bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of Molecular Mutations Causing Haemophilia B From China: A Single-Center Experience
Authors: Zhou Rong-Fu , Zhang Xian , Ouyang Jian , Yang Yonggong , Shao Xiao-Yan , Xu Jingyan , Zhang QiGuo
Published: October 2019

Abstract 4627

Objective:

To identify F9 gene mutations in patients with hemophilia B registered in Nanjing Drum Tower Hospital Hemophilia Registeration Center. Methods: One stage method was used to detect APTT, PT, TT, Fg and the activities of endogenous coagultation factors. Correction testing was employed to exclud the existence of inhibitor with mixed normal plasma. Genomic DNA was extracted from blood samples of 19 unrelated haemophilia B patients and their traceable family members. All exons and their flanking sequences of the F9 gene were amplificated by PCR and subsequently, the products were purified and sequenced directly. Results: APTT was significantly prolonged for all 19 cases of hemophilia B patients, but could be corrected by mixed normal plasma. According to the serial number, FIX:C was 3.7%, 3.5%, 1.9%, 1.9%, 2.2%, 2.0%, 1.9%, 3.2%, 3.5%, 10.8%, 7.8%, 2.2%, 3.8%, 2.3%, 1.6%, 1.4%, 3.7%, 7.8% and 3.5%, respectively. Thirteen different mutations of F9 gene were identified, including C 20518 T, T 6427 C, C 6460 T, C 31008 G, C 17761 T, A 17759 G, G 30150 A, G 31093 C, T 30930 C, G 20565 A, G 30987 A, A 6473 G and C 9 G, respectively. The mutations were composed of 10 missense mutations, one nonsense mutation, one a donor splice site mutation and one promoter mutation. Among them, mutations sites nt6460, nt17761, nt20518, nt30150 and nt31008 were located in CpG islands, belonging to mutation hot-spots. Mutations including C 9 G, C 31008 G and G 31093 C were firstly reported. Conclusions: No inhibitors are detected in the plasma of all patients. The F9 mutations are heterogenous and the missense mutations are the most prevalent gene defects in Chinese HB patients.

Disclosures:

No relevant conflicts of interest to declare.
Refractory Atypical Hemolytic Uremic Syndrome
Authors: Chao Ju-Hsien , Riley Beth C. , Redman Rebecca
Published: October 2019

Abstract 4635

Atypical hemolytic uremic syndrome is a rare but often lethal disease if not diagnosed and treated early. The mainstay of treatment has been plasma exchange, though eculizumab has recently been approved for treatment.

We report a 24-year-old Caucasian male whom presented from a rehab facility with weakness. Three months prior, he had sustained polytrauma from a motorcycle accident. At discharge, labs were normal. At admission, he was found to have anemia, thrombocytopenia, and elevated creatinine and bilirubin. Further testing revealed evidence of hemolysis. ADAMTS13 level was greater than 100% to assess for thrombotic thrombocytopenic purpura (TTP). Flow cytometry for CD59 was negative to assess for paroxysmal nocturnal hemoglobinuria (PNH). Shiga toxin was negative. He was diagnosed with atypical hemolytic uremia syndrome (aHUS). Patient was initially treated with steroids then underwent daily plasma exchange for 2 weeks with improvements in lab parameters and no evidence of hemolysis. Exchanges were changed to every other day for 2 weeks with plans to start eculizumab as outpatient therapy. After discharge, one dose of eculizumab 900 mg was given without further exchanges. Three days later, lab check showed decrease in hemoglobin and platelets, concerning for aHUS exacerbation. Patient was admitted for daily plasma exchange with eculizumab 100 mg boost after each procedure for about 2 weeks. Once his lab parameters were consistent with resolution, exchanges were spaced out to every other day for another 1 week then discontinued. Then he was started on monotherapy with eculizumab 900 mg once weekly with surveillance lab monitoring. He had no further exacerbation of his aHUS and was discharged back to rehab with weekly eculizumab and lab surveillance.

Early detection and treatment of aHUS is important to minimize the damages from the underlying thrombosis and prevent disease related mortality. Plasma exchange remains the standard treatment for this condition with close monitoring of lab parameters, though no set treatment schedule has been established. The recent approval of eculizumab offers another option in the management of this disease and appears equally efficacious.

Disclosures:

No relevant conflicts of interest to declare.
Treatment of Congenital Fibrinogen Deficiency; Global Development Plan for a Double Virus Inactivated Fibrinogen Concentrate
Authors: Schwartz Bruce A. , Knaub Sigurd
Published: October 2019

Abstract 4636

Congenital afibrinogenaemia and hypofibrinogenaemia are rare inherited disorders occurring in homozygotic patients with an estimated incidence of 1 in 106. Patients present with frequent severe bleeding episodes since birth or early childhood. Bleeding may occur after a minor trauma or a small surgical intervention, into the skin, mucosa, muscles, gastrointestinal tract, or the brain. Therapeutic substitution with human fibrinogen concentrate can correct the haemostatic defect and arrest the bleeding in patients with these fibrinogen deficiencies. Octafibrin is a highly purified, lyophilized, human plasma fibrinogen concentrate, without added albumin. Octafibrin is double virus inactivated using 2 dedicated virus inactivation/removal steps, solvent/detergent treatment, and nanofiltration. A plan for the global development of Octafibrin has been prepared taking into account discussions with European Regulators and the FDA. This plan also involves discussions with the European pediatric committee (PDCO) which oversees the inclusion of pediatric subjects into drug development under the new EMA guidelines.

The development plan calls for a prospective, randomized, open label, multinational, pivotal PK comparison of Octafibrin to an existing marketed product in 18 adult and adolescent patients, including comparison of a surrogate efficacy endpoint measured by TEG. In a second study the efficacy and safety of the product in bleeding and invasive procedures will be assessed in 24 adult and adolescent patients. Finally a pediatric PK, efficacy and safety study in patients below 6 years will be performed but because of the rarity of these patients this study will not need to be completed before review and approval by the regulatory agencies. Clinical studies are planned to be started later this year.

A double virus inactivated, plasma derived fibrinogen concentrate (Octafibrin) will be globally developed in an ultra rare congenital disease after harmonized discussions with EU and US regulators. Pivotal comparative PK data and interim efficacy and safety data will be available at time of regulatory submissions while the finalization of the pediatric study is deferred.

Effective management of congenital fibrinogen deficiencies in bleeding situations is necessary for the prevention of potentially life-threatening bleeding episodes. This clinical program will help to confirm that fibrinogen substitution is able to successfully control bleeding, increase the fibrinogen plasma levels, and reduce the amount of transfusions needed with allogeneic blood products. In addition, it will give additional information on the tolerability and overall safety profile of fibrinogen replacement therapy.

Disclosures:

Schwartz: Octapharma: Employment. Knaub:Octapharma: Employment.
Acquired Hemophilia After a Spider Bite
Authors: Vinayek Namita , Kloecker Goetz H , Riley Beth C.
Published: October 2019

Abstract 4637

A 59 year old white female with no history of bleeding diathesis presented with swelling of her left forearm two days after a brown recluse spider bite. Hematology was consulted for uncontrolled bleeding from the fasciotomy site after compartment syndrome. The hand surgeons did not see any vascular abnormality at the site of surgery. Due to ongoing bleeding for days from the surgical site, she required 8 PRBCs, 6 FFPs and 6 apheresis platelets units. PT, PTT, and platelet count were normal, ristocetin cofactor was 339% and vWF antigen 306% and vW Factor VIII binding capacity was normal. Her platelet functional assay was normal. Factor levels of II, V, VII, XI, X, XI and lupus anticoagulant were all with in normal range. A Bethesda assay however showed a low titer of antibody against factor VIII (1.25 BU). Factor VIII was low at 6% and remained low at 16% with no significant response to recombinant factor VIII.The bleeding however responded well to recombinant factor VIIa. She was maintained on high dose steroids for few months and tapered. She eventually had a skin graft to the site of injury and is now followed in our hemophilia clinic with no further bleeding complications.

To our knowledge only one case of spider bite resulting in factor VIII inhibitor has been documented in the literature. We hypothesize that direct antigenic stimulation by spider venom can transiently develop factor VIII inhibitor in individuals.

Disclosures:

No relevant conflicts of interest to declare.
Quantification of Immature Platelets with Abbott CD-Sapphire and Sysmex XE-5000 in Patients with Haematological and Malignant Diseases
Authors: Meintker Lisa , Haimerl Maria , Fritsch Juliana , Ringwald Jürgen , Krause Stefan W.
Published: October 2019

Abstract 4639

Background:

Quantification of immature, i.e. RNA-containing platelets (PLT) was introduced into routine diagnostics by Sysmex as immature platelet fraction (IPF) some years ago and recently by Abbott as reticulated platelets (rPLT). In this study, we evaluated the precision and agreement of both analyzers in normal and pathological samples and correlation of IPF or rPLT with clinical situations.

Methods:

Anonymized blood samples of cancer patients after chemotherapy, patients with PLT destruction (immune thrombocytopenia, ITP, or heparin-induced thrombocytopenia, HIT), and normal controls were analyzed with double measurements with both, the Abbott CD-Sapphire and Sysmex XE-5000. Furthermore, longitudinal evaluations after chemotherapy were performed and effects of PLT transfusions were analyzed. Precision and agreement were assessed with Bland-Altman analyses and Pearson correlation.

Results:

In a first set of experiments, a total of 220 blood samples were analyzed. We studied patients after chemotherapy with beginning thrombocytopenia (30 samples, median PLT count 23/nl) or recovering PLT counts (64 samples, PLT 48/nl), patients with PLT destruction (16 samples, PLT 84/nl) and normal controls (110 samples, PLT 249/nl). Both analyzers demonstrated acceptable overall precision (reproducibility) with lower precision in thrombocytopenia and a non-significant trend towards a better precision with XE-5000. Inter-instrument comparison showed a moderate correlation (Pearson r2=0.38) and a systematic bias of 1.04 towards higher IPF-values with the XE-5000 in the Bland-Altman analysis. Regarding different patient groups, the reference range (percentile 5 – 95) of normal controls was much narrower with the CD-Sapphire, leading to a clear separation of patients with recovering platelet counts after chemotherapy and IPT/HIT, both with increased rPLT as expected (figure). The same trend was visible for the XE-5000, however, with a much broader overlap to the normal range. In longitudinal studies, increased IPF or rPLT were frequently observed already during early thrombocytopenia with a decrease following platelet transfusions.

Conclusion:

Correlation between both analyzers for measurement of IPF and rPLT, respectively, was moderate. Precision of measurements was low in thrombocytopenic samples. Samples with abnormally increased IPF/rPLT could be indentified with both analyzers, however, separation from normal was better with the CD-Sapphire. Prediction of PLT recovery after intensive chemotherapy by the measurement of IPF/rPLT is questionable.

Disclosures:

Krause: Beckman Coulter: Research Funding.
Hyperbaric Oxygen Therapy Improves Post-Transplant Umbilical Cord Blood Engraftment
Authors: Aljitawi Omar S. , Yinghua Xiao , Lin Tara , Eskew Jeff , Parelkar Nikhil , Swink Megan , Radel Jeff , McGuirk Joseph , Li Linheng , Vielhauer George
Published: October 2019

Abstract 4663

Background:

Delayed engraftment following umbilical cord blood (UCB) transplantation is associated with increased transplant-related mortality. Earlier, we have reported that hyperbaric oxygen therapy (HBO) might have altered early UCB engraftment kinetics in a transplant animal model. Herein, we report use of HBO to improve long-term UCB engraftment.

Methods:

Six-to eight-week old NSG mice were sublethally irradiated 24 hours prior to CD34 selected UCB cell transplant. The irradiated mice were separated into a control group (where mice remained under normoxic conditions) and the HBO group (where mice received two hours of HBO therapy; 100% oxygen at 2.5 atmospheres absolute). Six hours after starting HBO therapy, both groups received 1×105 CD34 selected UCB cells via tail vein injection. The infused CD34 selected cells were transduced with a lentivirus carrying luciferase gene for in vivo imaging. Mice (n=4) from each treatment group were sacrificed after 3 and 4 months to assess long-term engraftment. Engraftment was measured as assessed by flow cytometry. Myeloid, B-cell, and T-cell subset engraftment was determined by flow cytometry.

Results:

At 3 and 4 months post-transplant, the mean percentage of human CD45 expressing cells in peripheral blood of HBO mice was significantly higher than controls (p=0.03 and p=.008 respectively). The mean percentage of human CD45 expression was higher in bone marrow of HBO mice at 3 months and 4 months, but only reached statistical significance at 4-month time point (p=0.04). At 3 months, the mean percentage of human CD19 expressing cells was significantly higher in peripheral blood of HBO mice (p=0.03) while no differences were noted between the two groups in terms of CD33 or CD3 expression. At 4 months, the mean percentage of human CD19 expressing cells and human CD3 expressing cells was significantly higher in peripheral blood of HBO mice (p=0.009 and p=0.0009, respectively) while no differences were noted between the two groups in terms of CD33 expression.

Conclusions:

HBO therapy given prior to UCB cell infusion significantly improved UCB's long-term engraftment. At 3 months, engraftment was skewed toward B-cell engraftment, while more balanced engraftment was noted at 4 months. Future studies are planned to confirm these findings and to determine the underlying mechanisms by which HBO improved UCB CD34 cell engraftment.

Disclosures:

No relevant conflicts of interest to declare.
Polyethylene Glycol - Polyethylenimine Nanoparticle-Mediated Transfection of Mir-150 Into the Leukemia Cells and Determination of the Expression Pattern Changes in Apoptosis and Cell Cycle Genes
Authors: Balci Tugce , Avci Cigir Biray , Ozcan Ipek , Susluer Sunde Yilmaz , Kayabasi Cagla , Saydam Guray , Gunduz Cumhur
Published: October 2019

Abstract 4679

The aims of this study are transfection of tumor suppressor miR-150 that is down-regulated in leukemogenesis into leukemia cells mediated by Polyethylene Glycol - Polyethylenimine (PEG-PEI) nanoparticle and determine the changes in gene expression pattern in chronic myeloid leukemia model cell lines; K-562 and KU812 and non-leukemia cell lines NCI-BL2347 and NCI-BL2171.

Characterization studies and stability studies of PEG-PEI copolymers were performed and by using these copolymers 9 nanoparticle formulations were prepared. Three copolymers (named T1, T3 and T9) were eligible for further analysis and nanoparticle complex (named F1, F2 and F3) formulations, particles that their size less than 300 nM have been evaluated.

Nanoparticle-mediated substitution of miR-150 reduced the expressions of cell cycle control genes CDK2 and CCNG2, CDK6 7-fold and 2-fold, respectively in K562 leukemia cell model.

Nanoparticle-mediated substitution of miR-150 has been evaluated KU812 leukemia cell model and reduced the expression of cell cycle control genes CHEK2, CDK5RAP1 and CCNG2 and CDKN1A 6- fold 5 –fold and 2-fold, respectively.

Substitution of deregulated tumor suppressor miR-150 to leukemia cells with non-viral transfection yielded promising results for the treatment of leukemia. Taking into account these results, it should be supported by preclinical studies in animal models, which would add benefit to current treatment protocols in clinical application.

Disclosures:

No relevant conflicts of interest to declare.
Normal Values for High Affinity Fc Gamma Receptor I (CD64) From Neonate to Young Adults
Authors: Matsushita Shoko , Asakura Yoshiko , Hayashi Yuko , Endo Mikiya , Nakatsuji Yukie , Ito Shigeki
Published: October 2019

Abstract 4685

The high affinity Fc gamma receptor I (Fc gamma RI, cluster of differentiation 64 [CD64]) is normally expressed to a very low extent by neutrophils; during bacterial infections, the expression has been shown to increase in adults and newborn infants. However, the normal range in steady status remains unknown. To ultimately develop the novel measurement technique and clinical application of CD64 expression as an early diagnostic marker for bacterial infection, normal CD64 expression levels were determined in normal neonates, infants, young children, and adults who had no symptoms of infection, stable temperature, normal leukocyte counts and normal C-reactive protein levels, and who were not using steroids, immunosuppressive drugs, aspirin, or other non-steroid anti-inflammatory drugs in this study. To quantify the expression levels of neutrophil CD64, mean fluorescence intensity (MFI) on neutrophils was measured by flow cytometry in 420 samples. Subjects were assigned to 7 groups by age: neonates (group A, n = 76); 1 – 11 months (group B, n = 51); 1 – 5 years (group C, n = 87); 6 – 10 years (group D, n = 76); 11 – 15 years (group E, n = 77); 16 – 20 years (group F, n = 33); and over 21 years (group G, n = 20). Each expression level in groups A, B, and C was significantly higher than that in group G (P < 0.01); no differences were found among groups D, E, F, and G. These results suggest that normal cutoff levels of CD64 should be determined according to age to apply to the clinical setting because CD64 expression has a tendency to increase according to age, especially over 21 years.

Disclosures:

No relevant conflicts of interest to declare.
Disseminated Tuberculosis with Hemophagocytic Lymphohistiocytosis (HLH)
Authors: Rao Suparna Ajit , Dutta Tarun K. , Naik Rakesh V , Krishnamurthy Aishwarya , Viswanath Vinod K.
Published: October 2019

Abstract 4686

A 38 year old woman presented with high grade fever and jaundice for one month. Patient also had reduced appetite and loss of weight for the same period. On examination, patient had significant pallor, icterus and pedal edema. Ultrasonogram showed enlarged liver (20 cms) and enlarged spleen (17.2 cms). Patient was empirically treated for malaria. Her subsequent investigations revealed Hb 35g/dl, total leucocyte count 2×109/l, differential leucocyte count - neutro 82%, lympho 18%, platelet count − 59×109/l, and red cell indices were as follows: MCV 71.6 fL, MCH 21.6 pg/cell, MCHC 30.2 g/dl. Her reticulocyte count was 0.5%. Peripheral blood smear showed pancytopenia with moderate anisopoikilocytosis. Her total bilirubin was 4.2 mg/dl and serum ferritin was found to be 1720 μg/L. In view of pancytopenia and non-response to antimalarials, patient was treated in line of septicemia with piperacillin and tazobactam, and simultaneously a bone marrow biopsy was performed. Bone marrow biopsy subsequently revealed a hypercellular marrow with erythroid hyperplasia. Number of macrophages was increased with some showing ingested red cells (hemophagocytosis) within them. In view of fever, splenomegaly, pancytopenia, hemophagocytosis and hyperferritinemia, a diagnosis of hemophagocytic lymphohistiocytosis (HLH) was made as per HLH 2004 diagnostic criteria. Bone marrow also revealed multifocal epithelial granulomas with caseation, pointing the etiology to that of disseminated tuberculosis. Patient expired before any anti-tuberculous treatment could be instituted.

Causes of HLH are broadly malignancy, collagen diseases and infections. Though malignancy and collagen diseases are common causes in the Western countries, tuberculosis is an important cause in a tropical country like India.

Disclosures:

No relevant conflicts of interest to declare.
Comparison of a Rotary Powered Bone Marrow Aspiration and Biopsy Device to the Traditional Manual Device in an Adolescent
Authors: Falcon Maria G. , Assanasen Chatchawin , Thomas Paul , Saldivar Victor
Published: October 2019

Abstract 4718

Bone marrow examination is important for the diagnosis of hematological malignancies and nonmalignant diseases in children. In patients with newly diagnosed lymphoproliferative diseases and certain non-hematopoietic malignancies, bone marrow examination is also part of the staging process. Core biopsy length has been found to be critical in diagnosing, predicting relapse or identifying residual disease following chemotherapy in patients. The larger the amount of marrow obtained increases the chance of finding a focal lesion. Unfortunately, the current practice of obtaining trephine biopsies and bone marrow aspirates in children via the manual method has a poor success rate for obtaining adequate specimens. In 2007, an FDA-cleared battery powered bone marrow aspiration and biopsy system (OnControl™ by Vidacare) was developed. Multiple studies have evaluated the use of the powered device in adults and found decreased time of procedure, decreased pain, and improved core biopsy specimens.

Here we present a direct comparison of the rotary powered device versus the traditional manual device (e.g. Jamshidi) when obtaining bilateral bone marrow aspirates and biopsies in a 17 year-old female with relapsed alveolar rhabdomyosarcoma. This patient required bilateral bone marrow biopsies to stage her disease and evaluate for bone marrow involvement. One aspirate and biopsy was obtained using the powered device from the right posterior superior iliac crest, and specimens were obtained from the left iliac crest using the traditional manual device. The endpoints measured were quality of the biopsy, length and width of the biopsy, time to obtain aspirate and biopsy, number of attempts to obtain the biopsy, post-procedural pain, and operator satisfaction with the device (O.S.).

Device Quality Rating Length (mm) Width (mm) Aspirate Time (sec) Biopsy Time (sec) Attempts Pain Score (0–10) O.S. (0–10) Powered 2 9 1.5 20 108 1 0 10 Manual 1 14 2 25 225 1 0 9

In conclusion, the powered device was superior to the manual device in terms of time to obtain the aspirate and biopsy and operator satisfaction with the device. It was found to be equivalent to the manual device in regards to number of attempts to obtain the biopsy, and post-procedural pain score. The manual device produced a biopsy that was longer, wider, and of higher quality than the biopsy obtained via the powered method. A randomized controlled trial in the pediatric population comparing the rotary powered device to the traditional device is currently underway as further studies are needed to evaluate the use of the powered bone marrow aspiration and biopsy device in children.

Disclosures:

Falcon: Vidacare Corporation: Research Funding. Assanasen:Vidacare Corporation: Research Funding.
Serum Fructosamine As an Alternative to Glycated Hemoglobin (HbA1c) for Glycemic Assessment in Diabetic Patients with Hemoglobin (Hb) Louisville
Authors: Lorenzo-Medina Mercedes , De-La-Iglesia Silvia , Quintana-Hidalgo Lucia , Ruiz-Garcia Lidia , Martin-Alfaro Ruth , Herrada Juan
Published: October 2019

Abstract 4771

Background:

HbA1c is widely used in the management of patients with diabetes mellitus (DM). Hb Louisville is an unstable hemoglobin that differs from HbA by the substitution of a phenylalanine residue for a leucine residue in position 42 of the b-chain, resulting in instability in the Hb molecule with increased erythrocytic destruction, clinically manifested as hemolytic anemia. The Hb Louisville heterozygotes suffer from a mild anemia, jaundice and hemolytic crisis. Abnormally low HbA1c concentrations are usually encountered in patients with high turnover rates of hemoglobin. Fructosamine is unaffected by disorders of red blood cells nor is influenced by hemoglobin variants, thus serving as an indicator of blood glucose concentrations of previous 2–3 weeks.

Methods:

Case report.

Results:

We report on three patients that belonged to the same family and had history of heterozygosity trait for Hb Louisville. All three patients had type 2 diabetes mellitus with falsely low HbA1c values (patient A: 2.8%, 2.9%, 3.2%; patient B: 4.4%, 3.8%, 3.7%; patient C: 4.2%, 4.6%, 3.7%). The patients had consistent elevation of fasting blood glucose values (patient A: 162, 171, 233 mg/dL; patient B: 262, 232, 235 mg/dL; patient C: 152, 130, 136 mg/dL). All three patients had evidence of hemolytic anemia, and their average hemoglobin levels were: patient A: 9.41 mg/dL, patient B: 10.9 mg/dL, patient C: 8.6 mg/dL. We therefore determined serum fructosamine levels, and the results were: patient A (patient deceased), patient B: 420 mcmol/L and patient C: 295 mcmol/L (Reference Range >285 mcmol/L).

Conclusion:

Fructosamine serum level determination is advocated for glycemic assessment in diabetic patients with Hb Louisville when discrepancies between glucose measurements and HbA1c values>are encountered.

Disclosures:

No relevant conflicts of interest to declare.
Changes in Regional Oxygenation At the Site of Sickle Cell Vaso-Occlusive Pain
Authors: Kato Roberta Miyeko , Nord Anne , McGuire Erin , Gardner Daniel , Bush Adam M , Detterich Jon , Khoo Michael , Meiselman Herbert J , Wood John C , Berdoukas Vasilios , Coates Thomas D.
Published: October 2019

Abstract 4773

Background:

The pathophysiology of sickle cell disease (SCD) is commonly thought to be in part a result of sickled red blood cells obstructing blood flow leading to hypoxic cellular injury. The most common SCD complication is painful vaso-occlusive crisis (VOC). However, the physiologic changes that occur at the site of VOC are poorly understood and there are no objective measurements of VOC. We hypothesized that regional oxygenation at the site of pain would be decreased from the patient's asymptomatic baseline and could be used as an objective measurement of the VOC process.

Methods:

Subjects were enrolled at the time of hospitalization for unilateral painful VOC. The subjects completed the adolescent pediatric pain tool (APPT) to assess the location, severity and quality of their pain. Regional oxygenation (RSO2) was measured by near infrared spectroscopy (NIRS) at the site that the subject identified as where they experienced the most severe VOC related pain. NIRS measurements were also made on the contralateral body site. NIRS measurements were made for 15 minutes with the subject in the supine position. Measurements were made in the first 24 hours of hospitalization. Blood pressure, heart rate, pulse oximetry, hematologic parameters and use of oxygen were determined. The subject returned 4 weeks after discharge for repeat testing to determine their physiologic post VOC RSO2 baseline. Mean values for the NIRS testing period were determined and the change in RSO2 during VOC from post VOC baseline were calculated.

Results:

5 female subjects with SCD were studied, mean age 15.8±3.0years. There was an average pain score of 7.4/10+1.9. Pain occurred in the back of 2 subjects and the lower extremities in the others. The RSO2% in the painful and not painful sites was increased from baseline by an average of 10.6±4.0% and 16.6±5.6%, respectively. The painful site had a decreased RSO2% in comparison to the contralateral side in 2 of 5 subjects (see figure). The RSO2% VOC baseline at the painful site post was higher in 3 of 5 subjects. There was no difference from baseline in blood pressure, heart rate, or arterial oxygen saturations during VOC. There were changes in the hematologic parameters consistent with VOC and active hemolysis.

Conclusions:

The SCD subjects demonstrated increased regional oxygenation at both the painful and contralateral not painful sites during VOC compared to the post VOC baseline. During VOC there was no predictable difference in regional oxygenation at the site of VOC pain contrary to our original hypothesis.

Disclosures:

Wood: Ferrokin Biosciences: Consultancy; Shire: Consultancy; Apotex: Consultancy, Honoraria; Novartis: Honoraria, Research Funding. Coates:Novartis: Speakers Bureau; Apopharma: Consultancy.
Second Cancers After Treatment with Stanford V Regimen in Eastern Cooperative Oncology Group (ECOG) Pilot Study E1492 At a Median Follow up of 17 Years
Authors: Advani Ranjana H. , Li Hailun , Hoppe Richard T. , Bartlett Nancy L. , Bennett John M. , Neuberg Donna S. , Cassileth Peter A , Kahl Brad S. , Horning Sandra J.
Published: October 2019

Abstract 4779

Purpose:

The Stanford V regimen is a combined modality approach for treatment of Hodgkin lymphoma (HL). E1492, an ECOG pilot study, consisted of 12 weeks of Stanford V chemotherapy followed by 36 Gy radiation therapy (RT) to sites > 5 cm or macroscropic splenic disease at diagnosis. Efficacy results were reported previously (Horning et al. J Clin Onc 18, 2000). The study now has a median follow up of 17 years and patients have been followed for overall survival (OS) and development of second cancers.

Methods:

47 eligible patients with stage bulky mediastinal (mass > one third of the maximum intrathoracic diameter) stage I-II or stage III-IV HL were enrolled between March 1992 and February 1995. Patients were followed every 3 months during the first year off therapy, every 6 months during years 2–5 and annually thereafter. The ECOG database was reviewed for OS and reported second cancers. Patient characteristics at baseline, type of second cancer and time to development of second cancer were assessed. RT summary forms were reviewed for patients with second cancers.

Results:

41 patients were treated with combined modality therapy and 6 with chemotherapy alone. The median age was 32 years (range 20–56 years). The 5 and 10 year OS are 96%, and 89% respectively. Seven second cancers were reported, as shown in the Table. The cumulative incidence for second cancers accounting for death as a competing risk is 0.02 (95% CI, 0–0.06) at 5 years, 0.07 (95% CI, 0–0.14) at 10 years, and 0.15 (95% CI, 0.04–0.27) at 15 years. Complete details of the exact location, histologic subtype and subsequent management of second cancers were not available for review.

Five of the 7 cancers [2 skin, 1 prostate, 2 acute myeloid leukemia (AML)] were not radiation-related. One patient developed AML after primary therapy as reported in the initial publication. A second patient developed AML 5 years after salvage therapy followed by autologous stem cell transplant for relapsed HL. It is likely the 2 cases of breast cancer were treatment related.

Conclusion:

Within the caveats of a retrospective analysis from a small cooperative group phase 2 trial, the mature 10 year OS of 89% and low frequency of secondary cancers are encouraging in comparison to historic treatment with combined modality treatment. Longer follow up of other Stanford V regimen data sets (i.e. United Kingdom National Cancer Research Institute Lymphoma Group Study ISRCTN 64141244 and the Eastern Cooperative Oncology Group E2496) are required to confirm these findings.

Disclosures:

No relevant conflicts of interest to declare.
A New Algorithm and Panel Construction for Pediatric Leukemia Immunophenotyping Using 10-Color Flow Cytometry
Authors: Keller Bettina , Radsak Markus P , Faber Joerg , Russo Alexandra
Published: October 2019

Abstract 4799

Background:

Rapid identification and quantification of abnormal cell populations in minimal specimen are crucial for diagnosis and longitudinal minimal residual disease (MRD) testing of childhood leukemia. So far, most standard immunophenotypic analyses are performed using antibody panels with up to five-colors and require high cell numbers. For infant and pediatric specimen, high-level multicolor analyses is highly desirable to gather sufficient data for initial diagnostic and follow up monitoring of pathologic populations.

Objective:

In this study, we aimed to establish a newly defined pediatric multicolor flow cytometric panel algorithm with high reliability yet minimal specimen requirement.

Results:

We defined a 10-color flow cytometric panel using the new violet laser dye “KromeOrange (KO)”. Applying CD45-KO/Side Scatter gating, combined with 2 additional backbone markers the panel is designed in two consecutive steps. In the first step, a single standardized 10-color-“screening tube” (FITC-HLA-DR, PE-CD15/CD56, ECD-CD5, PC5.5-CD33, PC7-CD13, APC-CD117, APC A700-CD34, APC A750-CD19, PB-CD3, KrO-CD45) is applied for initial orientation of specific lineage assignment. Based on results obtained with the screening tube, a specific multi-tube “classification panel” is used to complete detailed characterization of lineage specific malignancy and maturation stage. Suitable specimens include fresh blood, bone marrow and all body fluids. All samples are stained directly with monoclonal antibodies, followed by the lyses of erythrocytes and a short wash. Compared to standard five color panel previously used the application of greater numbers of informative antibodies in the screening tube and in the 2ndstep muti-tube classification panel is cost and time efficient and results in a more precise characterization of any single event.

Conclusion:

Our panel construction and algorithm definition for infant and pediatric leukemia immunophenotyping is one of the first 10-color flow cytometry panels described for this application. Advantages are the possibility to obtain highly specific information from minimal specimens with significantly improved laboratory efficiency. The overall performance is currently tested in a routine clinical setting.

Disclosures:

No relevant conflicts of interest to declare.
Src Family Kinase Inhibitor PP2 Has Different Effects On ATRA or Arsenic Trioxide-Induced Differentiation of Acute Promyelocytic Leukemia Cell Line
Authors: Kim Kyoung Ha , Cheong Hee-Jeong , Kim Sook-Ja , Jeon Young Woo , Yoon Jina , Kim Han Jo , Kim Se Hyung , Kim Hyun Jung , Lee Sang-Cheol , Bae Sang Byung , Kim Chan-Kyu , Lee Namsu , Lee Kyu Taeg , Park Sung Kyu , Hong Dae Sik , Hee-Sook Park , Won Jong-Ho
Published: October 2019

Abstract 4800

Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia (AML) with distinctive biologic and clinical features that is now highly curable. Leukemic promyelocytes have the unique ability to undergo differentiation after exposure to retinoic acid and both differentiation and apoptosis after exposure to arsenic trioxide (As2O3, ATO). Emerging evidence has implicated Src family kinases (SFKs) as regulators of proliferation and survival of myeloid lineage cells. Recent studies showed that inhibition of SFKs resulted in enhancement of retinoic acid-induced myeloid differentiation. In this study, we demonstrated that the SFK inhibitor PP2 enhanced the differentiation of NB4 cells when combined with ATO as well when combined with as all-trans-retinoic acid (ATRA) and the difference in the retinoic acid-induced gene expression between the cells treated with PP2 in combination with ATRA and the cells treated with PP2 in combination with ATO.

SFK inhibitor PP2 significantly enhanced ATRA- or ATO-induced differentiation of NB4 cells. The synergistic effect was significantly stronger when PP2 was combined with ATRA than when PP2 was combined with ATO. Flow cytometric analysis demonstrated a significant increase in CD11b-positive granulocytes up to 60.73% and 31.58%, respectively. These results were confirmed by morphologic analysis using Wright stain and NBT staining. These effects were not related to apoptosis. Annexin-V-fluorescein staining revealed that PP2 combined with ATRA or PP2 combined with ATO did not induce apoptosis in NB4 cells. Retinoic acid-induced gene expression was different in both groups. ICAM-1 expression was significantly increased in the cells treated with PP2 in combination with ATRA whereas cathepsin D expression was significantly increased in the cells treated with PP2 in combination with ATO. These findings suggested that the synergistic effect of SFK inhibitor PP2 in combination with ATRA was significantly stronger than that of PP2 in combination with ATO on NB4 myeloid leukemia cell differentiation and this difference was related to the disparate activation of retinoic acid-induced genes.

Disclosures:

No relevant conflicts of interest to declare.
Hepcidin Inhibition by Modified Heparins without Anticoagulant Activity
Authors: Poli Maura , Girelli Domenico , Naggi Annamaria , Campostrini Natascia , Finazzi Dario , Maccarinelli Federica , Castagna Annalisa , Arosio Paolo
Published: October 2019

Abstract 483

Background.

There is an increasing interest in the development of pharmacological agents able to modulate hepcidin, the peptide hormone that critically regulates iron metabolism. In particular, hepcidin antagonist may have a therapeutic role in the anemia of chronic diseases, where hepcidin levels are often increased by pro-inflammatory cytokines. We previously demonstrated that heparin is a potent inhibitor of hepcidin expression in hepatic cell lines, probably by interfering with BMP/HJV/SMAD signalling, and that it was also effective in reducing hepcidin expression in mice (Poli M, Blood 2011; 117:997–1004). Since the therapeutic use of heparin for hepcidin modulation is hampered by its strong anticoagulant activity, we were interested in evaluating modified heparins without such activity.

Methods.

Heparins modified to inactivate the antithrombin binding site, with different molecular weight and degree of sulfation, were supplied to hepatic cell lines and mice to evaluate their potential modulation of hepcidin expression. We analysed their interference with the BMP/SMAD signalling, as well as serum hepcidin levels in mice by mass spectrometry.

Results.

Over 20 modified heparins were initially screened by evaluating their dose-dependent suppression of hepcidin expression before and after BMP induction. All of them showed a certain degree of anti-hepcidin activity, with two glycol-split molecules being as potent as classical unfractionated heparin. These two molecules suppressed BMP/SMAD signalling in HepG2 cells at pharmacological concentrations with maximum inhibition after 6 hours. In mice, treatments with 20 or 60 mg/Kg did not affect coagulation but strongly reduced liver pSMAD, hepcidin mRNA and serum hepcidin. Again, the maximum effect on liver hepcidin expression was observed 6 hours after the injection. This effect was observed also in conditions of high hepcidin caused by experimental inflammation or iron overload.

Conclusions.

Some non-anticoagulant heparins have strong anti-hepcidin activity both in vitro and in vivo, and may represent promising hepcidin antagonist with potential therapeutic applications.

Disclosures:

No relevant conflicts of interest to declare.
Advanced Non Small Cell Lung Cancer Patients with Fewer Numbers of Blood Myeloid Dendritic Cells and CD4 Cells Prior to Treatment Have Inferior Progression-Free Survival: Implications for Cancer Immunotherapy
Authors: DeJoubner Nutan J. , Li Qunna , Harris Wayne A.C. , Wang Zhibo , Liu Yuan , Waller Edmund K.
Published: October 2019

Abstract 4837

Background:

The tumor microenvironment includes tumor cells, and host-derived endothelial cells, fibroblasts, innate and adaptive immune cells. Tumors may induce neo-vascularization that supports local tumor growth or immune suppression and tolerance that facilitates tumor metastasis. We hypothesized that the patients with higher numbers of circulating CD34+ endothelial progenitor cells (CD34+/CD146+/CD45-, CEC), a cellular bio-marker for vasculogenesis, would have worse post-treatment outcomes and patients with more hematopoietic progenitor cells (CD34+/CD45+/CD45dim/CD133+, HPC) and Immune cells including T-cells would have better outcomes.

Methods:

We analyzed blood samples from sixty-two patients with advanced NSCLC at 3 time points: before chemotherapy, after cycle one, and at completion of treatment or progression of disease, in an IRB-approved protocol. CEC, HPC, and immune subsets were measured by high throughput multi-parameter flow cytometry, 2.5,000,000 events were acquired using a lyse, no-wash method optimized for rare event detection. Primary outcomes were progression free survival(PFS) and Overall Survival(OS) from the time of study entry. The patient population was stratified into groups based on optimum cut-off point for each cell subset of interest. Statistical analysis was done with log-rank test and Cox regression.

Results:

Mean age at diagnosis was 64 (37–87 years), 30 events (death) occurred with median follow-up of 9.3 months. Forty-six patients (74%) had disease progression with a median follow-up of 4.7 months. At baseline lower numbers of WBC, Neutrophil lymphocyte ratio(NLR), CEC, HPC were associated with better PFS, while only WBC and Neutrophil lymphocyte ratio (NLR) were associated with a favorable OS. While lower numbers of Immune cells were associated with worse PFS and OS (increased HR death or relapse) in univariate analysis as noted in the Table. Only covariates that were significant and non collinear were entered in the Multivariable model adjusted for age, gender, smoking, race, TNM stage, pathology, and performance status at diagnosis. This showed that baseline numbers of CD4+ T-cell (HR 0.46; 95% CI 0.33–0.98; p= 0.045), Myeloid DC (HR 0.38; 95% CI 0.39–0.81; p=0.012), HPC (continuous variable) (HR 0.78; 95% CI 0.64–0.93; p= 0.008) were significant for disease progression, while NLR was significant for death after study entry (Figure; HR 3; 95% CI 1.45–6.25; p=0 0.003).

Conclusions:

In patients with advanced NSCLC, lower numbers of HPC and NLR were associated with improved PFS and OS respectively. Lower numbers of immune subsets at diagnosis were associated with inferior outcomes to treatment, supporting the role for immune-mediated disease control.

Disclosures:

No relevant conflicts of interest to declare.
Overcoming CD28-Mediated Multi-Drug Resistance With a Novel PIM2 Inhibitor
Authors: Baldino Carmen M. , Nair Jayakumar R. , Caserta Justin , Murray Megan , Dumas Stephane , Flanders Yvonne , Lee Kelvin P.
Published: October 2019

Drug resistance in multiple myeloma (MM) is the major cause of treatment failure and is significantly mediated by pro-survival interactions with bone marrow microenvironment. A key myeloma receptor involved in this interaction is CD28, which has been largely characterized as the prototypic T cell costimulatory receptor. However, CD28 expression on myeloma cells is significantly correlated with disease progression, worse prognosis, and is significantly higher in the poor prognosis t(14;16) MAF subgroup. We now report that CD28 signaling mediates significant drug resistance and protects MM against death from multiple chemotherapeutics with different mechanisms of action – including dexamethasone, arsenic trioxide, melphalan or bortezomib. Inhibition of specific signaling (PI3K or Akt) or targets (Foxo3A or Bim) downstream of CD28 activation abrogates this protection. Unexpectedly, we found evidence that the PIM2 kinase (which is largely uncharacterized in MM but is also significantly overexpressed in the MAF subgroup) may be a previously unreported component of the CD28 pro-MM survival pathway. The novel small molecule PIM2 inhibitor JP_11646 (IC50 0.5 nM) abrogates CD28-mediated protection against apoptosis in MM cell lines in vitro, which we have not previously seen for any chemotherapeutic tested. In addition, blockade of CD28 activation sensitized MM cells significantly to JP_11646-induced death. Altogether, these data suggested that PIM2 inhibitors can overcome a major mechanism of multi drug resistance in MM.

Jasco’s novel and selective pan-PIM inhibitor (JP_11646) has demonstrated biochemical IC50s of 24, 0.5 and 1 nM for PIM1, PIM2 and PIM3 respectively. The PIM mechanism of action has been confirmed through cell based transphosphorylation assays, where JP_11646 decreased PIM dependent phoshphorylation of the proapoptotic protein BAD at nM levels. JP_11646 increases apoptosis and decreases cell viability in multiple myeloma cell lines with the MAF translocations (<100 nM). JP_11646 is orally bioavailable and has demonstrated in vivo efficacy, inhibiting tumor growth by >80% in a MM1.S tumor xenograft study. These data provide solid rationale for further development of JP_11646 as a targeted therapy in MM, and specifically for patients exhibiting the MAF translocation.

Disclosures:

Baldino: Jasco pharmaceuticals: Employment, Equity Ownership, Founder and President Other, Membership on an entity’s Board of Directors or advisory committees. Caserta:Jasco Pharmaceuticals: Co-Founder Other, Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees. Dumas:Jasco Pharmaceuticals: Employment. Flanders:Jasco Pharmaceuticals: Employment. Lee:Jasco Pharmaceuticals: Research Funding.
Carfilzomib PLUS Melphalan and Prednisone (CMP) Is A Promising Combination Therapy For The Treatment Of Elderly Patients With NEWLY Diagnosed Multiple Myeloma: Results Of A PHASE I/II Trial In 68 CASES
Authors: Moreau Philippe , Kolb Brigitte , Hulin Cyrille , Caillot Denis , Benboubker Lotfi , Tiab Mourad , Touzeau Cyrille , Leleu Xavier , Roussel Murielle , Chaleteix Carine , Attal Michel , Facon Thierry
Published: October 2019

Melphalan-Prednisone + bortezomib (MPV) is one of the standard of care for the frontline treatment of patients with symptomatic multiple myeloma non eligible for high-dose therapy. In the pivotal VISTA trial for approval of MPV, the main toxicity was grade 3-4 peripheral neuropathy (PN) described in 14% of the cases. Carfilzomib (CFZ), the second-in-class proteasome inhibitor has shown promising activity and a favorable toxicity profile with low PN rates. Therefore, the option of combining CFZ with MP (CMP) is an attractive one. Therefore we designed a phase I/II study to determine the maximum tolerated dose (MTD) of CMP and to assess safety and efficacy. In the phase I portion of the trial, CFZ was started at 20mg/m2, then escalated to 27, 36, and 45mg/m2, administered IV over 30 minutes in 42-day cycles on D1/2/8/9/22/23/29/30 for 9 cycles. Melphalan 9mg/m2 and prednisone 60mg/m2 were given PO D1–4 of every 42-day cycle. MTD was based on dose-limiting toxicity (DLT) in cycle 1 defined as any grade 4 (G4) hematologic adverse event (AE), any hematologic AE preventing aministration of ≥ 2 CFZ doses except G4 thrombocytopenia without bleeding or G4 neutropenia ≤ 7days, ≥ G3 febrile neutropenia, or any ≥G3 nonhematologic AE.

As of July 6, 2013, 24 pts have been enrolled in phase I: 6 for each dose level. There were 2 DLTs at 45mg/m2 (fever plus hypotension) resulting in a MTD of 36mg/m2. In Phase II, 44 additional patients received CMP at 36mg/m2 CFZ for N=68 total PhI/II patients (50 patients overall treated at the dose pf 36mg/m2). The median age of the series was 72 years, with 36% of the patients presenting with ISS3. Overall response rate was 89.5% including 56% ≥ very good partial response. With a median follow-up of 12 months, the projected 2y OS was 87%, and the median event-free survival was 22 months. CMP was well tolerated and only 1 patient developed grade 3 PN. These promising results compare favorably to those of MPV, MP+Thalidomide, MP+lenalidomide (R), and R+dex in similar pts. CFZ 36mg/m2 + MP is tolerable and effective in elderly patients with symptomatic newly diagnosed MM. Treatment is ongoing, 20% of the patients are receiving their last cycles of CMP. Final safety and efficacy data will be presented during the meeting.

Disclosures:

Moreau: CELGENE: Honoraria, Speakers Bureau; JANSSEN: Honoraria, Speakers Bureau. Off Label Use: FRONTLINE TREATMENT WITH CARFIZOMIB. Hulin:CELGENE: Honoraria; JANSSEN: Honoraria. Leleu:CELGENE: Honoraria; JANSSEN: Honoraria. Roussel:CELGENE: Honoraria; JANSSEN: Honoraria. Attal:CELGENE: Honoraria, Speakers Bureau; JANSSEN: Honoraria, Speakers Bureau. Facon:CELGENE: Honoraria, Speakers Bureau; JANSSEN: Honoraria, Speakers Bureau.