bluuebunny/crossref_metadata_2025_split huggingface dataset:
Screening for Immunosuppressive Genes Responsible for Resistance Towards CAR-T Cell Therapy in Cancer Cells
Authors: Valiullina Aygul , Petukhov Alexey , Sayarova Regina , Zhuravleva Margarita , Kletukhina Sevindzh , Malanin Sergey , Bulatov Emil , Rizvanov Albert 
Published: October 2019

Abstract
Despite expanding areas of clinical application for CAR-T cell therapy there is still lack of complete information about genes responsible for tumor resistance to this type of therapy. Widely known anti-PD-1 antibody drug Nivolumab and anti-CTLA-4 antibody drug Ipilimumab represent immunotherapeutics that activate the immune system against advanced melanoma and have proven superior to cytotoxic chemotherapy. However, in addition to existing ligands (i.e. CD80, CD86, PD-L1, PD-L2) there are supposedly other surface-expressed ligands stipulating the ability of cancer cells to suppress CAR-T efficacy. It may turn out that tumor resistance to CAR-T therapy is caused by previously unknown immunosuppressive genes. Revealing such genes could improve clinical outcomes by engineering enhanced CAR-T cells and antibodies capable of overcoming the gene-mediated resistance.
In this study we propose the use of CRISPR technique and NGS sequencing to validate this hypothesis and identify novel genes that could presumably determine resistance towards CAR-T cells. The strategy is based on genome-wide screening using human CRISPR/Cas9 Synergistic Activation Mediator (SAM) pooled library that utilize an engineered protein complex for the transcriptional activation of endogenous genes. CAR-T cells were generated from healthy donor T cells genetically modified by lentiviral vectors and expanded ex vivo.
Main steps of the experiment include:Amplification of pooled SAM library and use of NGS to confirm library representation.Generation of pooled lentiviral vectors using SAM library and transduction of HeLa cells.Generation of CAR-T cells.Co-cultivation of CAR-T and modified HeLa cells, selection of resistant cells.PCR analysis of sgRNA-containing fragment, followed by cloning of amplicons and NGS of amplicon library.Validation of identified genes to confirm resistance towards CAR-T cells.
We generated a polyclonal CD19-positive HeLa cell line expressing dCas9-VP64 fusion and activation helper protein. This cell line was transduced with Human CRISPR Activation Library (SAM-3 plasmid system) and incubated with anti-CD19 CAR-T cells in order to select resistant cells and identify resistance-determining genes by NGS.
Work supported by RFBR grant 18-315-00049 to A.P. and performed in accordance with Program of Competitive Growth of Kazan Federal University.
References: A. Titov, A. Petukhov, A. Staliarova, D. Motorin, E. Bulatov, O. Shuvalov, S. Soond, M. Piacentini, G. Melino, Y. Zaritskey, N. Barlev. The biological basis and clinical symptoms of CAR-T therapy-associated toxicities. Cell Death & Disease (2018)

Disclosures
No relevant conflicts of interest to declare.
Genetic Mutations By Next-Generation Sequencing in Childhood Biphenotypic Acute Leukemia
Authors: Baek Hee Jo , Lee Jun Hyung , Lee Hyun Gyung , Kim Ho Sung , Shin Myung Geun , Kook Hoon 
Published: October 2019

Abstract
Introduction: Biphenotypic leukemia (BAL) or acute leukemias of ambiguous lineage (ALAL) is an uncommon manifestation of childhood acute leukemia. The aim of this study is to further assess clinicopathological characteristics and genetic mutation of de novo BAL or ALAL.
Materials and Methods: Among 377 patients ≤ 18 years old who were diagnosed with de novo Acute leukemia from 2001 to 2017, 12 patients (3.2%) satisfied the definition of BAL by the European Group for the Immunological Classification of Leukaemias (EGIL) criteria or ALAL by World Health Organization (WHO) criteria. By the EGIL criteria, 11 patients were diagnosed as BAL: B-cell/myeloid (B/My) phenotype, 5; T-cell/myeloid (T/My) phenotype, 5. By the 2008 WHO criteria, 7 patients (1.9%) remained as ALAL: acute undifferentiated leukemia (AUL), 2; MPAL, 5. The remaining 5 BAL patients were redirected as ALL (n=3) and AML (n=2). Except one case with ALL by WHO criteria, 11 cases were examined by next generation sequencing using 60 gene panels.
Results: In 5 patients with MPAL by the WHO criteria, a total of 31 distinct mutations were identified in 26 different genes. One case had BCR-ABL gene rearrangement. PTPN11 and NDS1 were recurrent mutation in 2 patients. In 2 patients with AUL, a total of 8 distinct mutations were identified in 8 different genes. In 2 cass of AML, 19 distinct mutations were identified in 15 differnet genes. FLT3-ITD was recurrent mutation. In 2 case of ALL, 17 distinct mutations were identified in 14 differrent genes. The median age of the patients at diagnosis was 8.6 years (range, 0 months-17.8 years). Six patients initially received AML-directed induction therapy, whereas 6 patients received ALL-directed induction therapy. Overall, 7 of the 12 (58.3%) patients achieved a complete remission (CR) after their initial induction therapy. ALL-directed induction chemotherapy was associated with higher chances of achieving a CR as compared to AML-directed regimens (100.0% vs. 16.7%; P =0.015). Nine patients except three cases (1, progression of disease; 1, intracranial hemorrhage during chemotherapy; 1, in continuous CR over 7 years chemotherpay only) underwent an allogeneic hematopoietic stem cell transplantation. With a median follow-up of 5 years, 5 year overall survival (OS) rate was 51.1±15.8% and event-free survival (EFS) rate was 51.9±15.7%. MPAL by WHO criteria seemed to have a better OS and EFS than AUL (both for 75.0 ± 21.7% vs. 0.0%; P=0.008).
Conclusions: BAL or ALAL contains heterogeneous genetic mutation. ALL-directed induction chemotherapy was much better in achieving CR than AML-directed induction chemotherapy. And, patients with MPAL as defined by 2008 WHO definition have better clinical contcome compared with patients with AUL.

Disclosures
No relevant conflicts of interest to declare.
Cost Analysis of Hematopoietic Autologous Transplantation with and without DMSO Cryopreservation
Authors: Sarmiento Mauricio , Beffermann Nicole , Solar Ximena , Veronica Jara , Katherine Soto , Arellano Silvana , Bertin Pablo , Pizarro Isabel , Lorca Carla , Rivera Elizabeth , Galleguillos Mauricio , Rodriguez Isabel , Ocqueteau Mauricio 
Published: October 2019

Abstract
Introduction.
The use of cryopreservation of hematopoietic precursors for autologous or allogeneic transplantation with DMSO is a procedure commonly used in many countries, but it is expensive and is not exempt of risks, since serious adverse events have been described. Our group recently published a comparative study between two centers and showed that avoiding DMSO cryopreservation favors better transplantation tolerance, significantly shorter hospitalizations, fewer episodes of febrile neutropenia and mucositis. In this report we show the financial analysis of both modalities of CRYO and Non CRYO preserved transplants.
Methodology
Database of the adult hematopoietic transplant program of our institution was retrospectively analyzed. Since Non CRYO modality was initiated in our institution on 2015, we compared 3 years before and after that date, assigning two groups (CRYO and Non CRYO). Costs data of mobilization, apheresis, hospitalization, freezing and blood banking were analyzed.
Results
Between 2013 and 2018, 90 autologous hematopoietic transplants were performed, 41 CRYO and 49 Non CRYO. The average cost of mobilization therapy with filgrastim and the use of plerixafor did not differ between groups (p = 0.26). The number of apheresis needed to achieve a satisfactory count was higher in CRYO (median 1.5 vs 1.2 p = 0.001) with 55% higher cost on average. The cost of transplant hospitalization including antibiotics costs, blood banking costs, analgesics requirements and nutritional parenteral support was lower in Non CRYO (minimum cost USD 8000, maximum USD 160000 average value USD 20.000) than in CRYO (minimum cost USD 11.000, maximum USD 200.000, average value USD 32000) (p = 0.001).
Conclusions
In addition to promoting better patient tolerance, Non CRYO modality in our country has lower cost. This information has important relevance for health systems of developing countries and can promote better access to transplant.

Disclosures
No relevant conflicts of interest to declare.
Use of Eculizumab for the Treatment of Hyperhaemolysis in Pregnancy in Sickle Cell Disease: A Case Report
Authors: Kirui Laura Chebet , Scully Marie , Mcqueen Norma , Porter John , Eleftheriou Perla 
Published: October 2019

Abstract
Hyperhaemolysis is a rare but well recognized complication of transfusion in sickle cell disease, associated with increased mortality. The pathogenesis is not well understood and is likely to be multifactorial. There is no gold standard management of hyperhaemolysis; immunosuppression with steroids and intravenous immuoglobulins has been the mainstay of treatment. More recently Rituximab and Eculizumab are increasingly used. Hyperhaemolysis in pregnancy is an even more complex problem with compounded risk to the mother and foetus.
This case reports a 29 year old female with HbSS who presented with hyperhaemolysis in pregnancy at 25 weeks gestation, 7 days after a red cell transfusion for a vaso-occlusive crisis. The patient received 1 g methylprednisolone and 2g/kg immunoglobulins in divided doses. Despite this, she required further blood transfusion as the haemoglobin was declining further and was symptomatic for her anaemia. Her haemoglobin reached a nadir level of 39 g/l. Therefore she was treated with Eculizumab 900 mg iv pre transfusion to prevent exacerbation of the haemolysis triggered by the transfusion. Her haemoglobin stabilised and the haemolysis resolved within 48 hours. She delivered safely at 34 weeks gestation.
The DAT remained negative throughout and no new red cell antibodies were detected. Urine HPLC confirmed the presence of HbA and HbS in the urine, confirming the diagnosis. The urine HPLC 48 hr post Eculizumab and 2 further red cell units was clear, demonstrating no further haemolysis with the additional red cells given.
This is the first case report detailing the use of Eculizumab for the management of hyperhaemolysis in pregnancy in sickle cell disease. The patient tolerated the treatment which resulted in resolution of the haemolysis, with safe delivery at 34 weeks gestation. The possible role of complement activation in hyperhaemolysis is discussed. The use of Eculizumab in other case reports of hyperhaemolysis is reviewed. The ideal dosing schedule remains uncertain. This report strongly suggests that Eculizumab should be accessible and considered in severe cases of hyperhaemolysis which are refractory to standard treatment. It also suggests safe usage in pregnancy in sickle cell patients.

Disclosures
Scully: Novartis: Honoraria, Other: Member of Advisory Board, Speakers Bureau. Porter:Cerus: Honoraria; Novartis: Consultancy; Agios: Honoraria.
Pneumococcal Vaccination (PCV13) Prior to Initiation of Immunosuppressive Therapy in Patients with Hematological Malignancies Is Associated with Decrease Morbidity and Decrease Hospitalizations
Authors: Draliuk Regina , Mintsman Iveta , Shnaider Alla , Viky Shuras , Benami Rotem , Salem Noga , Valkovsky Olga , Ballan Haj Mouna , Shabad Evelyne , Kotler Antonio , Mishchenko Elena , Preis Meir 
Published: October 2019

Abstract
Introduction: Pneumococcus is a common bacterium that can cause serious infections, such as pneumonia, meningitis or blood stream infection in patients receiving immunosuppressive therapy. Infection by pneumococcus bacteria can be life-threatening leading to hospitalizations, complications and even death. In this work we have established a system base intervention that provides pneumococcal vaccine (Prevenar 13) for patients before starting chemotherapy and/or biological treatment.
Methods: The intervention program included a set of measures: 1. Standard operating procedure (SOP) for vaccination of patients with hematological disorders. 2. Increasing the awareness of the medical and nursing staff regarding the importance of administering the vaccine before chemotherapy/biological treatments. 3. Purchasing vaccines by the hospital. 4.Coaching patients about the importance of receiving the vaccine before starting treatment. 5. Building a computerized record for monitoring and controlling the administration of the vaccine before starting treatment. 6. Transferring the information about the vaccine to the patient outpatient providers. 7. Documentation of the vaccine in the patient's electronic medical record. 8. Monthly review of new patients and vaccination before starting treatment.
Results: In 2015, before intervention, only about 25% of patients were vaccinated appropriately against pneumococcus. With the start of the quality improvement project in April 2016, there was a gradual increase in the number of vaccinated patient before starting treatment. In December 2016, all new patients before starting treatment were properly vaccinated against pneumococcus. Although there was a significant increase in the number of patients at the institute in 2017 there was a 35% decrease in the number of patients hospitalized for pneumonia or sepsis compared to 2015. In addition, there was a 41% decrease in the number of hospitalizations with pneumonia or sepsis in the institute's patients. There was also a significant trend towards decrease in mortality (11% vs. 7%).
Conclusions: The intervention program included: The creation of cooperation at several levels, medical, nursing and pharmacological that created cooperation between the medical center and the outpatient community clinics which led to the creation of a system that ensures that the patient will be vaccinated prior to starting immunosuppressive therapy. As a result of the intervention at the Institute and the data that was examined over 3 years, there has been a significant decrease in the rate of hospitalizations of patients due to pneumonia or sepsis.

Disclosures
No relevant conflicts of interest to declare.
A Human Fibrinogen Variant with Impaired StaphylococcusAureus Binding Function Promotes Host Survival during Septicemia in Mice
Authors: Negron Oscar , Koopman Jaap , Weggeman Miranda , Huisman Linda , Stang Amanda , Flick Matthew J. 
Published: October 2019

Abstract
Staphylococcus aureus is a common gram-positive bacterium that is a causative agent for a number of life-threatening diseases. The emergence of antibiotic resistant strains of S. aureus (i.e., methicillin and vancomycin resistant) emphasizes a renewed need to better understand host-pathogen interactions for defining novel strategies to combat infection. The coagulation factor fibrinogen is a major target of S. aureus virulence factors as numerous fibrinogen-binding proteins are produced by the bacterium. Our previous studies, using FibgΔ5 mice that express a fibrinogen mutant lacking the binding motif for clumping factor A (ClfA) indicated that binding to this site is a major mechanism of pathogen virulence during acute septicemia. Specifically, in vivo studies showed that FibγΔ5 mice have improved survival following S. aureus bacteremia suggesting that this interaction is critical for S. aureus virulence. To investigate a potential clinical translation of these findings, we utilized variants of fibrinogen γ', an alternatively sliced form of fibrinogen (comprising 8-15% of total plasma fibrinogen) that has structural similarities to fibrinogenγΔ5. Specifically, we analyzed human fibrinogen γ' heterodimer (hFib γ-γ') and recombinant fibrinogen γ' homodimer (rFib γ'-γ'). For bacteria under stationary phase growth conditions, ClfA mediated binding to immobilized fibrinogen whereas under exponential growth conditions binding to fibrinogen was largely independent of ClfA. Interestingly, hFib γ-γ' supported both ClfA dependent and independent binding to fibrinogen. However, studies performed with rFib γ'-γ' showed that this variant almost completely abolished ClfA dependent and independent binding to fibrinogen. Similar results were observed in analyses of fibrinogen-dependent 'clumping' in solution. Fibrinogen-dependent 'clumping' was fully dependent on ClfA in both stationary and exponentially growing S. aureus. Importantly, reconstitution of fibrinogen-deficient mice or WT mice with hFibγ' provided a significant prolongation in host survival relative to mice reconstituted with hFibγ indicating that hFibγ' confers protection alone or when present with other fibrinogen species. These findings provide the proof-of-concept that naturally occurring fibrinogen variants could offer significant therapeutic potential against infection and potentially other diseases.

Disclosures
Koopman: Fibriant: Research Funding. Weggeman:Fibriant: Research Funding. Huisman:Fibriant: Research Funding.
Evidence for an Alternative Mechanism Suppressing Hepcidin during the Recovery from Hemorrhage-Induced Anemia
Authors: Sardo Ugo , Perrier Prunelle , Billore Benjamin , Cormier Kevin , Kautz Leon C. 
Published: October 2019

Abstract
Introduction
The liver-produced hormone hepcidin regulates the body iron stores. Its expression is induced by iron and inflammatory cytokines but repressed by the erythroid regulator erythroferrone (ERFE) when erythropoietic activity intensifies during anemia. Although Erfe-deficient mice fail to appropriately suppress hepcidin during the first 24h following hemorrhage, these mice still recover from anemia with a few days delay suggesting that another mechanism compensates for the absence of ERFE. We therefore decided to study the kinetic of hepcidin during the recovery from anemia induced by bleeding in Erfe-deficient mice.
Material and methods
Six week-old C57BL/6 WT and Erfe-deficient mice were phlebotomized (500 μL) and analyzed 1, 2, 3, 4, 5 and 6 days after phlebotomy until full recovery.
Results
Liver hepcidin mRNA expression was suppressed 5-fold one to five days after phlebotomy in WT mice. In contrast with the sustained inhibition of hepcidin, serum ERFE concentration progressively decreased after 24 hours to reach its baseline at day 4. Interestingly, although hepcidin levels were unchanged after 24 hours, Erfe-deficient exhibited significantly reduced hepcidin levels after 48 hours. Hepcidin mRNA and protein levels were comparable to those of WT mice 2 to 5 days after phlebotomy. Interestingly, the repression of hepcidin occurred without any change in phosphorylation of the effectors Smadd1/5/8 and in hepatic expression of the BMP/SMAD target genes Atoh8, Smad7 and Id1. Similarly, mRNA expression of the proposed negative regulators of hepcidin Gdf15, Twsg1 and Gdf11 was not increased in the spleen and the bone marrow of phlebotomized mice compared to control mice. Finally, disruption of the erythroid compartment by irradiation or injection of carboplatin prevented the suppression of hepcidin in WT and Erfe-deficient mice.
Conclusion
An alternative mechanism regulates hepcidin independently of iron and ERFE during stress erythropoiesis. Our data suggest that a second yet unknown erythroid regulator of hepcidin may exist.

Disclosures
No relevant conflicts of interest to declare.
Implications of TP53 alterations for Therapy Response in Multiple Myeloma
Authors: Munawar Umair , Barrio Santiago , Roth Markus , Einsele Hermann , Bargou Ralf C , Stuehmer Thorsten , Kortum Martin 
Published: October 2019

Abstract
Deletion of the tumor suppressor gene TP53is significantly associated with an unfavorable clinical course of Multiple Myeloma (MM). In addition, point mutations that abrogate p53 function similarly shorten survival. Most recently 'double hit', bi-allelic TP53inactivated MM was identified as an ultimate high risk feature of MM, affecting 3.7% of newly diagnosed MM patients (NDMM, Walker et al., Leukemia 2018).
For TP53genetic analysis we combined data from two targeted sequencing M3P cohorts with targeted sequencing and FISH data, one of NDMM (n=142, Kortüm et al. Blood Cancer J. 2016), and one of multirefractory patients (rMM, n=40; Kortüm et al. Blood 2016). We also included two independent cohorts with paired NDMM and rMM, one with mutational data (n=43; Corre J et al 2017) and one with paired FISH analysis (Merz M et al. Haematologica 2017). We confirmed an increase of mutations in TP53 (8% NDMM / 16.9% rrMM), as well as del17p (12.6% / 22%). Similarly, mono- (12.7% / 22.5%) and bi-allelic events (5.6% / 17.5%) both demonstrated a significant increase over time (Figure, top). Importantly we observed deletion after mutation and vice versa in our cohorts.
Next we established an AMO-1 MM cell line model (TP53+/+) mimicking mono and bi-allelic TP53-inativation. After CRISPR/Cas9-mediated TP53destruction, we introduced a modified Sleeping Beauty (SB) vector with two separate expression cassettes (p53 wt / wt and mutant p53 (R282W or R175H). Functionality of the p53 system was confirmed using nutlin-3, as inhibitor of MDM2-p53 interaction, as described elsewhere.
Results: Doxorubicin and Melphalan are commonly used compounds in the treatment of MM. The IC50 of Melphalan in AMO1 naïve cells was 5µM and 5.5 µM after the reintroduction of 2 copies of wt TP53 in the KO model, with cell viability at 10µM of 30% and 31% respectively, measured by AlamarBlue Assay. Strikingly, we observed significant resistance induction in our hemizygous systems (del/wt p53; cell viability at 10µM 60% and mut/wt (62%). This furthermore increased within our homozygous models (TP53 del/del (85%); TP53 mut/del 80%) (Figure, bottom). Similar results were observed under doxorubicin treatment. Remarkably, this effect was absent against proteasome inhibition.
Conclusions
Here we present first evidence of TP53 inactivation impacting drug response to Melphalan and Doxorubicin, which might lead to the clonal selection of MM subclones harboring increased risk. The fact, that response to proteasome inhibition was not affected in our model might, at least in part, might explain their ability to confine high risk in MM.
Figure. Figure.

Disclosures
No relevant conflicts of interest to declare.