 1x107 CB Treg cells, tagged with CellTrace Violet dye, administered on days +4, +7, +11, +18. Mice were followed every other day for weight, GvHD score and survival. Serial blood draws were performed to analyze for cell compartment and cytokine assays.

Results: We examine whether the addition of rux impacts the suppressor function of CB Treg cells. Varying concentration of rux including 0.01, 0.05, 0.1 and 0.5 µM were added at 24, 48 and 96 hours of the cell suppression assay. The addition of rux 0.05 µM at 48 hours of the cell suppression culture led to a restoration of poor cell function (Figure A). Lowest GVHD score was reported in the rux+CB Treg combination arms at day +14 when compared to rux alone or CB Treg alone arm which (Figure B) translated into a superior survival in the rux +CB Treg arm (Figure C). Furthermore, an increase in the hemoglobin level (Figure D) and the platelet count (Figure E) was demonstrated in the rux+CB Treg arm.

Addition of rux led to longer persistence of the injected CB Treg cells on day 14 (data not shown) which correlated with the increase in the plasma level of the pro-Treg cell signaling markers including IL-7 (Figure F) and IL-15 (Figure G). A decrease in the IL-4 production supported the increased Treg cell function (Figure H). A synergistic suppression of inflammatory cytokines including IL-17 (Figure I) and IL1A (Figure J) was evident in the rux+CB Treg arm.

Conclusion: The combination of CB Tregs with ruxolitinib leads to improved overall survival, decreased inflammatory cytokines and improved hematologic parameters. Such combination should be explored in a clinical setting

Figure

Disclosures

Sadeghi: Cellenkos Inc.: Current Employment. Parmar:Cellenkos Inc.: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.
Traumatic Hyphema in a Type 1 Von Willebrand Patient: Clinical Case and Management
Authors: Lewis Jason , Dhami Jatinder , Langue Michael , Smink Gayle
Published: November 2020

Von Willebrand disease (vWD) is the most common inherited congenital bleeding disorder. Type 1 is a deficiency, but not absence, of von Willebrand factor (vWF) and is the most common type. Bleeding episodes in patients with type 1 vWD vary and are related to the relative amount of von Willebrand factor produced. We report a case of a 15-year-old male with mild von Willebrand disease type 1 (vWF: 35%) who presented with a right eye grade lll hyphema and elevated intraocular pressure (IOP) following a traumatic injury. Despite medical management with Diamox, ophthalmic steroids, vWF replacement with Humate-P, the patient had a repeat bleeding event and worsening of his elevated intraocular pressure. Due to concern for corneal blood staining, the patient required surgical intervention with an anterior chamber washout. Von Willebrand factor replacement was given on presentation and prior to the procedure with goal von Willebrand factor (vWF) activity and Factor Vlll (FVlll) levels of 80-100 IU/dL. Factor replacement for a traumatic hyphema is necessary to reduce bleeding, but could lead to increased risk of thrombosis formation and increase risk of corneal staining. Balance between bleeding and thrombosis can lead to treatment challenges in patients with bleeding disorders including vWD.

Disclosures

Smink: Forma Therapeutics:Membership on an entity's Board of Directors or advisory committees;Highmark Insurance:Membership on an entity's Board of Directors or advisory committees.
Efficacy of Isatuximab on Surrogate Endpoints in Multiple Myeloma; A Systematic Review
Authors: Ammad Ud Din Mohammad , Hussain Syed Ather , Farrukh Hizra , Pasha Fajar , Abdullah Syed Maaz , Waris Imtisaal , Rehman Ebad , Setarehaseman Alireza , Ullah Asmat , Anwer Faiz
Published: November 2020

Introduction:

Isatuximab (ISA) is an anti-CD38 monoclonal antibody that has shown benefit in patients with relapsed/refractory multiple myeloma (RRMM) in combination regimens. This study focuses on the clinical utility and tolerability of ISA based therapies in patients with ISS stage III disease.

Methods:

Literature search on PubMed, EmCare, Clinicaltrials.gov, Cochrane, and Google Scholar was performed from inception to July 1, 2020, for the use of ISA in MM patients. A total of five clinical trials, including phase Ib/II and phase III trials, were selected for a systematic review out of 109 studies (n=556 patients).

Results:

The median age was 61-71 years. The addition of ISA to pomalidomide-dexamethasone (PD) in previously treated patients was evaluated by Attal et al. (2019). International Staging System (ISS) stage III disease was present in 28% of the study population at baseline. Patients treated with ISA-PD had a meaningful increase in progression-free survival (PFS) (11.53 versus 6.47 months, P = 0.001, Hazard Ratio (HR): 0.596; 95% confidence interval (CI): 0.436-0.814. PFS benefit was maintained in subgroup analysis based on renal involvement, refractoriness to lenalidomide, and proteasome inhibitors. However, PFS benefit was not statistically significant in ISS stage III disease (HR: 0.64; CI: 0·36-1·11). Martin et al. (2017) evaluated the addition of ISA to lenalidomide and dexamethasone in 57 patients. 22% of the patients had ISS stage III disease. Mikeal et al. also included 9% and 37.1% of patients with ISS stage III MM in their phase Ib and II trials, respectively. All studies demonstrated a significant prolongation of PFS in patients refractory to lenalidomide with ISA administered at 10 mg/kg weekly/every two weeks. Iida et al. (2019) also reported statistically significant improvement in PFS with ISA monotherapy in heavily pretreated Japanese patients (PFS 4.7 months; 95% CI 3.75-NC). In patients with newly diagnosed MM, Ocio et al. (2017) showed encouraging results with an ISA based regimen in 17 patients. ISS stage III disease was present in 88% of the population. At the time of data cut-off, the overall response to treatment was 87%, and 82.4% continued to remain on treatment (see Table 1).

Conclusion:

ISA as monotherapy and in combination therapy improved surrogate endpoints without increased toxicity in patients refractory to previous lines of treatment. Limited data suggest an improvement in PFS for ISS stage III disease. Long-term results on efficacy in MM with high-risk cytogenetics are awaited.

Disclosures

Anwer: Incyte, Seattle Genetics, Acetylon Pharmaceuticals, AbbVie Pharma, Astellas Pharma, Celegene, Millennium Pharmaceuticals.: Honoraria, Research Funding, Speakers Bureau.
Photodynamic Therapy in Refractory Mycosis Fungoides: A Prospective, Open-Label Study
Authors: Brumfiel Caitlin M , Severson Kevin J , Patel Meera H , Cumsky Helen J L , Ginos Brenda F , Kosiorek Heidi E. , Janeczek Monica C , Besch-Stokes Jake , Rule William , DiCaudo David , Rosenthal Allison C. , Pittelkow Mark R , Mangold Aaron R
Published: November 2020

Background:Case reports suggest photodynamic therapy (PDT) with 5-aminolevulinic acid (ALA) is an effective treatment for refractory mycosis fungoides (MF). No prospective trials have examined the use of ALA-PDT in MF.

Objective:We aimed to assess the efficacy of ALA-PDT in refractory MF.

Methods:This was a prospective study at Mayo Clinic in Scottsdale, Arizona in patients ≥ 18 years with plaque-stage treatment-refractory MF. Monthly sessions of ALA with blue light PDT were administered for up to six months. Responses were measured by Composite Assessment of Index Lesion Severity (CAILS) and Physician Global Assessment (PGA). Ad hoc analysis with modified CAILS (mCAILS) was performed, eliminating hyperpigmentation from scoring.

Results:Eleven patients (30 total lesions) were treated. Six patients completed the trial per protocol. Objective response rates were 36.4% by PGA (10.9-69.2%), 18.2% by CAILS (2.3-51.8%), and 36.4% by mCAILS (10.9-69.2%).

Limitations:Five patients did not complete all six PDT cycles per protocol, possibly accounting for reduced efficacy.

Conclusion:ALA-PDT with blue light irradiation was moderately effective and well tolerated in refractory plaque stage MF. Lower response rates compared to similar trials may be partially explained by utilization of strict lesional assessment criteria and other variations in methodology.

Disclosures

Mangold: MiRagen:Research Funding;Sun Pharma:Research Funding;Elorac:Research Funding;Kirin:Membership on an entity's Board of Directors or advisory committees;Solagenix:Research Funding.

OffLabel Disclosure:

Photodynamic therapy is a two-step therapy in which a drug that acts as a photosensitizer is administered to target a diseased tissue, followed by illumination with visible light to activate the drug and destroy the target tissue. PDT has been used to treat internal cancers as well as treatment of cancer and precancer of the skin.
DCE-MRI for Detection of Acute Myeloid Leukemia Derived Bone Marrow Vascular Dysfunction
Authors: Da Silva Gomes Ana , Siow Bernard , Passaro Diana , Bonnet Dominique
Published: November 2020

Acute Myeloid Leukemia (AML) is the most common acute leukemia in adults. While the clinical presentation is quite uniform, it is a highly heterogeneous disease at the genetic level. Using intravital two-photon microscopy in AML xenograft models, we have previously showed that AML patient-derived samples induced a common pathologic bone marrow (BM) vascular phenotype. To understand the translational potential of our findings we have optimized dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) for the assessment of BM vascular dysfunction. We quantified non-model based parameters from DCE-MRI scans: Contrast Enhancement (CE); Wash-in Rate (WiR); and Wash-out Rate (WoR). CE reflects tissue vascular density and is a measurement of the proportion of fully functional vessels per pixel area, WIR reflects blood flow and tissue perfusion, and WoR reflects vascular permeability and tissue clearance capacity. We first measured DCE parameters in control mice. We observed no gender-related differences in BM DCE parameters, while we found significant age-related changes. As mice age, BM vascular density decreases together with increased vascular permeability and reduced tissue clearance capacity. When compared to age- matched controls, mice injected with AML cell lines showed decreased vascular density, decreased blood inflow, increased vascular permeability and reduced tissue clearance capacity. There was a progressive decline in BM vascular function as the disease burden increased, particularly in WiR and WoR parameters, with even low levels of leukemia significantly altering BM vascular function. When analyzing mice injected with AML patient samples, changes in BM vascular function were more dependent on the AML sample than on the engraftment. Low disease burden did not significantly change DCE vascular parameters. At high disease burden, patient-derived xenografts show different degrees of vascular disruption, spanning from global altered DCE parameters to milder phenotypes. Upon AraC treatment, altered vascular parameters remained unchanged in most of the cases, suggesting inability to fully rescue BM vascular function. In conclusion, we showed that DCE-MRI is able to detect consistent changes associated with BM vasculature induced by aging and leukemia. Despite the heterogeneous nature of the disease, our method captures different degrees of vascular alterations, with WIR having the highest diagnostic potential. Our results further suggest that the detection of a pathologic vascular phenotype in the BM of AML patients could be of use in the clinical scenario.

Disclosures

No relevant conflicts of interest to declare.
Fiirst-2: Prospective, Randomized Study Comparing Administration of Clotting Factor Concentrates with Standard Massive Hemorrhage Protocol in Severely Bleeding Trauma Patients
Authors: Da Luz Luis , Callum Jeannie L , Beckett Andrew , Peng Henry T , Engels Paul , Parry Neil , Tien Homer , Nathens Avery , Schwartz Bruce A. , Karkouti Keyvan
Published: November 2020

Introduction: Acute trauma coagulopathy (ATC) is associated with severe hemorrhage. ATC etiology is often multifactorial, with acquired fibrinogen deficiency and consumption of clotting factors recognized as major factors. The FiiRST-2 study will determine the impact of early co-administration of fibrinogen concentrate (FC) and prothrombin complex concentrate (PCC) on the total number of allogeneic blood products (ABPs) transfused compared to the current standard of care (ratio-based plasma resuscitation).

Methods: FiiRST-2 is a multicenter (8 Canadian centers), pragmatic, randomized, parallel-control, superiority trial with a two-armed, two-stage design with an adaptive interim analysis. Research ethics board approval has been obtained and the study complies with the Declaration of Helsinki. Trauma patients >16 years old at risk of massive hemorrhage will be randomized to receive FC + PCC or standard of care (ratio-based plasma resuscitation + FC in response to low fibrinogen levels) (Table 1) until all units in the second massive hemorrhage protocol (MHP) pack have been administered, or earlier if the MHP is terminated. The primary endpoint is to demonstrate superiority with respect to the number of ABP units (RBCs [red blood cells] + plasma + platelets) transfused within 24 hours of arrival at the trauma bay/emergency department. Secondary endpoints include RBC units transfused within 24 hours, incidence of thromboembolic events, and ventilator-free days up to Day 28. Safety endpoints include all adverse events (AEs) and serious adverse events (SAEs) up to Day 28. It is projected to enroll 360 patients depending on the results of the Interim analysis.

Results: An interim analysis will be performed after 120 patients have completed the study. The study is expected to start late 2020 and enrollment is expected to require about 2 years.

Conclusions: FiiRST-2 will determine if early hemostatic therapy with FC + PCC is superior to standard MHP packs in bleeding trauma patients. Results could have a major impact on clinical practice and improve management and outcomes in this high-risk group of patients.

Disclosures

Da Luz: Octapharma Canada: Research Funding. Callum:Octapharma: Research Funding; Canadian Blood Services: Research Funding. Schwartz:Octapharma: Current Employment. Karkouti:Canadian blood services: Research Funding; Octapharma: Research Funding.

OffLabel Disclosure:

PCC and fibrinogen concentrate for acquired coagulopathic bleeding (off-label in the USA)
Hematopoietic Jagged1 Is Required for the Transition of Hematopoietic Stem Cells from the Fetal Liver to the Adult Bone Marrow Niche
Authors: Shao Lijian , Paik Na Yoon , Pajcini Kostandin V.
Published: November 2020

Notch signaling is known to play important roles in hematopoietic development and differentiation. Notch1 is required for emergence of the definitive hematopoietic stem cells (HSCs) from the hemogenic endothelium, and we have previously shown that Notch signaling is essential for survival and function of HSCs in the fetal liver. Activation of canonical Notch signaling requires direct cellular contact; thus, the identity of the ligand and the ligand-presenting cell during hematopoietic development would provide valuable information of the Notch signaling mechanism in HSCs as well as the identity of key niche cells that drive the expansion and cell fate decisions of embryonic HSCs. In the present study, we have taken a comprehensive approach to determine the ligands and cells that initiate Notch signaling in the mouse fetal liver. To this end, we have performed single-cell PCR analysis for all Notch signaling proteins in E14.5 fetal HSCs and compared the findings to the adult bone marrow HSCs. We also have analyzed fetal liver endothelial cells for surface expression of all Notch ligands. We determined that Jagged1 (Jag1) is highly expressed in both endothelial cells as well as in fetal HSCs but not adult HSCs. We have performed conditional loss-of-function analysis of Jag1 in fetal endothelial cells using inducible Ve-cadherinCreERT2 as well as in fetal hematopoietic lineages using constitutive VavCre. Our results indicate that while loss of endothelial Jag1 has severe effects in embryonic vascular development, loss of hematopoietic Jag1 allows for normal fetal morphology, yet severely impedes the functional ability of fetal liver HSCs to expand and differentiate both in vitro and in vivo. Fetal to adult transplantation of VavCre+Jag1f/f HSCs indicated a defect in reconstitution potential of fetal HSCs that lack Jag1 expression. Our findings indicate that hematopoietic Jag1 is essential for maturation of HSCs in the fetal liver and for homing and reconstitution potential of HSCs into the post-natal bone marrow microenvironment.

Disclosures

No relevant conflicts of interest to declare.
Development and Validation of a Risk Assessment Model for Early Infection during the First 3 Months in Patients with Newly Diagnosed Multiple Myeloma
Authors: Shang Yufeng , Wang Weida , Liu Minghui , Chen Xiaoqin , Xia Zhongjun , Zhou Fuling
Published: November 2020

PurposeEarly infection was an important cause of mortality in patients with multiple myeloma (MM). The study aimed to assess factors affecting early infection and identify patients with high risk developing infection.

MethodsDuring January 2010 to June 2019, patients with MM were analyzed, retrospectively. The data was divided into training and independent validation cohort. The least absolute shrinkage and selection operator (LASSO) regression model was used for data dimension reduction, feature selection, and model building.

ResultsOf 745 confirmed MM patients, 540 eligible cases were included in final analyses. In total, 165 patients (30.6%) suffered infections, while 110 patients (20.4%) occurred early infections during the first 3 months after diagnosis. Bacteria and the respiratory tract were the most common pathogen and localization of infection, respectively. In training cohort, PS≥2, HGB<100g/L, β2MG≥6.0mg/L and GLB≥80g/L were identified associated with early infections by LASSO regression. Based on the four factors, an early infection risk model of MM (IRMM) was established to define high- and low-risk groups, which showed significantly different rates of infection (35.3% vs. 9.4%,P<0.001, HR=4.381 [95% CI, 2.802-7.221]). IRMM displayed good discrimination (AUC=0.756) and calibration (P=0.94).

ConclusionWe determined risk factors for early infection and established a predictive model to help clinicians identify patients with high-risk infection. It can help clinicians to determine whether to adjust monitoring and treatment strategies, or apply prophylactic interventions to high-risk patients.

Disclosures

No relevant conflicts of interest to declare.
Selectively and Efficiently Expanding Natural Killer Cells in Vitro
Authors: Lu Xuzhang , Liu Jie , Zhuxia Jia , Ouyang Xiaoqing , Chen Baoan
Published: November 2020

Natural killer (NK) cells are the main innate immune cells, and have powerful antitumor activity in vitro, but NK cells based immunotherapy does not often get satisfactory results in clinical practice due to insufficient number and poor activity of NK cells.. Therefore, how to improve expansion efficiency and killing activity of NK cells in vitro is critical. Here we report an in vitro culture system culturing peripheral blood mononuclear cells (PBMC) with irradiated LCL as feeder cells in 10%FBS/RPMI1640 culture medium in the presence of 100U/ml IL-2 and 5ng/ml IL-15 for 14 days could selectively expand NK cells. With this culture system, the purity of NK cells increased from 11.5%± 7.01% to 75.3±10.9% (P< 0.01), and the expansion efficiency was about 625±63 times than the original PBMC. Compared with the fresh NK cells isolated from PBMC, there was no significant change in the expression of inhibitory receptors on the surface of expanded NK cells, while the expression of activating receptors was significantly increased (p<0.05). The cytotoxicity of expanded NK cells against leukemia cell line K562 was significantly higher than that of freshly isolated NK cells (72± 5.6% vs 8.7±2.3%, E: T=1, P<0.01), expanded NK cells also showed stronger cytotoxicity to LCL (22±3.6%, E: T=1) and primary leukemia cells (42±5.2%, E:T=1). In summary, we could selectively expand NK cells with this in vitro culture system, the expanded NK cells gained higher expression of NCRs and NKG2D receptors, and displayed greater cytotoxicity against tumor cells, therefore could be readily applied to anti-tumor treatment in clinical treatment.

Figure

Disclosures

No relevant conflicts of interest to declare.
Frequency of Venous Thromboembolism in 6513 Patients with COVID-19: A Retrospective Study
Authors: Crowther Mark , Garcia David A. , Hill Jason , Savage Bryan , Peress Shira , Chang Kevin , Deitelzweig Steven
Published: November 2020

Background: Patients infected with coronavirus 2 (SARS-CoV-2) appear to be at increased risk for venous thromboembolism (VTE), especially if they become critically ill with coronavirus disease 2019 (COVID-19). Some centers have reported very high rates of thrombosis despite anticoagulant prophylaxis.

Methods: The electronic health record (EHR) of a New Orleans-based health system was searched for all patients with PCR-confirmed SARS-CoV-2 infection who were either admitted to hospital or treated and discharged from an emergency department between March 1 and May 1, 2020. From this cohort, patients with confirmed VTE (either during or after their hospital encounter) were identified by administrative query of the EHR.

Results: Between March 1, 2020 and May 1, 2020, 6,153 patients with COVID-19 were identified; 2,748 of these patients were admitted, while 3,405 received care exclusively through the emergency department. Data on patient outcomes were determined up until and including May 21, 2020. In total, 637 patients required mechanical ventilation and 206 required renal replacement therapy. Within the hospitalized cohort, the overall mortality rate was 24.5% and VTE occurred in 86 patients (3.1%). In the 637 patients who required mechanical ventilation at some point during their hospital stay, 45 developed VTE (7.2%). After a median follow-up of 14.6 days, VTE had been diagnosed in 3 of the 2,075 admitted who were discharged alive (0.14%).

Conclusions: Among 6,153 patients with COVID-19 who were hospitalized or treated in emergency departments, we did not find evidence of unusually high VTE risk. Pending further evidence from prospective, controlled trials, our findings support a traditional approach to primary VTE prevention in patients with COVID-19.

Disclosures

Crowther: Precision Biologicals: Membership on an entity's Board of Directors or advisory committees; Hemostais Reference Laboratories: Honoraria; Pfizer: Speakers Bureau; CSL Behring: Speakers Bureau; Alnylam: Divested equity in a private or publicly-traded company in the past 24 months; Servier Canada: Membership on an entity's Board of Directors or advisory committees; Diagnostica Stago: Speakers Bureau; Asahi Kasei: Membership on an entity's Board of Directors or advisory committees.
Epidemiological and Survival Analysis of Primary Intestinal Non-Hodgkin's Lymphoma Patients from SEER Database
Authors: Singh Vinit , Agrawal Pooja , Gupta Varsha , Prakash Amulya , Du Doantrang T.
Published: November 2020

Introduction: Gastrointestinal tract is the most common site of extranodal non-Hodgkin's lymphoma (EN-NHL). Most of the data published so far has been on the gastric NHL and very limited data is available on primary intestinal - Non-Hodgkin's Lymphoma (PI-NHL). Here, we are presenting the epidemiological and survival data for PI-NHL from the SEER 21 database.

Methods: Data for all the EN-NHL and Intestinal NHL are collected for the period 2000-2015 from the SEER 21 database based on the WHO ICD-O3 classification. PI-NHL data has been extracted from EN- NHL and intestinal datasets and then cross-matched for accuracy of the desired patient subset selection. A total of 9,290 PI-NHL cases common to both the lists were selected and used for analysis. Patients with incomplete staging and survival data were excluded from the survival analysis. Survival analysis variables include gender (male or female), ethnicity (white or non-white), early age of onset (≤ 50 years), late age of onset (>50 years), location (small or large intestine), the staging of the tumor as early-stage (stage 1 and stage 2) or late-stage tumors (stage 3 and stage 4), and history of prior malignancy (first primary tumor or second/later primary tumor). Survival analysis is done using a cox-proportional hazard regression model.

Results: The percentage of PI-NHL of all the intestinal cancers and extranodal non-Hodgkin's lymphoma is 1.42% (1.40 - 1.45, 95% CI) and 10.52% (10.32 - 10.72, 95% CI) respectively. The demographic and clinical characteristics of the patients are described in Figure 1. In the survival analysis (Figure 2), the risk of overall mortality is higher in the late-onset cancers (HR - 1.16, 1.08 -1.24, P-value<0.001), non-white population (HR- 1.13, 1.05 - 1.22, P-value 0.002) and second or later primary tumors (HR - 1.10, 1.01 - 1.20, P-Value - 0.036). Gender, site, and stage are not significantly related to mortality but the trend towards higher mortality is seen in the late-stage tumor (HR - 1.04, 0.97 - 1.12, P-Value - 0.29).

Conclusion: PL-NHL is a rare type of intestinal malignancy. Our study showed that the risk of overall mortality is higher in late-onset cancer, non-white population, and in second or later primary tumors. Gender, site, and stage are not significantly related to mortality. The analysis was done for overall survival and this may have affected the outcome of the survival variables. Therefore, a survival analysis of cause-specific mortality data will give a more elaborate picture of the relationship between the mortality data and these variables.

Disclosures

No relevant conflicts of interest to declare.
Sickle Cell Disease Model Mice Lacking 2,3-Dpg Show Reduced RBC Sickling and Improvements in Markers of Hemolytic Anemia
Authors: Knee Kelly M. , Barakat Amey , Tomlinson Lindsay , Ramaiah Lila , Wenzel Zane , Kapinos Brendon , Ahn Youngwook , Lintner Nathanael G. , Field S. Denise , Jasuja Reema , Pettersen Betty A.
Published: November 2020

Sickle cell disease (SCD) is a severe genetic disorder caused by a mutation in hemoglobin (b6Glu-Val), which allows the mutant hemoglobin to assemble into long polymers when deoxygenated. Over time, these polymers build up and deform red blood cells, leading to hemolytic anemia, vaso-occlusion, and end organ damage. A number of recent therapies for SCD have focused on modulating the mutant hemoglobin directly, however, reduction or elimination of 2,3-DPG to reduce Hb S polymerization and RBC sickling has recently been proposed as a therapeutic strategy for SCD. Current clinical studies focus on activation of pyruvate kinase to reduce 2,3-DPG, however, direct targeting of the enzyme which produces 2,3-DPG; Bisphosphoglycerate Mutase (BPGM) may also be possible.

In this study we evaluate the impact of elimination of 2,3-DPG on SCD pathology by complete knockout of BPGM in Townes model mice. Animals with complete knockout of BPGM (BPGM -/-) have no detectable 2,3-DPG, while animals that are heterozygous for BPGM (BPGM -/+) have 2,3-DPG levels comparable to Townes mice. Western Blot analysis confirms that BPGM -/- animals completely lack BPGM, while BPGM -/+ animals have BPGM levels that are nearly equivalent to Townes mice. As expected from the lack of 2,3-DPG, BPGM -/- animals have increased oxygen affinity, observed as a 39% decrease in p50 relative to Townes mice.

Complete elimination of 2,3-DPG has significant effects on markers of hemolytic anemia in BPGM -/- mice. Mice lacking 2,3-DPG have a 60% increase in hemoglobin (3.7 g/dL), a 53% increase in red blood cell count, and a 29% increase in hematocrit relative to Townes mice. The BPGM -/- mice also have a 57% decrease in reticulocytes, and a 61% decrease in spleen weight relative to Townes animals, consistent with decreased extramedullary hematopoiesis. Consistent with the reduction in hemolysis, BPGM -/- animals had a 59% reduction in red blood cell sickling under robust hypoxic conditions. BPGM -/+ animals had hemoglobin, RBC, and hematocrit levels that were similar to Townes animals, and a similar degree of RBC sickling to Townes mice. Liver phenotype was similar across all variants, with areas of random necrosis observed in BPGM -/-, BPGM -/+ and Townes mice. Higher percentages of microcytic and/or hyperchromic RBCs were observed in BPGM -/- animals relative to BPGM -/+ or Townes animals.

These results suggest that modulation of 2,3-DPG has a positive effect on RBC sickling and hemolytic anemia, which may have therapeutic benefits for SCD patients. However, the lack of improvement in organ damage suggests that modulation of 2,3-DPG alone may not be sufficient for complete elimination of SCD phenotypes, and further investigation of this therapeutic avenue may be necessary.

Disclosures

No relevant conflicts of interest to declare.
Bclw Overexpression Predicts Aggressive Disease in B-Cell Lymphomas
Authors: McBride Amanda , Adams Clare M , Mitra Ramkrishna , Eischen Christine M
Published: November 2020

B-cell lymphomas encompass a phenotypically and genetically heterogenous subgroup within hematologic malignancies. Despite this heterogeneity, the ability to evade apoptosis is a unifying feature across B-cell lymphomas, and alterations within the Bcl-2 family of apoptosis regulatory proteins is a key mechanism for this evasion. While overexpression of the anti-apoptotic Bcl-2 family member BCL2 has been widely described in multiple B-cell lymphomas, altered expression of other anti-apoptotic proteins within this family, including BCLX, MCL1, A1 and, in particular, BCLW has been under-investigated. This is highlighted by clinical trials of the BCL2 inhibitor, venetoclax, which found poor single agent activity in B-cell lymphomas that were thought to be highly BCL2-dependent. Therefore, we investigated the contributions of anti-apoptotic Bcl-2 proteins in B-cell lymphomas. Analysis of gene expression profiling datasets of patient samples of major B-cell lymphoma subtypes as compared to normal B-cells demonstrated differential overexpression of anti-apoptotic Bcl-2 proteins, with BCL2, BCLX and BCLW being significantly overexpressed in multiple B-cell lymphoma subtypes. Focusing on aggressive B-cell lymphoma subtypes, BCLW was consistently overexpressed across all major B-cell lymphoma subtypes. Notably, BCLW overexpression was maintained in high grade follicular lymphoma compared to low grade, whereas BCL2 levels decreased as grade increased. These findings indicate that BCLW overexpression is selected for in more aggressive B-cell lymphoma subtypes. BCLW overexpression in aggressive B-cell lymphoma cell lines conferred resistance to the non-specific BH3 mimetic navitoclax as well as to venetoclax. Further, analysis of diffuse large B-cell lymphoma (DLBCL) cell lines demonstrated that the combination of high BCLW/low BCL2 in GCB DLBCL and high BCLW/moderate BCL2 in non-GCB DLBCL is also associated with decreased navitoclax sensitivity, suggesting that high BCLW may confer treatment resistance. In accordance with this, analysis of patient outcomes in DLBCL demonstrates that patients exhibiting high BCLW levels with low BCL2 levels have poorer survival than those with low BCLW. Moreover, Cox regression analysis showed BCLW was an independent determinant of overall survival in DLBCL. Together, our results indicate that BCLW predicts for more aggressive disease in B-cell lymphomas and increased resistance to therapy.

Disclosures

Eischen: AbbVie Inc.:Research Funding.
COVID-19 in Relation to Hematological Events (Thrombosis/Bleeding) and Mortality in Inpatient Setting
Authors: Gadde Saikrishna , Manjunath Nikhil , Badari Ambuga , Cole John T. , Saeed Rula
Published: November 2020

COVID-19 is a newly diagnosed entity that has been identified for about 6 months. With the US now being the prevalent nation affected, we aimed to identify factors that were relevant to survival and hematological events in COVID patients. Our group had primarily been assessing COVID-19 confirmed patients as per PCR at Ochsner Medical Center in Louisiana; we primarily studied patients who were admitted between January 2020 to May 2020. Our main events that are currently being studied include factors such as mortality, length of stay, and bleeding or thrombotic events. Secondary endpoints included factors such as influence on blood type and also amount of transfusions required by these patients.

Our overall analysis of 1773 patients yielded a number of 60 patients who were able to identify as having an active cancer diagnosis. This disproportionate number of patients may be related to factors such as reluctance to visit the hospital among cancer patients, a phenomenon noted similarly in Cardiology where the number of admissions for cardiac causes had declined suddenly during this pandemic timeline. Our half of our population was African American and included an even proportion of males and females. The age range of patients was between 35-93 years old.

The most common malignancies that were noted to co-occur with COVID-19 in our population seemed to be lung, prostate, and hematological malignancies. Although final analyses regarding mortality are to be completed, the proportion of patients who had died with a diagnosis of cancer was around 50% per collection of our current data. Twenty three out of 60 (38%) patients required blood transfusions. In comparison to other reports which had reported bleeding events and thrombotic events, our report had revealed a much less rate of bleeding events (5/60) and thrombotic events (5/60). We plan to repeat our analysis to assess for any confounders in identifying these events, as noted, since some earlier literature had reported between ¼ to ⅔ of patients having a thrombotic event.

Our analysis also looks at other descriptive variables such as use of anticoagulant and antiplatelet agents, absolute counts of neutrophils and lymphocytes, platelets, and coagulation markers. Also, our assessment includes a study in assessment of any delays noted in chemotherapy dates for these patients. Analysis of assessment of bleeding, thrombosis, and mortality for the entire patient group is currently in process.

Disclosures

No relevant conflicts of interest to declare.
A Novel Assay Using Enzyme Capture - ELISA for Accurate Determination of Factor XIII Activity
Authors: Waller Amanda P. , Durda Michael A. , Franz Kennedy , Abdelghani Eman , Kerlin Bryce A.
Published: November 2020

Background: The currently available, clinically approved factor XIII (fXIII) activity assays rely on indirect measurement of activated fXIII transglutaminase activity by detecting "ammonia-release" during an intermediate step of the reaction. Unfortunately, this assay is also sensitive to fXIII-independent ammonia-producing reactions that may take place physiologically in plasma, rendering it prone to overestimate fXIII activity, especially when levels are <10% (0.1 IU/mL). High quality quantitative fXIII activity assays are required for the accurate and timely diagnosis of fXIII deficiency. fXIII circulates in complex with fibrinogen, and activated fXIII retards fibrinolysis by incorporating α2-antiplasmin (α2AP), a potent antifibrinolytic, into the forming clot via intermolecular α2AP:fibrin crosslinks. We have thus developed a new fXIII transglutaminase activity assay that directly measures α2-AP incorporation onto captured fibrin(ogen):fXIII complexes. We hypothesized that this novel assay has high sensitivity in detecting FXIII activity, especially at the low end of the dynamic range (<10%; <0.01 IU/mL).

Methods: A standard curve was established by diluting known concentrations of recombinant factor XIII-A (rFXIII-A; Tretten) into (1) fXIII-A congenitally deficient and (2) fXIII immunodepleted plasma to determine if our assay is able to detect variable fXIII concentrations from 1 to 200% (0.01 - 2 IU/mL).

Results: We demonstrated that α2-AP incorporation is dependent upon rfXIII-A concentration in the starting sample and was linear from 1-12% (0.01 - 0.12 IU/mL; Figure). Further expansion of the standard curve to include all concentrations within the normal physiologic range demonstrated that the assay remains strongly linear from 1-20% (0.01 - 0.2 IU/mL; R2 0.966). Moderate linearity (R2 0.908) extended to 50% (0.5 IU/mL). The curve plateaued between 50%-200% (0.5 IU/mL- 2 IU/mL). Thus, the dynamic range of the Enzyme Capture-ELISA is expected within 1-50% (0.01 - 0.5 IU/mL).

Conclusion: We conclude that Enzyme Capture-ELISA is a promising novel method for accurate detection of FXIII activity and is expected to improve sensitivity at low levels (<20%; <0.02 IU/mL), which are most clinically relevant. Further experiments are needed to refine the upper limit of assay linearity and thereby refine the dynamic range of the assay. With further optimization and validation, the EC-ELISA method may provide an improved diagnostic assay.

Disclosures

No relevant conflicts of interest to declare.
Diagnostic Utility of Targeted Next Generation Sequencing in Patients with Vascular Anomalies
Authors: Eng Whitney , Dilek Sophie , Ward Abigail , Kozakewich Harry PW , Al-Ibraheemi Alyaa , Janeway Katherine , Adams Denise M , Church Alanna
Published: November 2020

Background: Vascular anomalies are diverse entities and can range in severity from self-limiting to life-threatening. Diagnosis and care of these patients is challenging due to overlapping clinical and histologic features. Recently, it has been established that many vascular anomalies arise from somatic mutations in cancer genes (PIK3CA, AKT, NRAS). Use of cancer genomics in patients with vascular anomalies may establish a genetic diagnosis and expand use of targeted medical therapies. We evaluated the utility of targeted next generation sequencing for vascular anomalies patients at a single pediatric center.

Methods: Using OncoPanel, a hybrid-capture and massively parallel sequencing assay that surveys DNA sequences of 447 genes implicated in cancer, we analyzed genetic variants in lesional tissue from vascular anomalies patients evaluated at Boston Children's Hospital between 5/2/2017 and 3/23/2020.

Results: A total of 276 patients were consented and sequenced under the Dana Farber Cancer Institute Profile protocols DFCI 11-104 (n= 68) and DFCI 17-000 (n= 208). Clinical diagnoses prior to testing were varied and 11 patients (7%) had an unknown diagnosis. Tissue was analyzed for 138 patients. Targeted sequencing resulted in diagnostically significant alterations in 80 of 138 (57%) of patients and therapeutically significant alterations in 58 of 138 (42%) patients. To date, 18 patients in our cohort have been treated with medical therapy informed by their genetic diagnosis. Several more await enrollment on clinical trials. For patients with diagnoses previously categorized as unknown (n=11), sequencing led to identification of a genetic variant in 6 patients (54%). Additionally, 8/138 patients had variants requiring further evaluation for potential germline involvement.

Discussion: Next generation sequencing in vascular anomalies patients identified actionable variants in a large proportion of the patients in our cohort. The mTOR inhibitor sirolimus has been used to treat a variety of vascular anomalies, but not all patients respond to this treatment. Targeted therapies based on specific genotypes hold promise as clinical trials in vascular anomalies are emerging. Additionally, sequencing in this cohort identified several variants suggesting a germline cancer predisposition requiring follow-up. Use of next generation sequencing has clinical utility and increased use of this testing may improve diagnosis, prognosis, and treatment for patients with vascular anomalies.

Disclosures

Adams: Novartis: Consultancy; Venthura: Consultancy.

OffLabel Disclosure:

Sirolimus is used off-label for the treatment of vascular anomalies.
An Improved Animal Model of Multiple Myeloma Bone Disease
Authors: Mehdi Syed , Zangari Maurizio , Yong Donghoon
Published: November 2020

Multiple myeloma (MM) is a plasma cell malignancy that represents an accumulation of terminally differentiated monoclonal plasma cells (PCs) in the bone marrow (BM), accompanied by increased osteoclasts and decreased osteoblasts in areas adjacent to myeloma cells, leading to MM associated bone disease (MMBD). Osteolytic bone disease is one of the defining features of MM. During the disease, over 90% of patients are developing MMBD. Many of MM animal models have been developed and enable us to interrogate the mechanisms of MM tumorigenesis. Most MMBD models were derived by intratibial injection of myeloma cells. In these models, osteolysis occurs locally at the site where myeloma cells were injected. Mouse myeloma cells, 5TGM1 transplanting C57BL/KaLwRij mouse via the tail vein develops and shows MMBD features close to human MMBD. Even in this model, the MMBD levels on each mouse are widely varied. Lack of appropriate in vivo MMBD model hampers our understanding of the disease and developing therapy. We try to establish a murine model for MMBD to study its pathophysiology and test a novel treatment.

1x106 luciferase-expressing 5TGM1 (5TGM1-Luc) cells were injected into 8-12 week old NOD SCID gamma mouse (NSG) and C57BL/KaLwRij mouse via the tail vein. Myeloma progression was weekly assessed by in vivo bioluminescence (BL) imaging using IVIS-200 (Perkin Elmer). Mice were sacrificed when they showed endpoint signals such as significant weight loss, hindlimb paralysis, etc.. At postmortem, the micro-computer tomography (micro-CT) was performed for bone histo-morphometric analyses using micro CT400, Scano medical, Inc.

The median survival was 56 days in NSG, while 42 days in C57BL/KaLwRij. In vivo BL image analysis showed that myeloma slowly develops in NSG mouse in comparison to C57BL/KaLwRij mouse. Histomorphic analyses found that severe osteolytic lesions occur at the lumbar spine in NSG mouse compared to C57BL/KaLwRij mouse, but no significant difference at the femur of both strains. At the lumbar spine, trabecular thickness (p < 0.0004) and trabecular space (p < 0.0014) were significantly increased in NSG mouse compared to C57BL/KaLwRij mouse. On the contrary, trabecular number (p < 0.0002) and bone volume density (p < 0.0005) were significantly decreased in NSG mouse compared to C57BL/KaLwRij mouse.

In conclusion, we found that the systemic 5TGM1 injected NSG mouse slowly progresses myeloma and develops more severe MMBD than C57BL/KaLwRij model. This model will serve a better MMBD model to evaluate the therapeutic effects of MMBD targeted drugs.

Disclosures

No relevant conflicts of interest to declare.
Zebrafish ALL Driven By HumanMYCand a New Technique to Biopsy Living Fish
Authors: Park Gilseung , Burroughs-Garcia Jessica , Foster Clay , Sinha Arpan A. , Frazer Kimble John
Published: November 2020

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer; B-lineage ALL (B-ALL) represents ~85% of cases. Transgenic humanMYC(hMYC), an oncogenic transcription factor in many blood cancers, is known to induce T-ALL in zebrafish. Until recently robust zebrafish B-ALL models did not exist, but we discovered fish of this transgenic line develop B-ALL also. To distinguish T-ALL from B-ALL, we use a transgenic marker,lck:GFP, where T-ALL fluoresce brightly while B-ALL fluoresce dimly. To define B- and T-ALL incidence, we monitored >600 fish by serial fluorescent microscopy and found 100% of fish developed ALL (64% T-ALL, 23% B-ALL, both 13%) by 10 months. To find transcriptional programs active in both ALL types, we performed RNA-seq on 10 T- and 14 B-ALL and compared these to T- and B-ALL induced by murineMyc(mMyc). Unexpectedly, we found B-ALL occur in distinct lineages in these related lines, withhMYCB-ALL expressingighzheavy chains, butmMycB-ALLighm. To investigate glucocorticoid responses, a key agent in ALL, we treated fishin vivowith dexamethasone (DXM).hMYCB-ALL were highly DXM-sensitive, but many recurred rapidly after treatment ceased.

Studies lymphocytes or ALL cells from zebrafish typically require fish euthanization; it would be useful to obtain cells without euthanasia. Thus, we developed biopsy techniques in living fish to obtain lymphocytes from the thymus, kidney-marrow, and even scales. We found B and T cells can be obtained from thymus, with B cells abundant in scales. We can also recover ALL cells from scales of fish with B- or T-ALL. Analyses of normal lymphocytes and ALL cells from these anatomic regions prove that GFPhi cells express T cell genes, and GFPlo cells express B cell genes. These novel biopsy methods will permit studies ofex vivosamples from live fish. Overall, these new results and methods expand the utility of zebrafishhMYC-driven ALL, a model of the most important pediatric cancer.

Disclosures

No relevant conflicts of interest to declare.