bluuebunny/crossref_metadata_2025 huggingface dataset:
Kinetics of Immune Reconstitution after CD19 CAR-T Cell Therapy in ALL Patients
Authors: Wang Ying , Cao Jiang , Zhiling Yan , Qiao Jianlin , Li Deipeng , Li Zhenyu , Xu Kailin
Published: November 2019

Background: Chimeric antigen receptor (CAR)-T cell therapy has achieved significant efficacy in relapsed or refractory(R/R) hematologic malignancies. CD19 CAR-T cells, which kill B lymphoblasts, but also target normal B lymphocytes, resulting in dysplasia of B cells and suppression of humoral immunity. There were no detailed reports on the profile of immune reconstitution in patients after CD19 CAR-T cell treatment. Our study focused on the kinetics of lymphocyte subsets and immunoglobulin reconstruction in 21 patients with acute lymphoblastic leukemia (ALL) after CD19 CAR-T cell therapy.

Methods: Patients with R/R ALL received CD19 CAR-T cell therapy who obtaining complete response at 1 month after CAR-T cell infusion from April 1, 2016 to Feb 28, 2019 were enrolled (Clinical Trials: NCT02782351). Blood was collected before lymphodepletion therapy and at intervals after CAR-T cell infusion for analysis of complete blood counts, lymphocyte subsets, immunoglobulin, and the amplification of CAR T cells.

Results: We found that the reconstitution of different immune cell subsets occurred at different rates after CD19 CAR-T cell infusion: CD8+ cells were the first to recover, followed by CD16+CD56+ cells and CD3+ cells, and finally CD4+ cells. CD4/CD8 ratio was inverted, sustaining for at least 1 year. B cell dysplasia occurred in all patients and CD19+ cells returned to normal 79 days after CAR-T cell infusion, which may be related to CAR-T cell depletion. IgG and IgM recovered on day 184 and 242 after CAR-T treatment, respectively. IgA recovered slowly and sustained longer at a low level compared with IgG and IgM, and did not return to normal 1 year after CAR-T cell treatment. A total of 9 infections occurred in 6 (28.57%) patients, including 6 cases with grade 2 infection and 3 cases with grade 3 infection. No patients died of severe infection. In patients with late relapse, IgG, IgA, and IgM were generally normal before CAR-T cell therapy, and were higher than those with early relapse, suggesting that patients with normal immunoglobulin levels before treatment may have longer remission time.

Conclusion: Our results showed the kinetics of lymphocyte subsets and immunoglobulin reconstruction in R/R ALL patients after CD19 CAR-T cell therapy. The recovery of CD8+ cells was fast, whereas the recovery of the CD4+ cell was delayed. All patients developed B cell dysplasia, and it took about 3 months to recover. IgG recovered firstly, followed by IgM and IgA, the later would not recover at least 1 year.

Disclosures

No relevant conflicts of interest to declare.
Dead/H-Box Helicase 11 (DDX11) Mutations Correlate with Increased Relapse Risk in Persons with Acute Myeloid Leukaemia and Promote Proliferation and Survival of Human AML Cells in Vitro and in Immune Deficient Mice
Authors: Zhou Ya-Lan , Wu Li-Xin , Gale Robert Peter , Wang Zi-Long , Li Jin-Lan , Jiang Hao , Jiang Qian , Jiang Bin , Cao Shan-Bo , Sun Ying , Lou Feng , Wang Chengcheng , Liu Yan-Rong , Wang Yu , Chang Ying-Jun , Xu Lanping , Zhang Xiaohui , Liu Kaiyan , Ruan Guorui , Huang Xiaojun
Published: November 2019

Background About one-half of persons with acute myeloid leukemia have normal cytogenetics but have diverse outcomes which might be explained, at least in part, by specific mutations. We focused on DDX11, the budding yeast ortholog of ChlR1, which encodes an ATP-dependent RNA- and DNA-helicase involved in diverse cell processes such as sister chromatid exchange cohesions and implicated in other cancers and which is associated with telomere shortening.

Methods DNA from 359 consecutive, newly-diagnosed adults with AML and normal cytogenetics were interrogated by deep target regional sequencing (TRS). Average effective sequencing depth was 1678.5X (1000.0-1800.0X) and median coverage, 99.9% (98.0, 100.0%). Outcomes studied included cumulative incidence of relapse (CIR) and event-free survival (EFS). DDX11 mutants were transfected into a human AML cell line which was studied in vitro and in immune deficient mice.

Results We identified 2909 non-synonymous somatic variants. 284 subjects (79%; 95% confidence interval [CI], 75, 83%) had ≥5 mutations with a median of 8 mutations per subject (range, 1-20). We identified mutations at 4 new loci including DDX11 in 29 subjects (8%), NPIPA5 in 15 (4%), CYP2F1 in 13 (4%), and MED12 in 9 (3%). In multi-variable analyses DDX11 mutation was independently associated with CIR (Hazard Ratio [HR]= 2.276 [1.318, 3.931]; P=0.003) and EFS (HR=2.319 [1.391, 3.867], P=0.001). Two missense DDX11-mutant alleles (DDX11P316L/WT or DDX11Q363K/WT) were generated and introduced to the kasumi-1 leukemia cells by lentivirus transfection. Both mutants showed proliferative and anti-apoptotic activities in functional analyses and increased resistance to killing by Cytarabine and Daunorubicin in vitro and in transplanted immune deficient mice.

Conclusions Subjects with AML with normal cytogenetics who have a DDX11 mutation have a higher CIR and worse EFS compared with subjects with wild-type DDX11. Functional studies of a transfected DDX11 human AML cell line provide a functional explanation of our clinical findings.

* Correspondence

Profs. Guo-Rui Ruan and Xiao-Jun Huang

Peking University Peoples Hospital and Institute of Hematology

No.11 Xi-Zhi-Men South Street, Beijing 100044, China

T 86-10-88324672

F 86-10-88324672

E ruanguorui@pkuph.edu.cn OR huangxiaojun@bjmu.edu.cn

Disclosures

No relevant conflicts of interest to declare.
Analysis of a New Haematopoietic Stem/Progenitor Cells with Megakaryocyte Differentiation Potential
Authors: Yang Mo , Chen Shichao , Li Liang , Xin Hongwu , Chong Beng H , Yang Liuming
Published: November 2019

Thrombocytopenia is a common clinical condition and requires more effective treatments. Our previous original work confirmed that 5-HT contributes to the expansion of cord blood stem/progenitor cells (CD34+/CD41+CD61+) and haematopoietic reconstitution in NOD/SCID mice, and through 5-HT2R and Erk1/2 pathway, 5-HT remodel F-actin organization, promoting megakaryocytes to pro-platelet formation (Stem Cells, 2007; 2014). It is suggested that there is a subtype of haematopoietic stem cells (CD34+5HT2R+) with the potential to differentiate into megakaryocytes (CD41+5HT2R+). We studied the specific functions of cells through subsequent experiments, including receptor expression and signaling pathways. Results showed that: (1) The presence of 5-HT2 receptors on CD34+ and CD41+ cells; (2) 5-HT activated the PI3K/AKT pathway and the Wnt/β-catenin pathway via 5-HT2R in CD34+ and CD41+ cells, with anti-apoptotic and pro-proliferative functions; (3) These CD34+5HT2R+ differentiated CD41+5HT2R+ cells, 5-HT acted on 5-HT2R and Erk1/2 pathways, and promoted the formation of pro-platelets by F-actin reorganization; (4) CD34+5HT2R+ haematopoietic stem cells implanted in NOD/SCID mice reconstituted blood function, especially on thrombopoiesis. This study provides a more scientific basis for haematopoiesis and drug development, especially for the treatment of thrombocytopenia.

Disclosures

No relevant conflicts of interest to declare.
"Prevalence and Outcome of Avascular Necrosis of the Hip (AVN) Among Young Omani Patients with Sickle Cell Disease"
Authors: Al Fadhali Iman , Al-Kindy Farah , Alshibli Naema , Alkindi Salam , Al-Khabori Murtadha , Al Rawas Abdulhakim , Al Shezawi Asma , Mahfouz Yasser , Mevada Surekha Tony , Wali Yasser
Published: November 2019

Background:

Sickle cell disease is a common hemaglopinapthy worldwide and in Oman. Avascular necrosis (AVN) of the hip causes significant morbidity to patients with sickle cell disease and has a profound impact on their quality of life. This study aims to identify the prevalence and outcome of AVN among young Omani patients with sickle cell disease.

Methods:

The is a cross sectional study done in the main tertiary care and referral facility in Oman, Sultan Qaboos University Hospital. Out of 3000 registered patients 85 patients found to have clinical and radiological proven AVN, between June 2017-January 2019.

Data obtained included demographics, the affected joint, uni or bilateral), radiological staging by FICAT (MRI), hydroxyurea use, dose and duration as well as laboratory data at time of diagnosis.

Results:

The prevalence of AVN among SCD Omani children and young adults is 2.8 %. Their age ranges between 5-25 (Mean 14.6 +3.4). Male to female ratio was 1.6:1. Based on FICAT system score, most of the cases (82%) are AVN grade 3 and above at diagnosis. Thirty-six patients (42%) were diagnosed to have stage IV AVN. Regarding the hydroxyurea use, 43 patients (50.6%) were already started on HU before the development of AVN. Their mean duration of HU use was 5.3 years, with an average dose of 15.7 mg/kg/day. Fifteen patients developed AVN and were started on HU as part of their management. The follow up MRI of 4 of them showed improvement of their AVN stage (from 4 to 2). Fifteen patients (17.6%) underwent joint replacement because of chronic pain and disability and almost half of them (7 patients) were on hydroxyurea.

Conclusion:

Prevalence of AVN in young patients with SCD in the main tertiary care referral facility in Oman is 2.8% which is less than the internationally reported. Patients are diagnosed at later stages (3 and above) indicating a possibility of underdiagnosis of asymptomatic stage one and 2 patients. Hydroxyurea use has improved the severity of the AVN in few patients.

Disclosures

Al-Khabori: SOBI: Honoraria; AstraZeneca: Honoraria; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Servier: Membership on an entity's Board of Directors or advisory committees; Shire (Takeda): Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; NovoNardisk: Membership on an entity's Board of Directors or advisory committees. Wali:Sultan Qaboos University Hospital: Employment.
RFWD2 Induces Cellular Proliferation and Proteasome Inhibitor Resistance By Mediating p27 Ubiqutinaiton in Multiple Myeloma
Authors: Yang Ye , Guo Mengjie , Gu Chunyan
Published: November 2019

Purpose: In recent years, with the emergence of targeted proteasome inhibitors (PIs), the treatment of multiple myeloma (MM) has made great progress and significantly improves the survival rate of patients. However, MM remains an incurable disease, mainly due to the recurrence of drug resistance. The constitutive photomorphogenic 1 (RFWD2, also known as COP1), is closely related to the occurrence and development of tumors, but its role in MM is largely unknown. This study was aimed to explore the mechanism of RFWD2 on cell proliferation and resistance to proteasome inhibitor in MM.

Experimental Design: Using gene expression profiling (GEP) samples, we verified the relation of RFWD2 to MM patients' survival and drug-resistance. The effect of RFWD2 on cell proliferation was confirmed by MTT and cell cycle analysis in RFWD2-overexpressed and RFWD2-knockdown MM cells. MTT and apoptosis experiments were performed to evaluate whether RFWD2 influenced the sensitivity of MM cells to several chemotherapy drugs. MM xenografts were established in immunodeficient NOD/SCID mice by injecting wild-type or RFWD2 over-expression MM cells with drug intervention. The mechanism of drug resistance was elucidated by analyzing the association of RFWD2 with E3 ligase of p27. Bortezomib-resistant RPMI 8226 cells were used to construct RFWD2 knockdown cells, which were injected into NOD/SCID mice to assess the effect of RFWD2 on bortezomib resistance in vivo.

Results: RFWD2 expression was closely related to poor outcome, relapse and bortezomib resistance in MM patients' GEP cohorts. Elevated RFWD2 induced cell proliferation, while decreased RFWD2 inhibited cell proliferation and induced apoptosis in MM cells. RFWD2-overexpression MM cells resulted in PIs resistance, however, no chemotherapy resistance to adriamycin and dexamethasone was observed in vitro. In addition, overexpressing RFWD2 in MM cells led to bortezomib resistance rather than adriamycin resistance in myeloma xenograft mouse model. RFWD2 regulated the ubiquitination degradation of P27 by interacting with RCHY1 ubiquitin ligase. The knockdown of RFWD2 in bortezomib-resistant RPMI 8226 cells overcame bortezomib resistance in vivo.

Conclusions: Our data demonstrate that elevated RFWD2 induces MM cell proliferation and resistance to PIs, but not to adriamycin and dexamethasone both in vitro and in vivo through mediating the ubiquitination of p27. Collectively, RFWD2 is a novel promising therapeutic target in MM.

Disclosures

No relevant conflicts of interest to declare.
The Splenic Microenvironment Is a Novel Site of Relapse Following ABT-199 Treatment
Authors: Di Grande Alessandra , Piers Sofie , Van Vlierberghe Pieter , Ni Chonghaile Triona
Published: November 2019

T-cell acute lymphoblastic leukaemia (T-ALL) is an aggressive hematologic malignancy arising from the transformation of immune T-cell lymphocytes. Early T-cell progenitor (ETP-ALL) is a subgroup particularly associated with a poor prognosis and a high risk for relapse. While the leukaemia initially develops in the thymus it spreads in the blood to the bone marrow, lymph nodes and often the spleen. Interestingly, splenomegaly was previously associated with a poor prognosis in leukemic patients. Recently, it was shown that ETP-ALL is dependent on the expression of the anti-apoptotic protein BCL-2, and is sensitive to inhibition with ABT-199, a BCL-2 specific BH3 mimetic. However, one issue with targeted agents, like ABT-199, is the development of resistance to treatment. Our aim was to determine potential in vivosites of resistance/relapse following ABT-199 treatment using a xenograft model of ETP-ALL.

We confirmed that the ETP-ALL LOUCY cell line is BCL-2 dependent and then labelled it with luciferase to enable visualisation of the leukaemia in vivo. Following establishment of the leukaemia in NOD/SCID gamma mice, as assessed by hCD45+, the mice were randomised to receive vehicle control or 50 mg/kg ABT-199 by oral gavage daily for two weeks. While the mice were initially sensitive to ABT-199, the leukaemia started to progress while on treatment. Interestingly, there appeared to be a selective redistribution of the leukaemia to the spleen following ABT-199 treatment. Indeed, LOUCY cells isolated from the spleen of the mice had a reduced BCL-2 dependence, as assessed by BH3 profiling. The reduced BCL-2 dependence correlated with reduced BCL-2 expression at both the mRNA and protein level.

Next, we confirmed that human splenic fibroblasts (HSF) co-cultured with the LOUCY cell line in vitro also altered BCL-2 dependence and expression using BH3 profiling and Western blotting. To identify potential splenic cytokines involved in the regulation of BCL-2 protein expression in ETP-ALL we performed a screening cytokine array. Upon co-culture of the LOUCY cells with HSF there was an increased expression of IL-6, this was confirmed using ELISA. Using an IL-6 receptor antibody we confirmed that blocking IL-6 receptor reversed the change in BCL-2 dependence in the presence of the splenic microenvironment.

Lastly, we confirmed in a T-ALL patient-derived xenograft, that is BCL-2 dependent, that the splenic microenvironment alters the mitochondrial apoptotic threshold. Currently, there are reports in the literature of ETP-ALL patients being treated with ABT-199. While there have been numerous studies lately describing cell autonomous events leading to ABT-199 resistance, our novel finding that the splenic microenvironment is a site of relapse is potentially of great clinical importance for BCL-2 dependent leukemia's.

Disclosures

Ni Chonghaile: AbbVie: Research Funding.
Preemptive Administration of Ruxolitinib Rapidly Ameliorate Gvhd after Allo-Hematopoietic Stem Cell Transplantation: A Single-Center Retrospective Study
Authors: Lin Kangni , Chen Minmin , Li Qian , Ren Jinhua , Zhu Haojie , Xu Jingjing , Zheng Rong , Chen Zhizhe , Hu Jianda , Yang Ting
Published: November 2019

Objective:

Clinical benefit with ruxolitinib as a salvage therapeutic agent in steroid-refractory acute graft-versus-host disease (aGVHD) has been reported. The purpose of this study is to explore the efficacy and safety profile of preemptive administration of ruxolitinib for patients with GVHD after hematopoietic stem cell transplantation (HSCT), and to determine the optimal timing of start of ruxolitinib treatment.

Methods:

We conducted a single-center retrospective study to analyze the overall response rate at day 28 and the amelioration of GVHD after preemptive treatment of ruxolitinib in patients with GVHD post-HSCT. A total of 35 patients (26 males/ 9 females) with GVHD (30 aGVHD/ 5 cGVHD) treated at Fujian Medical University Union Hospital (Fuzhou, China) from July 2016 to April 2019 were enrolled. All patients received conditioning regimens including ATG, cyclosporine A, methylprednisolone, methotrexate mycophenolate mofetil w/o basiliximab for prevention of GVHD. Ruxolitinib was administered orally at 10 mg twice daily for patients ≥ 40 kg or 5 mg twice daily if <40 kg. The patients were divided into 3 groups according to the starting time of GVHD treatment with the preemptive ruxolitinib: shorter than 3 days (≤3 day,13, 37.1%), between days 3-7 (3-7 days,10, 28.6%) and more than 7 days(>7 days,12, 34.3%). Treatment responses of ruxolitinib and the safety are compared among groups.

Results:

These three groups were well matched demographically. There were no significant differences in conditioning regimens, donor types, CD34+ cells infusion, GVHD prophylaxis regimens and primary treatment among 3 groups. The median time to response of ≤3 days group, 3-7 days group and >7 days group was 20.5 (6-43) , 19.5 (2-107) and 20.5 (9-60) days, respectively. The ≤3 d group showed a best overall response rate (ORR) and complete remission rate (CRR) at day 28 after treatment than 3-7 d group and >7 d group (ORR: 92.3% vs 90.0% vs 83.3%, P=0.361; CRR: 46.2% vs 40.0% vs 25.0%, P=0.512 ). The cumulative complete remission rates among 3 groups were 58.5% (≤3 d group), 86.7% (3-7 d group) and 25% (>7 d group), respectively (P=0.096). The severity of GVHD were significantly ameliorated at day 28 in ≤3 d group than the other 2 groups ( 92.3% vs 70.0% vs 50.0%, P=0.064), and moreover, the clinical GVHD scores dropped significantly in ≤3 d group than >7 d group (-1.54 grade vs -0.75 grade, P=0.041) at day 28 after treatment. Incidence and severity of adverse reactions did not differ significantly between groups (P>0.05).

Conclusion:

When preemptive administration ruxolitinib is initiated rapidly, remarkable efficacy on amelioration of GVHD can be achieved without significant adverse effects. Larger randomized trials are needed to confirm efficacy.

Disclosures

No relevant conflicts of interest to declare.
Cerebral Venous Thrombosis Case Series at a Tertiary Care Hospital: Exploring Treatment Safety and Efficacy of Direct Oral Anticoagulants
Authors: Alsheef Mohammed Abdullah , Alomar Mukhtar , Zaidi Abdul Rehman Z. , Kullab Ghaydaa Juma , AlHazzaa Mohammed , Bafaraj Asaad , Badahman Abdullah
Published: November 2019

Background:

Cerebral venous thrombosis (CVT) is an uncommon cause of stroke that mainly affects young adults and children. Initial treatment with heparin followed by wafarin is the mainstay of treatment. Only insufficient experience is available for direct oral anticoagulants (DOACs).

Aims:

The study aims to demonstrate the efficacy and safety of DOACs such as (Rivaroxaban and Dabigatran) in patients with objectively confirmed CVT.

Methods:

Data of 46 cases of CVT collected using a standardized case report form. Inclusion criteria were patients diagnosed with CVT, confirmed by CT or MRI imaging.

Results:

The total number of patients was 46 (9 males and 37 females). The mean age of the patients was 35.2± 5 years. The most common clinical manifestations among our patients were headache followed by seizure. 52% of cases were unprovoked, while 48% were provoked by pregnancy and oral contraceptive pills. Superior sagittal sinus (55%) and transverse sinus (44.9%) were the most common sites. Involvement of more than three venous sinuses was 34.8%. Thrombophilic abnormality was detected in 21.7% of patients. Initiation of anticoagulation (AC) was mostly low molecular weight heparin (LMWH) (80%), followed by unfractionated heparin (UFH) (17.7%) and fondaparinex (2%). Maintenance AC with Rivaroxaban after heparin (LMWH/UFH) was in 63% of our patients, the rest were switched from Warfarin to Rivaroxaban (34.8%), and one was treated by Dabigatran (2%). CVT recurrence was observed in one patient. Major bleeding (according to ISTH criteria) was not reported in our case series.

Conclusions:

DOACs demonstrated good safety and efficacy profile and can potentially replace warfarin in CVT patients.

Disclosures

No relevant conflicts of interest to declare.
Combined Thalidomide and Recombinant Human Interferon-α-1b and Interleukin-2 for Acute Myeloid Leukemia of Various Disease Status: A Multi-Center Prospective Study
Authors: Mi Ruihua , Chen Lin , Wei Xudong , WANG Xiaojiao , Song Yongping
Published: November 2019

AML (acute myeloid leukemia) patients with relapsed refractory diseases or in poor physical conditions have very limited choices of appropriate treatment regimens, and most alternative options are costly. Here, we reported that an affordable regimen with combined recombinant human interferon-α-1b (IFN-α1b), thalidomide, and recombinant interleukin-2 (IL-2) (the ITI regimen) achieved varying degrees of therapeutic effects in AML patients of various disease status and vulnerabilities. ITI regimen was administrated as follow: a subcutaneous injection of IFN-α1b 60 μg qod, IL-2 1 million unit qod, and 200mg thalidomide tablet orally taken every night before sleep.

Group A Sixty-eight patients who were with relapsed or refractory AML were enrolled, sixty finished at least one course, a response rate (CR+CRi) of 16.7% (10/60) was observed, and, and 7 (11.7%) patients achieved partial remission.(Table 1)

Group B Eighteen patients with morphologically complete remission and consistently positive MRD were enrolled, each patients underwent ITI regimen for 2 months at least. According to the criteria of the WHO risk stratification, 7 were with a favorable risk, 8 were with a intermediate risk, and 3 were with a high risk. Patients with FLT3-ITD mutations were treated with oral sorafenib during early induction and consolidation treatment. These 18 patients received conventional dosage of ITI regimen. Seven patients had a negative MRD after 1 or 2 months. The MRD levels of 3 patients significantly decreased after one or two months. Five patients suffered a Morphological relapse. Another 3 patients failed to the conventional dosage of ITI regimen with an increased MRD rate, but it decreased after we made a modification of IFN-α1b and IL-2 administration daily. The response rate of the 18 patients in Group B was 72.2%, where MRD < 0.01% was defined as the negative threshold. (Table 2)

Group C Eighty-eight patients with MRD-negative AML after consolidation were enrolled. Among the 88 patients with initial CR, 11 (12.5%) relapsed during the maintenance period (Figure 1). All these 11 patients relapsed within 2 years, with a median recurrence period of 20 months, of which 2, 4, and 5 patients, respectively, were with favorable, intermediate, and high risk. The indicated that this regimen effectively reduced the relapse rate.(Table 3)

Disclosures

No relevant conflicts of interest to declare.
An Institutional Survey of Thrombophilia Evaluation in the Setting of Venous Thromboembolism (VTE)
Authors: Garrahy Ian , Pawar Tushar , Donato Anthony , Stevenson Amber , Forman Daniel
Published: November 2019

Introduction:

There has been increasing recognition of various laboratory markers of thrombophilia that are associated with increased risk of thrombosis either through hereditary (i.e. Factor V Leiden, prothrombin G20210A mutation, and protein C, S, and antithrombin deficiencies) and/or acquired means (i.e. antiphospholipid antibodies) over the past decades. Availability of thrombophilia biomarkers has the potential to lead to inappropriate testing and treatment.

Methods:

An online anonymous questionnaire was sent to all providers in the Tower Health System asking them about their practice, specialty, degree, years in practice, and basic questions regarding their comfort and frequency of prescribing anticoagulants. The questionnaire also asked the providers how they would manage ten various clinical scenarios related to anticoagulation management. One question specifically asked whether a hereditary thrombophilia panel should be ordered for a 65-year-old with provoked VTE and no concerning family history of VTE. Analysis of variance was used to compare the scoring means between groups while linear regression and Pearson's correlation coefficient measured the relationship between years of practice and test scores.

Results:

Out of 404 responders, 170 (42.1%) inappropriately ordered a hereditary thrombophilia panel on the question (see Figure 1) that addressed this topic. 53.6% and 35% of the outpatient and inpatient physicians answered the question incorrectly which was statistically significant (p value 0.026).

Conclusion:

This project suggests a professional practice gap in which overtesting might be ordered. In particular, this study found a statistically significant difference between inpatient and outpatient providers in the ordering of a thrombophilia panel. Whether just-in-time reminders or other educational interventions could close this gap is a matter for further study.

Figure 1

Disclosures

No relevant conflicts of interest to declare.
Development of a Low-Cost Electrical Impedance-Based Microflow Cytometer
Authors: Dieujuste Darryl , Liu Jia , Du E. , Alvarez Ofelia A.
Published: November 2019

Sickle Cell Disease (SCD) is a genetic condition caused by a mutated hemoglobin molecule (HbS) found in red blood cells (RBCs). HbS polymerizes in low oxygen environments and contribute to painful vaso-occlusion in patients. Laboratory diagnosis of SCD is typically made by detection of the presence of sickle cells by peripheral blood smear, and presence of HbS by electrophoresis and high-performance liquid chromatography. Recently, flow cytometry technique in companion with sickling assays has demonstrated the capability in quantitative measurements of sickle cells at single-cell level, using software algorithm for cell-imaging analysis (Van Beers et. al. American Journal of Hematology 2014), and electrical impedance (Liu et. al. Sensors and Actuators B: Chemical 2018). Here, we show a portable, cost-efficient electrical impedance-based sensor and its capability to be used in conjunction with microfluidics-based sickling assay for microflow cytometry of sickle cells.

The impedance microflow cytometer is based on a commercially available integrated circuit (IC), the AD5933. Using a microcontroller and additional circuitry on a custom designed printed circuit board, we are able to produce sinusoidal signals of up to 100kHz in frequency and sample up to 200 data points per second, at a cost under $60 in materials to create. The impedance measurement range is optimized to work in companion with microfluidic chips in general. In order to measure sickle cells, the impedance microflow cytometer is used in companion with our unique Polydimethylsiloxane (PDMS) microfluidic cell sickling assay (Du et. al. PNAS 2014). Cells are suspended in phosphate buffered saline (PBS) medium and move in the microchannel using a pressure driven flow. Impedance measurement is achieved using two Ti/Au electrodes embedded in the microchannel as cells flow past the electrodes. Data is captured and made available for post processing using a customized MATLAB script. RBCs from healthy donors and SCD patients were used to demonstrate the capability of the developed system. The results showed that our system can separate between normal RBCs and sickle cells, as well as between sickled and unsickled cells. The performance in detection of sickle cells is comparable to a commercial impedance analyzer. This proof-of-concept design aims to minimize the physical space needed for cytometry as well as bring affordable and reliable cytometry results within its given limitations.

Figure

Disclosures

Alvarez: Forma Therapeutics: Consultancy; Novartis: Consultancy.
Clinical Efficacy of L-Annamycin, a Liposomal Formulated Non-Cross-Resistant and Non-Cardiotoxic Anthracycline in Relapsed/Refractory AML Patients
Authors: Gil Lidia , Shepard Robert C. , Silberman Sandra L , Zak Ewa Maria , Priebe Waldemar
Published: November 2019

Introduction: An anthracycline combined with cytarabine remains the backbone for induction therapy in the treatment of AML. De novo and acquired multidrug resistance (MDR) limits efficacy of clinically used anthracyclines. L-Annamycin, a novel anthracycline formulated in multilamellar liposomes, has been shown to overcome MDR in preclinical in vitro and in vivo doxorubicin resistant models as well as in clinical studies. Furthermore, preclinical toxicology studies comparing L-Annamycin to doxorubicin determined that the drug displayed dramatically reduced cardiotoxicity. These promising properties justified initiation of Phase I/II clinical trials in both the US and Europe using L-Annamycin for the treatment of R/R AML.

Aims: The aim of the Phase I component is to determine the recommended Phase 2 dose (RP2D) of L-Annamycin for the treatment of R/R AML. The expansion Phase IIa studies are designed to assess L-Annamycin efficacy leading to a Phase II registration study for accelerated approval.

Methods: The Phase I component is a standard 3+3 design to determine the RP2D. An additional 21 patients expansion phase will be enrolled at the RP2D to confirm efficacy. A DMC will monitor all data and decide if the study should continue and if any changes to the protocol are needed. Cardiotoxicity is being closely studied with stress 2-D echocardiography and biomarkers such as troponin.

Results: This study is ongoing in both Europe and the USA. Even at a relatively low starting dose, two patients had sufficient response from a single 3-day course of L-Annamycin to qualify for a potentially curative bone marrow transplant. In total, 11 patients have completed at least one course of L-Annamycin with no signs of cardiotoxicity. Adverse events included one instance of Grade 2 mucositis, which resolved quickly.

Summary/Conclusion: Despite progress in the treatment of AML most patients will relapse and die from refractory disease. Properties of L-Annamycin, including the ability to overcome MDR, a critical mechanism of resistance, and the lack of cardiac toxicity, are highly promising and fully justify a Phase II clinical study designed to demonstrate L-Annamycin efficacy in all patients with R/R AML agnostic of mutational status.

Disclosures

Shepard: Moleculin Biotech Inc: Employment, Equity Ownership. Silberman:Trovagene: Consultancy; Moleculin Biotech: Employment. Priebe:MOLECULIN BIOTECH INC: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.
A 19-Year Retrospective Analysis of Venous Thromboembolism Trends in Chemotherapy-Induced Anemia: Red Blood Cell Transfusion Versus Erythrocyte Stimulating Agent Administration
Authors: Bryer Emily , Kallan Michael , Chiu Ting-Shaun , Scheuba Katerina , Henry David H.
Published: November 2019

Introduction: Anemia is a common and unfortunate consequence of chemotherapy; patients receiving a variety of chemotherapy regimens often develop chemotherapy-induced anemia (CIA), which contributes to poor outcomes including increased mortality. Prompt and effective treatment of CIA is essential to prevent fewer chemotherapy dose delays and reductions. Optimal therapy of CIA is controversial and involves the solitary and combined use of intravenous iron, red blood cell (RBC) transfusions, and erythropoietin stimulating agents (ESAs). Despite the baseline coagulopathies present in patients with malignancy, administration of both RBC transfusions and ESAs is independently associated with venous thromboembolism (VTE). It remains unknown whether the risk of VTE in patients with CIA is greater among patients who receive RBC transfusions or ESAs.

Methods: A retrospective study analyzed 13,334 patients in the University of Pennsylvania Health System with malignancies of various type, stage, and histopathology who developed CIA between 1998-2017. Using multivariate Cox regression, we determined adjusted hazard ratios (and corresponding 95% confidence intervals) of VTE development after adjusting for RBC and ESA intervention (all during the 90 days following CIA diagnosis).

Results: (Table 1) Among the 13,334 patients with CIA, 10,948 patients did not receive any therapy during the 90-day period for their anemia (neither RBC nor ESA), 1,892 received RBC transfusion, 368 received ESA, and 126 patients received both. Among all patients, 2,620 (19.7%) developed a VTE within the 90-day period. VTE risk following RBC transfusion (HR=2.18, 95% CI 1.98-2.39, p<0.001) was approximately four-fold the VTE risk following ESA administration (HR=0.55, 95% CI 0.42-0.72, p<0.001).

Conclusion: While both RBC transfusion and ESA administration are associated with VTE, our data suggests a greater risk of VTE development with RBC transfusion as compared with ESA administration.

Disclosures

No relevant conflicts of interest to declare.
A Comprehensive Analysis of Rhoa Mutation Positive and Negative Angioimmunoblastic T-Cell Lymphomas By Targeted Deep Sequencing, Expression Profiling, and Single Cell Digital Image Analysis
Authors: Butzmann Alexandra , Sridhar Kaushik , Jangam Diwash , Kumar Jyoti , Sahoo Malaya K , Shahmarvand Nahid , Warnke Roger A , Rangasamy Elumalai , Pinsky Benjamin A , Ohgami Robert S.
Published: November 2019

Angioimmunoblastic T-cell lymphoma is a uniquely aggressive mature T-cell neoplasm. In recent years, recurrent genetic mutations in RHOA, TET2, DNMT3A and IDH2 have been identified which characterize this disease. However, a deep molecular study assessing both DNA mutations and RNA expression profile, combined with digital image analysis is lacking.

We designed a study to assess the significance of molecular and morphologic features by high resolution digital image analysis in cases of AITL. Eighteen separate tissues from ten patients with AITL were identified and analyzed in this study.

Our results confirm recurrent mutations in RHOA, TET2, DNMT3A, and IDH2, and demonstrate increased DNA mutations in coding, promoter and CTCF binding sites in RHOA mutated AITLs versus RHOA non-mutated cases as well as increased overall survival in RHOA mutated patients. In addition, single cell computational digital image analysis morphologically characterized RHOA mutated AITL cells as distinct from cells from RHOA mutation negative patients. Computational analysis of single cell morphological parameters revealed RHOA mutated cells have reduced eccentricity (more circular) compared to RHOA non-mutated AITL cells.

Overall, this study expands our understanding of AITL and demonstrates that there are specific cell biologic and morphologic manifestations of RHOA mutations in cases of AITL.

KeyPoints:

1) RHOA mutated AITL show distinctly increased and more deleterious mutations than RHOA unmutated cases

2) RHOA mutated T-cells have distinct morphologic features from RHOA unmutated T-cells

Disclosures

Ohgami: Agilent technologies: Other: received support/funding.
Complementary Transplantation Improved Results of Both Peripheral Blood Stem Cells and Unrelated Cord Blood Transplants in Thalassemia: A Multi-Center Study from China
Authors: Li Chunfu , Liu Sixi , He Yuelin , Wang Xiaodong , Liao Jianyun , Li Shan , Zheng Xiaolin , Wu Xuedong , Li Changgang , Feng Xiaoqin , Wen Feiqiu , Liu Huaying , Zhou Xiaohui , Peng Zhiyong , He Lan , Bu Chaoke , Wing Leung
Published: November 2019

Background: Unrelated cord blood (UCB) transplant (UCBT) is not recommended in patients with thalassemia major (TM) so far. Post-transplant (PT) Cyclophosphamide (PTCy) with long pre-transplant immunosuppression therapy have improved haploidentical peripheral blood (PB) stem cell transplant (haplo-SCT) survival in TM patients but with 2/31 primary rejection. So, we designed a novel dual transplantation of UCBT following haplo-SCTwith PTCy(NF-14-TM-CT protocol).

Aim:To improve results of haplo-SCT and UCBT in patients with TM.

Patients and method: NF-14-TM -CT protocol was termed as double-insurance dual transplantsincluding a haplo-SCT and an UCBT, in which conditioning regimen consisted of ATG (at -10 to -8 day), Cy (-7), Fludarabine (-6 to -2), Busulfan (-6 to -4) Thiotepa (-3), haplo-PB (0), PTCy (+3, +4) and UCB (+6). PTCy serve as GVHD prophylaxes after haplo-SCT and as conditioning before UCBT. In total 131 patients with TM from three pediatric center in China received NF-14-TM-CT protocolfrom June, 2014 to April, 2019, with a median follow-up of 13 (2-59) months and a median age of 8 (3.5-17) years.

Results Final haplo-PB engrafted(group1)in 76 patients with mean PBSC-MNCof 22.49 (±5.36) x108/kgand UCB nuclear cells (NC) of 5.95 (±3.39) x107/kg and final UCB engrafted (group 2) in 55 patients with mean PBSC-MNC of 21.78 (±5.68) x108/kg and mean UCB-NC of. 5.43 (±2.32) x107/kg. The 4-year overall survival (OS), thalassemia-free survival (TFS), graft rejection (GR), and transplant related mortality (TRM) were 97.6%, 96.0%, 1.5%, and 2.4%, respectively (Fig. A), in total. The corresponding rates for group 1 were 98.3%, 96.9%, 1.7% and 1.8% and for group 2 were 95.5%, 93.8%, 4.5% and 1.4%, respectively. No statistic significant difference was found in OS, TFS, GR and TRM, respectively, when comparing group 1 with group 2 (Fig. B. C, D, E).The incidence of grade II-IV acute GVHD, III-IV acute GVHD, mild chronic GVHD, moderate/severe chronic GVHD, VOD, PT cystitisand PT hemolysis of the entire cohort was 16.8%, 6.87%, 9.92%, 1.52%, 4.60%, 31.3% and 14.5, respectively.

Summary:Current study proved that the novel CT improved the results of haplo-SCT and UCBT in patients with TM.

Disclosures

Wing: Miltenyi Biotec: Employment.
1q21 Gain May Challenge the Role of t(4;14) As an Adverse Prognostic Marker of Multiple Myeloma
Authors: Du Chenxing , Gang An , Xu Yan , Mao Xuehan , Yan Yuting , Liu Jiahui , Fan Huishou , Sui Weiwei , Deng Shuhui , Zengjun Li , Li Chengwen , Yi Shuhua , Hao Mu , Zou Dehui , Zhan Fenghuang , Tai Yu-Tzu , Anderson Kenneth C. , Qiu Lugui
Published: November 2019

In the era of bortezomib, traditional prognostic markers in multiple myeloma (MM) may be suspect. The outcome of patients with t(4;14) is largely improved by bortezomib based treatment. Whether the outcome of patients with t(4;14) is still homogeneously inferior remains unknown. In this long follow-up non-randomized clinical trial, we observed no significant difference in the PFS and OS of t(4;14) positive or negative patients in the bortezomib arm. Interestingly, t(4;14) was highly correlated with the presence of 1q21 gain. In t(4;14)-positive patients, the median OS of patients with and without 1q21 gain was 38.8 and 57.2 months (P=0.034), respectively. In t(4;14)-negative patients, the median OS of the two subgroups was 43.0 and 89.4 months (P<0.001), respectively. In contrast, no statistical difference in PFS or OS was observed in 1q21 gain patients with versus without t(4;14) aberration. We then discovered that 1q21 gain facilitated the acquisition of new chromosome abnormalities and contributed to the genomic instability, evidenced by the strong correlation between 1q21 gain and complex karyotypes or the acquisition of more than two cytogenetic aberrations. Moreover, 1q21 gain and/or del(17p) were powerful enough to discriminate the high-risk patients. The combination of 1q21 gain with International Staging System (ISS) was useful to divide MM patients with respect to their survival. Finally, although bortezomib might benefit patients with 1q21 gain, it could not completely overcome its adverse effects, suggesting the necessity of more effective therapies for these patients.

Figure

Disclosures

Zhan: BIPHARM LLC: Consultancy, Other: % Allocation of Profit. Anderson:Gilead Sciences: Other: Advisory Board; Janssen: Other: Advisory Board; OncoPep: Other: Scientific founder ; Sanofi-Aventis: Other: Advisory Board; C4 Therapeutics: Other: Scientific founder .
Epidemiology, Diagnosis and Management of Neonatal Thrombosis
Authors: Shaik Mohammed , Patel Dhiman , Mihir Bhatt , Chan Anthony K.C. , Paes Bosco
Published: November 2019

Introduction:

Current research on neonatal thrombosis is limited. It relies heavily on individual case reports and much of the data is extrapolated from the adult scientific literature. The aim of this descriptive study is to review the epidemiology, diagnosis and management of neonatal thrombosis at a single tertiary care institution.

Methods:

This is a retrospective study of apatients admitted to McMaster Children's Hospital, neonatal intensive care unite from 2005 to 2018. Data on patient demographics, diagnostic imaging, thrombophilia testing, co-morbidities, treatment, outcomes were collected from health records.

Results:

A total of 242 neonatal patients were included. The distribution of 251 thrombotic episodes was: 144 portal vein, 15 cerebral sino-venous, 12 cardiac, 2 pulmonary embolisms, 5 renal vein, 40 extremity and central venous and 13 arterial. Additionally, 20 cases categorized as miscellaneous, consisted of clots in sites such as the basilic vein or umbilical artery, among other locations. Male and female percentages were 55% and 45%, respectively. Preterm to term infant ratio was 1.5:1. The average age at diagnosis was 13.2 days. 70% of thrombotic episodes were line-related, while infection and surgery were associated with 19% and 12% of episodes, respectively. 52% of the patients were blood group O. 30% of the patients received initial anticoagulation treatment, while the rest were managed conservatively. There was a trend towards higher rate of complete clot resolution in patients who received anticoagulation (48%) compared to those who did not (40%).

Conclusion:

This is one of the largest, single institution, epidemiologic studies of neonatal thrombosis. In our group of patients, portal vein thrombosis was most prevalent compared to other sites of thrombosis and catheterization was the commonest risk factor. There was a trend towards improved resolution with anticoagulation. Further prospective studies are required to confirm these findings.

Disclosures

No relevant conflicts of interest to declare.
Identification of Novel Therapeutic Targets in Atm-Deficient Lymphomas Using a Whole Genome CRISPR/CAS-9 Screen
Authors: Stankovic Tatjana , Davies Nicholas , Tee Louise J , Beggs Andrew D , Taylor Malcolm
Published: November 2019

ATM is a principal DNA damage response protein that synchronises a complex network of cellular responses to double stranded DNA breaks. ATM gene is recurrently mutated in a wide range of lymphoid malignancies, including B-cell chronic lymphocytic leukemia (CLL), T-prolymphocytic leukaemia (T-PLL), mantle cell lymphoma (MCL) and diffuse B cell lymphoma (DLBCL). ATM pathway is utilized by many DNA damaging agents and consequently inactivation of this pathway can lead to chemoresistance. Furthermore, in the absence of ATM tumour cells exhibit genomic instability that can lead to clonal selection and evolution even under current targeted treatments. Consequently there is clear need to understand dependency pathways in ATM-deficient tumours and apply tailored targeted therapies that will specifically eliminate those tumour cells.

We have previously presented a novel murine model of ATM-deficiency that spontaneously generate lymphoid tumours, mostly DLBCL. These tumours have been successfully propagated both in recipient mice and in vitro, where several cell lines have been generated.

Genome editing methods, such as CRISPR/CAS-9, permit the targeted disruption of specific genes. Protocols for genome wide screens have been developed based on this technology which can be used to identify genes that are essential for cellular survival. As such, these screens can be used to identify dependency pathways for tumours with specific genetic lesions.

Using lentiviral transduction we established two cell lines that stably expressed CAS-9. We then performed a genome wide CRISPR screen using the GeCKO library to identify novel therapeutic targets in these Atm-deficient tumours. This library consists of 130,209 unique single guide RNA (sgRNAs), targetting 20,611 genes including 1176 miRNAs.

A comparative analysis was performed of sgRNA drop-out following 15 cellular doublings. This revealed a number of pathways including those already known to be synthetically lethal with ATM deficiency, such as ATR and PARP. Pathway analysis of the top genes from this drop-out analysis identified oxidative phosphorylation, the spliceosome, ribosome biogenesis, N-glycan biosynthesis, pyrimidine metabolism and purine metabolism as the most significantly affected pathways. Furthermore, the drop-out screen revealed a number of miRNAs, including MiR-3470a, Mir-3971, MiR-669f and MiR-719.

These data provide a unique molecular assessment of the dependency of ATM-deficient lymphomas and provide a number of novel putative therapeutic targets for treating such tumours.

Disclosures

No relevant conflicts of interest to declare.
UAF1 DNA Binding Activity Is Critical for RAD51-Mediated Homologous DNA Pairing
Authors: Liang Fengshan , Miller Adam S , Tang Carolilne , Sung Patrick , Kupfer Gary M.
Published: November 2019

Background: In the Fanconi anemia (FA) DNA repair pathway, DNA damage induces the mono-ubiquitination of the FANCI-FANCD2 (ID2) heterodimer by the FA core complex through its inherent E3 ligase activity. The timely deubiquitination of ID2 by USP1-UAF1 deubiquitinase complex is also critically important for the FA DNA repair. UAF1 has a DNA binding activity, which is required for FANCD2 deubiquitination. UAF1 also enhances RAD51-mediated homologous DNA pairing in a manner that is dependent on complex formation with RAD51AP1. UAF1 deficient cells are impaired for DNA repair by homologous recombination (HR).The biochemical and cellular functions of UAF1 DNA binding activity in HR remain elusive.

Methods:UAF1 wild type and DNA binding mutant proteins were purified and used to define its biochemical properties in HR. In vitroD-loop formation and synaptic complex assembly assay were performed to discover the DNA binding of UAF1 in RAD51 recombinase enhancement. U2OS-DR-GFP cell lines with impaired UAF1 or RAD51AP1DNA binding were generated to examine HR efficiency and DNA damage resistance.

Results:UAF1 preferentially binds an HR-intermediate-like DNA substrate (D-loop, Fig.1). The DNA binding deficient mutant of UAF1 is unable to stimulate RAD51AP1 promotion of RAD51-mediated D-loop (Fig. 2) and the ability to recruit homologous DNA to form the presynaptic complex formation in HR (Fig. 3). In cells, the UAF1 DNA-binding mutant is compromised for the ability to repair DNA damage and to implement HR (Fig. 4). Such activity correlates with the ability to confer resistance to DNA cross linking agents such as mitomycin C (Fig. 4). The DNA binding of UAF1 and RAD51AP1 have a coordinated role in HR-directed DNA damage repair (Fig. 5).

Conclusions: UAF1 DNA binding activity is indispensable for its function in enhancing RAD51-mediated homologous DNA pairing within the context of the UAF1-RAD51AP1 complex. UAF1 DNA binding deficiency causes DNA damage sensitivity and impairs HR efficiency in cells.

Translational Applicability:Our findings reveal a critical role of UAF1 DNA binding in DNA repair and genome maintenance. The identification of UAF1's role in repair will enable targeted efforts to improve molecular approaches for FA therapy.

Disclosures

No relevant conflicts of interest to declare.