bluuebunny/crossref_metadata_2025 huggingface dataset:
The Molecular Genetic Analysis of Coagulation Factor XI (FXI) Deficiency in Two Chinese Patients
Authors: Zhou Rong-Fu , Li Qian , Zhou Min
Published: October 2019

Objective: To investigate the molecular pathogenesis of two coagulation factor XI (FXI) deficiency patients.

Methods: The diagnosis was validated by coagulant assays: APTT and correct test, PT, INR and coagulation factors activities. Coagulation factor activity were tested with clotting assay. The patients' DNA were extracted and all exons and flanking sequences of FXI gene were amplified using PCR. After purified, the products were sent for sequencing directly, the mutations were detected by comparing with wild sequences and analyzed using some bioinformatics software.

Results: The two patients were diagnosed as coagulation factor XI deficiency due to prolonged APTT and low activities of coagulation factor FXI. The results of APTT, FXI:C was 88.1s, 1.1% and 107.1s, 3.8% , respectively. Genetic analysis found that compound heterozygous mutations g.1251-1G > A and g.1271delT in the first patient and the sequencing results of TA plasmid clones showed that the two mutations were located on different single strands of chromosomes. Double heterozygous mutations g.1070A >G and g.1446C > G were detected in the second patient, resulting in Lys357Arg and Cys482Stop. Software analysis indicated the mutations probably brought amino acid sequence changed, protein features affected and splice site changed.

Conclusion: Compound heterozygous mutations g.1251-1G > A, g.1271delT and g.1070A > G , g.1446C > G had been identified in two coagulation factor XI deficiency patients, which might be the cause of their prolonged APTT and low FXI:C. To the best of our knowledge, the four mutations are reported for the first time in the literature.

Figure. Figure.

Disclosures

No relevant conflicts of interest to declare.
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

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

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

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

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

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

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

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

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.
The Optimal Cut-Off Level for Hemoglobin A2 to Differentiate between Sickle Cell Disease Genotypes
Authors: Al Shueili Fayiz , Al-Khabori Murtadha K. , Al-Kindi Salam , Wali Yasser , Al-Zadjali Shoaib
Published: October 2019

Objectives: Hemoglobin A2 (HbA2) is elevated in the presence of beta thalassemia trait and it is used as an indicator of its presence. High-Performance Liquid Chromatography (HPLC) overestimates HbA2 in patients with Sickle Cell Disease (SCD) due to the co-elution of HbS in HbA2 column. The optimal cut-off level of HbA2 to indicate the presence of beta thalassemia trait has not been well established in patients with SCD. We aim to define the optimal cut-off level of HbA2 to differentiate between SS and S/Beta SCD genotype variants.

Methods: In this cross-sectional study, we included 241 patients with SCD who have either SS or S/Beta genotype based on their HPLC and the Sickledex test®. The diagnoses were confirmed by the direct sequencing of PCR amplified products of all exons, exon-intron junctions and promoter region of the beta globin gene. We retrieved the following clinical and laboratory variables from the electronic health records: age, gender, Hemoglobin (Hb), Mean Corpuscular Volume (MCV), Red Blood Cell (RBC) count and HbA2. We used the receiver operating curve (ROC) analysis to obtain the optimal cut-off level of HbA2 using the maximum sensitivity and specificity (Youden criteria). All descriptive and analytical statistics were performed using R program. The ROC analysis was performed with the "OptimalCutpoints" package available in R program.

Results: Among the 241 patients included in the analysis, SS and S/Bthal patients were 81% and 19% respectively. Male to female ratio was 126:115. Fifty-two percent were using hydroxyurea. The median HbA2 level was 4.5% (Range: 0.0-6.5) in the SS group and 6.5% (Range: 3.5-8.2) in the S/Bthal group. The median Hb, MCV and RBC count was 9.4 g/dL (Range: 5.3 - 15.0), 75 fL (55 - 111) and 3.8 *1012/L (1.9 - 6.3) respectively for the SS group; while it was 9.7 g/dL (6.6 - 12.3), 68 fL (57 - 86) and 4.2 *1012/L (2.5 - 5.4) respectively for the S/Bthal group. The optimal cut-off level for HbA2 was estimated to be 5.7% using a sensitivity of 91% and a specificity of 92%. The positive and negative predictive values were 75% and 98% respectively. The discrimination estimated using the Area Under the Curve (AUC) was 0.936 (95% Confidence Interval: 0.878-0.994).

Conclusions: The optimal cut-off HbA2 level to differentiate SCD with the S/Bthal genotype from the SS genotype is 5.7% with a high sensitivity, specificity and discrimination. The unexpected overlap in the MCV and the RBC count is likely related to the high rate of Alpha thalassemia trait in the analyzed population. Incorporation of the presence of alpha thalassemia trait in the analysis may improve the discrimination of MCV and RBC count.

Disclosures

No relevant conflicts of interest to declare.
Proteomics Analysis Reveals Protein Panels That Are Associated with Prediction to Tyrosine Kinase Inhibitors Response, Bone Marrow Transplant, Survival and Disease Outcome of Chronic Myeloid Leukemia Patients
Authors: Alaiya Ayodele , Aljurf Mahmoud , Shinwari Zakia , Alsharif Fahad Z. , Alzahrani Hazza A. , Ahmed Syed Osman , Owaidah Tarek , Hanbali Amr Suleiman , Shaheen Marwan , Al Fraih Feras , Elfakih Riad , Alhayli Saud , Hashmi Shahrukh , Rasheed Walid , Almhareb Fahed , Chaudhri Naeem A.
Published: October 2019

Clinical and molecular diagnosis of most hematological malignancies including Chronic Myeloid Leukemia (CML) can be accurately made. However, prediction of treatment response and estimation of disease survival period eludes the currently available tools for patient care. Quantitative expression proteomics can potentially be developed as effective tool to monitor therapy response towards achieving personalized medicine for CML patients.

We have over 10 years follow up period for some of the CML patients, and the majorities of them are alive and doing well on Imatinib. On the other hand, a small fraction of the patients were switched to alternative Tyrosine Kinase Inhibitors (TKI) and some underwent bone marrow transplants (BMT).

Our follow up results stratified all 37 analyzed newly diagnosed CP-CML patients into 5 distinct cohorts including 52% on Imatinib, 12% on Nilotinib, 9% on Dasatinib, and 15% BMT, while 12% of the patients had disease-related mortality. Kaplan-Meier survival curve showed no significant difference between all patients on TKI and BMT that were alive under a follow up period of 4-11 years compared. On the other hand 3 of 4 patients had disease related deaths less than 2 years of diagnosis. Only one patient survived for almost 10 years (Figure 1). Besides survival analysis, peripheral blood samples obtained from the patients at time of diagnosis were subjected to expression proteome analysis using label-free quantitative liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). A subset of significantly differentially expressed proteins was able to distinctively discriminate samples into their respective treatment response and disease outcome groups based on unique protein expression signatures (P<0.05 and > 2- ∞- fold change, (Figure 2). Some of the identified proteins were implicated in hematological diseases including CML as potential biomarkers using Ingenuity Pathway Analysis.

These protein signatures once validated in larger sample cohorts might be capable of prediction of molecular response, choice of therapy and disease outcome for CML patients. Therefore; allowing for identification of would be high risk patients that might potentially benefit from aggressive treatment at point of diagnosis pre initiation of conventional therapy. Altogether our findings indicate that analysis of panel of protein markers have the potential of clinical utility for prediction of response to therapy, disease survival and objective prognostication of disease outcome, thus bringing personalized medicine closer to CML patients.

Disclosures

No relevant conflicts of interest to declare.
Assisted Administration of Subcutaneous Rituximab to the Patient's Home. Significant Reduction of Indirect Costs Incurred By Care Recipients and Unpaid Caregivers Together with a Total Adherence to the Treatment Schedule and Same Safety
Authors: Iuliano Francesco , Iuliano Eleonora , Rossi Marco
Published: October 2019

Dec 2018, 7 consecutive patients, ( CLL n = 1; FL n = 3; DLBCL n= 1 AEA n=1,ITP n=1), were enrolled in the study. Median age of the patients was 68 years (range, 28-87). The pharmacoeconomic analysis included assessment of direct costs ( hospital inpatient, physician inpatient, physician outpatient, emergency department nursing home care, specialists' and other health professionals' care, diagnostic tests, prescription drugs and drug sundries, and medical supplies),indirect costs incurred by care recipients and unpaid caregivers, including time, productivity and travel cost.

Results Rituximab subcutaneous formulation was administered at a fixed dose of 1400 mg with a frequency related to the type and the phase of the treated disease. Before starting MabThera subcutaneous injections, all patients received beforehand, a full dose of Rituximab by intravenous infusion. Premedication with an anti-pyretic an antihistaminic and prednisone , was given orally in the evening before and the morning of the subcutaneous administration the infusion was carried out by the staff of the home care unit together with a doctor. All the procedure was controlled by the hospital specialist through a smart-phone. Median age of the patients was 58 years (range, 32-83). Median number of courses delivered to each patient was 6 (range, 4- 12) .Adherence to treatment was 100%.No Infusion related adverse reactions have been observed.

Conclusion

In our experience, despite the high percentage of elderly patients of whom 36% living in rural area, it was possible to give treatment to all patients with high adherence and satisfaction. There has been a reduction in direct medical costs due to less use of hospitalization, a reduction of indirect costs by 70% due to the lower number of working days lost and a drastic reduction of travel costs, with the same safety of administration.

Disclosures

No relevant conflicts of interest to declare.
Therapeutic Disassembly of Leukemogenic Enhancer Complexes
Authors: Forbes Lauren , Isaac Krasnopolsky , Minuesa Dinares Gerard , Kharas Michael G. , Kentsis Alex
Published: October 2019

Despite a detailed understanding of the genetic mechanisms of acute myeloid leukemia (AML), treatments remain deeply inadequate. Aberrant activity of hematopoietic transcription factors and their co-activators has been implicated in the expression of leukemogenic genes, providing a compelling therapeutic target. Hematopoietic transcription factor MYB and its co-activator CREB-binding protein/P300 (CBP/P300) are required for the initiation and maintenance of a variety of acute leukemias. Using protein engineering methods, we have developed cell-penetrant peptidomimetic inhibitors that interfere with this interaction by targeting the KIX domain of CBP/P300. A second-generation inhibitor based on the chimeric interaction of MYB and CREB, termed CRYBMIM, binds CPB/P300 KIX with 10-fold higher affinity and exhibits logarithmic cell killing of most leukemia subtypes, while sparing healthy human hematopoietic progenitor cells (Figure 1). Comparative gene expression, chromatin mapping, and protein-protein interaction mapping elucidate the molecular mechanisms by which CRYBMIM dismantles oncogenic enhanceosomes, leading to the downregulation of leukemogenic gene expression in leukemia but not normal cells. Finally, we have developed a peptide-protein conjugation strategy for in vivo delivery of peptidomimetics by polyvalent reversible conjugation to transferrin (TF). We found that TF-peptide conjugates are readily taken up by AML cells and accumulate in AML nuclei, supporting their translation into new therapeutic strategies for patients. Taken together, this work advances an effective therapeutic strategy for therapeutic disruption of leukemogenic enhancer complexes, and defines mechanisms of aberrant gene expression control in AML.

Disclosures

No relevant conflicts of interest to declare.
Efficacy of Maintenance Therapy after Autologous Hematopoietic Stem Cell Transplantation in High-Risk Aggressive Lymphoma: A Single-Center Prospective Non-Randomized Study
Authors: Chen Yongquan , Zheng Zhihong , Luo Xiaofeng , Ren Jinhua , Geng Haili , Issa Hajji Ally , Chen Zhizhe , Hu Jianda , Yang Ting
Published: October 2019

OBJECTIVE: Relapse after autologous hematopoietic stem cell transplantation (ASCT) is still challenging for high-risk aggressive lymphoma. This study was to investigate the efficacy and safety of maintenance therapy post-ASCT.

METHODS: From June 2009 to March 2018, patients with high-risk aggressive lymphoma in our hospital were treated with maintenance therapy post-ASCT according to the patient's wishes, and then assigned to maintenance group or observation group. The end point of follow-up was disease progression or death. The 3-year overall survival (OS) and disease-free survival (DFS) were compared between these two groups.

RESULTS: A total of 79 patients were enrolled, with a median age of 38 years (8-64 years), 50 males and 29 females. IPI score ≥2 points in 28 cases, stage III-IV in 58 cases, B symptoms in 21 cases, Extranodal lesions ≥ 2 in 17 cases, bone marrow infiltration in 15 cases, and 24 cases with partial remission (PR) prior to ASCT. In addition to 2 cases of early death, the remaining 77 patients with lymphoma were underwent successfully transplantation and achieved CR post-ASCT. 54 patients were assigned to the observation group, and 23 patients to the maintenance group, including 17 with rituximab and 6 with DPP/DCEP-G alternation regimen. There were no s ignificant differences in gender, age, pre-transplant disease status, and hematopoietic reconstitution between the two groups. The main causes of death were disease recurrence. No serious adverse reactions occurred during the maintenance treatment period. After a median follow-up of 616 days (12-2854 days), the relapse rates of the observation group and the maintenance group were 49.1% vs 12.9%, and the DFS of the 1st, 2nd, and 3rd years were 62.8% VS 93.8%, 48.1% VS 87.1%, and 45.3% VS 87.1% (P = 0.007), respectively. The OS of the 1st, 2nd, and 3rd years were 89.3% VS 100%, 83.2% VS 92.9%, and 76.2% VS 92.9% (P=0.212), respectively.

Conclusion: Maintenance therapy post-ASCT could reduce the risk of relapse and promote disease-free survival, which deserves further investigation.

Disclosures

No relevant conflicts of interest to declare.
Contribution of Flow Cytometry Immunophenotyping in Diagnostic of Acute and Chronic Leukemias
Authors: Paiva Aldair Sousa , Paiva Hugo Diogenes De Oliveira , Cavalcanti Geraldo Barroso , Silveira Lenilton Silva , Silva Lorena KF , Gil Erica Aires , Vasconcelos Roberto C , Bahia Frank , Freitas Rodrigo Villar , Silva Dany GKC , Sales Valéria SF , Jardim Alessandra Suelen , Soares Victor lima , Marciel James Farley
Published: October 2019

BACKGROUND: Today immunophenotyping by flow cytometry is an useful adjunct to cytomorphologyc analysis to correct diagnostic of leukemias. It provides objective and quantitative data allowing for a high level of sensitivity detection and better characterization of acute and chronic leukemias. The purpose of this study was to demonstrate the contribution of the immunophenotyping by monoclonal antibodies (Mo.Ab.) in leukemic cells from patients with acute and chronic leukemias. METHODS: Analyzed 76 patients with leukemias before the treatment. The diagnostic of leukemias was based on the morphological and immunophenotyping analysis of leukemic cells from peripheral blood and bone morrow. The cytomorphologycal analysis was based on French - American - Britsh criteria (FAB classification) in blood and bone marrow films stain by leishmann and the immunophenotyping by flow cytometry with a panel of Mo.Ab. specific to acute leukemias as: CD1a, CD2, CD3, CD4, CD5, CD8, CD7, CD10, CD13, CD19, CD20, CD103, CD22, CD33, CD34, CD117, CD38, HLADR, TdT, anti-mieloperoxidase, anti-IgM and anti-kappa and lambda light chain. Further clinical and laboratory information as age, sex, presence of tumoral mass, lymphadenopathy, hepatosplenomegaly, hemoglobin determination, total and specific white cell count and cytomorphological analysis of blood film and bone morrow smear. RESULTS: Thirty four patients presented acute myeloid leukemia (AML), twenty acute lymphoblastic leukemia (ALL), nineteen B-cell chronic lymphocytic leukemia (B-CLL), two T-cell chronic lymphocytic leukemia and one case was hairy cell leukemia (HCL). Males were more frequently found in all types of leukemias. ALL were more observed in children (age < 15 years old) and in AML however were more frequently in adults patients. The chronic leukemias were presented in patients with 50 years old or more in all cases. The correlation between the immunophenotyping and clinical pathological profile of these leukemias demonstrated that ALL were more associated to lymphadenopathy, AML to hepatosplenomegaly, and CLL to lymphadenopathy and high count of white cells in peripheral blood. The thrombocytopenia and anemia were found in more cases of acute leukemia. CONCLUSION: This date suggest that today the immunophenotyping by flow cytometry is an important methodology to diagnostic and classification of leukemias.

Disclosures

No relevant conflicts of interest to declare.
Direct Oral Anticoagulant Vs Vitamin K Antagonist As Anticoagulant Therapy in Patients with Inherited and Acquired Thrombophilia
Authors: Olivera Pável E , Campoy Desirée , Velasquez César A , Flores Katia , Ene Gabriela S , Canals Tania , Artaza Gonzalo , Subira Maricel , Vila Sofía , Sierra Jorge , Salinas Ramón
Published: October 2019

BACKGROUND

The patients with venous thromboembolism (VTE) and thrombophilia have historically been treated with vitamin K antagonist (VKA) therapy. Nowadays, direct oral anticoagulants (DOAC) are broadly used in the treatment and prevention of VTE. However, the use of DOAC in the treatment of inherited and acquired thrombophilia remains unclear.

AIMS

To assess the safety and effectiveness in both cohorts according to DOAC or VKA treatment in a cohort of patients with VTE and thrombophilia.

METHODS

We included adult patients with VTE and thrombophilia treated with DOAC or VKA (2016-2018) from the beginning of treatment. Patients in the VKA group received acenocoumarol. Demographic, laboratory, thrombophilia diagnosis data and clinical presentation of VTE were collected. All patients with history of at least one VTE episode were included. The diagnosis of VTE was established by a positive finding of duplex sonography. Pulmonary embolism was diagnosed based on positive results of high resolution computed tomography. Inherited trombophilia included homozygous and heterozygous Factor V Leiden and prothrombin G20210A, natural anticoagulant deficiencies (protein C, protein S, or antithrombin) or combined defects. Patients with antiphospholipid syndrome (APS) were considered to have acquired thrombophilia. Bleeding events were classified according to ISTH criteria.

RESULTS

Among 181 patients, 96 (45.5%) were treated with DOAC (6 Dabigatran, 45 Rivaroxaban, 12 Apixaban and 33 Edoxaban) and 85 were treated with VKA. Among patients receiving VKA, the time in therapeutic range was 58.5%. Mean Follow - Up (FU) were 8.1 and 6.8 months (DOAC vs VKA, p:0.77). Demographic characteristics, thrombophilia subtypes and clinical features of VTE are summarised in table 1. The most frequent previous anticoagulant treatment was low molecular weight heparin (DOAC 71.6% vs VKA 98%, p:0.15). During FU, 9 recurrent VTE episodes were reported: 2 (2.1%) vs 5 (5.9%) [DOAC vs VKA, p: 0.57] and 5 (5.2%) vs 17 (20%) major and clinically relevant non-major bleeding events [DOAC vs VKA, p: 0.001] occurred. There were 2 arterial thromboembolic events in the VKA group (APS and antithrombin deficiency patient's in the periprocedural setting).

CONCLUSIONS

In our study, the effectiveness of DOAC treatment for VTE in patients with thrombophilia had a similar profile to VKA and caused significantly less major and clinically relevant non-major bleeding. This suggests that DOAC could be a good treatment option in this clinical setting. However, additional evaluation of DOAC use in patients with thrombophilia is necessary in order to provide more evidence.

Disclosures

No relevant conflicts of interest to declare.
Bimonthly Medical Visits Reduce Sickle Cell Admissions
Authors: Imahiyerobo Allison A , Billings Daniel D , Mora Erika , Eisenberger Andrew , Diuguid David L.
Published: October 2019

Background: Sickle cell disease (SCD) is a chronic medical disease that in some patients is characterized by frequent vasoocculsive crisis (VOC). A minority of sickle cell patients comprises a majority of hospital VOC admissions, and the transition to adult care is a particularly high risk time period. We sought to identify if increased frequency of outpatient medical care led to a reduction of hospital days following intervention and if there were improvements in daily function, and reduction of premature requests for opiate refills.

Methods: Retrospective review of 11 patients, average age of 25 (range 19-37 years old), 63% were female. Genotype of SCD included 8/11 with SS, 2/11 with SC and 1/11 with SBeta Thal. Sickle cell standard of care medical visits were monthly at our institution. Two nurse practitioners and 2 hematologists at the medical center implemented the intervention of increased frequency of medical visits to bimonthly follow up for between 5 and 48 weeks, with a mean length of 12 weeks of increased frequency of medical visits.

Results: In the 6 months preceding intervention there were a total of 300 hospital days of admission for VOC (2 patients <10 hospital days, 6 patients with 10-20 hospital days, 3 patients with over 20 hospital days; range 27-111 days). During the intervention 7/11 patients had no hospitalizations, 2/11 were discharged from the emergency department and never admitted. Another patient had 7 days of admission over 34 weeks of bimonthly medical visits and 1 patient had increase to days 37 days over 22 weeks of bimonthly medical visits. The average length of follow up after the intervention was 25 weeks: 4/11 had reduction of hospital days, 2/11 had the same number of hospital days and 4/11 had more hospital days. Secondary benefits were 3/11 patients were able to restart and maintain employment, 2/11 were able to re-enroll in educational programs, and 3/11 had a reduction in premature requests for opiate refills.

Conclusions: Increased frequency of outpatient medical visits leads to reduced hospital admission time for some patients and subjective improvement in daily function as measured by the ability to work or go to school. Future research is needed address the small subset of young adults who had an increase in hospital admission.

Figure. Figure.

Disclosures

No relevant conflicts of interest to declare.
The Predictive Value of Thromboelastogram in the Evaluation of Patients with Suspected Acute Venous Thromboembolism
Authors: Abu Assab Tareq , Raveh-brawer David , Abramowitz Julia , Naamad Mira , Rowe Jacob M. , Ganzel Chezi
Published: October 2019

The diagnosis of venous thromboembolism (VTE) is being done by imaging studies but can be ruled out by clinical probability assessment together with a D-dimer blood test. Thromboelastogram (TEG) is usually used for the evaluation of bleeding tendency but can demonstrate also hypercoagulable states. Our hypothesis was that TEG may estimate the presence of VTE more accurately than D-dimer. This prospective study recruited patients that presented to the emergency room or to the vascular laboratory with signs/ symptoms that raise the suspicion of acute VTE. TEG parameters that were examined were: Reaction time(R), Clot time formation (K), Alpha angle (α), Maximal amplitude (MA), Clot viscoelasticity (G), Coagulation Index (CI) and Clot lysis at 30 minutes (LY30). The expected values for hypercoagulable state include: short R and K and high α, MA, G, LY30 and CI.

Between April and October 2016, a total of 109 patients were enrolled in the study with a median age of 55.7 (21-89) years. Their characteristics are summarized in table 1. Forty-eight percent of the patients had at least one risk factor for development of VTE. According to the Well's criteria, 54 (49.5%) patients had low probability, 46 (42.2%) - moderate and 9 (8.3%) - high probability for developing VTE. Eighteen patients were diagnosed with VTE; 12 with DVT, 7 with PE and one with both.

Analyzing the different TEG parameters, both as continuous (table 2) or categorical variables (according to their normal range), did not reveal a statistically significant difference between VTE positive and negative patients. Combining different TEG parameters or dividing the cohort according to: gender, clinical suspicion of VTE based on the Well's criteria or different levels of D-dimer did not change the results of the analysis.

In conclusion, the current study could not demonstrate a significant value of any TEG parameter as a predictor of VTE in a general population of patients who came to the emergency room or vascular laboratory with signs/ symptoms that raise the suspicion of VTE.

Disclosures

No relevant conflicts of interest to declare.