bluuebunny/crossref_metadata_2025 huggingface dataset:
Recovery From Hemoglobin Of 2g/Dl Without Prbc Transfusion: A Lesson Learned From a Jehovah’s Witness With Cold Agglutinin Disease
Authors: Gohari Pouyan , Chandra Abhinav B. , Slatkin Richard , Lipshitz Jay S.
Published: October 2019

Supportive management of critically ill patients with anemia often involves packed red blood cell transfusion. Jehovah’s Witness’ refusal of blood products because of their religious beliefs can render these situations challenging. Successful adaptation of medical management in Jehovah’s Witness’ with severe anemia provides physicians with a distinct opportunity to question common transfusion practices and learn about anemia. We present a case of a 43 year-old Jehovahs Witness who presented with autoimmune hemolytic anemia – cold agglutinin disease. His hemoglobin nadired at 2g/dL. The patient was successfully managed to recovery with corticosteroids, rituximab, folic acid and an erythropoeitin stimulating agent. To our knowledge this is the lowest Hgb that a patient with life threatening cold agglutinin disease survived without the use of any blood product transfusion. Such cases afford a deeper appreciation of the body's ability to adapt to and recover from anemia without the use of exogenous blood.

Disclosures:

No relevant conflicts of interest to declare.
Systematic Evaluation Of Chronic Organ Damage In Adult Sickle Cell Patients. A Seven-Year Follow-Up Study
Authors: van Tuijn Charlotte F.J. , Schimmel Marein , van Beers Eduard J. , Biemond Bart J.
Published: October 2019

Introduction

The occurrence of organ damage in sickle cell disease (SCD) is a crucial determinant for both medical treatment and prognosis. In a previous study, we systematically analyzed the prevalence of SCD related organ damage and complications in adult sickle cell patients in a tertiary teaching hospital in the Netherlands. We now describe a seven-year follow-up of this patient cohort, to provide an insight into the course of the various forms of organ damage and SCD related complications.

Methods

At baseline in 2006, 110 adult patients visiting the outpatient clinic of our hospital were enrolled. All enrolled patients from the primary analysis were included for follow-up. Patients were screened for sickle cell related manifestations during visits to the outpatient clinic biannually. Various forms of sickle cell related organ damage and complications (presence of microalbuminuria, renal failure, pulmonary hypertension retinopathy, iron overload, cholelithiasis, avascular osteonecrosis, leg ulcers, acute chest syndrome, stroke, priapism and admissions for painful crises) were routinely screened according to international guidelines. Patients with genotype HbSS/HbSβ0 and HbSC/HbSβ+were grouped for further analysis.

Results

Of all originally included patients (N=110), nine were lost to follow-up (N=9). The mean age of the current study cohort is 37 years (IQR 27-46). Overall, 59 patients (58%) developed a new form of organ damage or new complication since baseline analysis, including eight patients who deceased (7 due to a sickle cell disease related death). In the HbSS/HbSβ0 genotype group (N=60) we found an increase in the percentage of patients who have had an Acute Chest Syndrome (29% to 47%) or have been diagnosed with avascular osteonecrosis (15% to 24%), retinopathy (23% to 34%) or pulmonary hypertension (31% to 48%). In the HbSC/HbSβ+ (N=35) group we found an increase in the occurrence of avascular osteonecrosis (9% to 14%), retinopathy (59 % to 70%) and pulmonary hypertension (9% to 19%). Furthermore, the use of hydroxycarbamide increased in both genotype groups and the frequency of admissions for painful crises remained stable for both genotype groups.

Conclusion

In the past period of seven years 58% of the patients in a previously well descript cohort of adult SCD patients developed a new sickle cell related complication. For some forms of organ damage or complications a substantial increase occurred dependent of a patient’s genotype.

Disclosures:

No relevant conflicts of interest to declare.
Thrombosis Risk Factors Assessment In Children Treated For Acute Lymphoblastic Leukemia
Authors: Smalisz-Skrzypczyk Katarzyna , Klukowska Anna , Pawelec Katarzyna , Matysiak Michal
Published: October 2019

The aim of the study was to evaluate factors predisposing to thrombosis in children treated for acute lymphoblastic leukemia according to ALL IC BFM 2002.

An analysis of 30 cases of thrombosis in 210 children (14%) treated due to ALL in the Department in years 2007-2011 was carried out. Age at onset, location, cause and treatment methods were taken into consideration. All analyzed patients underwent screening for congenital thrombophilia.

The age of patients ranged from 1.5 to 16 years with the median of 7.5 years. Arterial thrombosis occurred in 1 person (3%), venous thrombosis in 29 (97%) patients. Thromboembolism was usually related to the central venous catheter (n = 27, 90%). Massive thrombosis occurred in 6 patients (20%). Relapses occurred in 2 children.

The most common causes of thrombosis were: the presence of the central catheter (n = 27, 90%) and L-asparaginase treatment (n = 16, 53%). The factor V Leiden mutation was diagnosed in 1 patient, protein C deficiency in 1 patient as well, and elevated levels of factor VIII were detected in 3 cases.

Five children (16%) underwent systemic thrombolysis with recombinant tissue plasminogen activator and 17 patients (52%) had local thrombolysis. Anticoagulation with warfarin following low-molecular-weight heparin discontinuation was used in 6 patients (20%). The complete resolution of the thrombus was observed in the whole group of patients.

Conclusions

1. A risk factor for thrombosis in children with ALL is the treatment of L-asparaginase and the presence of central venous catheter.

2. Screening for congenital thrombophilia in children treated for ALL should not be done routinely since the coexistence of this two conditions is rare.

Thrombosis in children with ALL responds well to treatment.

Disclosures:

Klukowska: Octapharma AG: Investigator Other.
Convergent Squint- a Symptom Of Thrombosis
Authors: Laguna Pawel , Szymanska Krystyna , Milewska Kamila , Sawionek Iwona , Pawelec Katarzyna , Matysiak Michal
Published: October 2019

Anticoagulative treatment of children has always presented problems due to a lack of generally approved clinical standards. We present the case of a 5 -year-old boy with bilateral convergent strabismus. It was preceded by otitis, which was treated with antibiotics for a few days before admission to hospital. In the last two days before admission his condition worsened, he complained of a headache and started vomiting. In neurological examination on the second day of his stay, the boy presented with decreased spontaneous activity and hypersomnia. This muscle tone, reflexes were decreased and he had, poor motor coordination. There were no abnormal reflexes and cerebellar testing showed no aberrations. There was 1 cm nuchal rigidity. Examination of cranial nerves showed sixth nerve palsy on both sides. Ophtalmological examination revealed bilateral papilledema. Neuroimaging showed thrombosis of the transverse and sigmoid sinus. A lumbar puncture was not performed because anticoagulative treatment, had been started. The final diagnosis was: bilateral sixth nerve palsy due to sinovenosous thrombosis as a complication of otitis in a patient with a genetic disorder The patient received anticoagulative, antiinflammatory, antibiotic and anti - edemal treatment. He responded to therapy, showing clinical improvement followed by normalisation in neuroimaging. On examination after 1.5 months his general condition was good. Motor coordination was poor and muscle tone was lowered as before. His comprehension was good. Speech was limited to the first syllables of words, as before. He had convergent strabismus in his left eye, without sixth nerve palsy symptoms. Movements of the right eye were normal. In thrombophilic investigation the presence of factor V Leiden mutation was found. The boy is now in outpatient care. He is undergoing vision therapy and speech therapy. Convergent squint can be a symptom of thrombosis in children. Neuroimaging is essential in diagnosis.

Disclosures:

No relevant conflicts of interest to declare.
Rapid and Effective Isolation Of (T) Cell Populations By a New Method Avoiding Magnetic Beads
Authors: Stadler Herbert , Przibilla Sabine , Dreher Stefan , Stemberger Christian , Germeroth Lothar , Busch Dirk
Published: October 2019

In the last two decades, conventional isolation of therapeutic cells has been carried out using high affinity antibodies and magnetic bead technology. Although good purity and reasonable yields can be obtained in many cases, major disadvantages remain comprising biological interference of non-reversible selection reagents (e.g. stimulation, receptor blockade etc.), the difficulty to purify complex multi-parametric cell populations by positive selection, time consuming protocols as well as limitations in high-throughput processing. We report here on the development of a new non-magnetic and fast cell selection technology applying immune affinity chromatography. Therefore, a matrix consisting of beads coated with Streptactin and low-affinity recombinant Fab-fragments (Fab-Streptamers) directed against defined T cell surface antigens has been generated. Stable and specific target cell retention is achieved by passing whole blood or PBMCs over the affinity matrix. After binding and washing, target cells can be gently retrieved by D-biotin administration. Eluted cells are then passed over a second matrix removing D-biotin and free Fab-Streptamers, subsequently yielding in a label-free authentic cell population for further use. Most importantly, total cell processing times can be kept extremely short; depending on the sample size even down to several minutes. Sequential isolation steps are possible and allow positive selection of complex cell populations like regulatory T cells or central memory T cells defined by several markers in high yields and purities. We are currently integrating this approach into a fully closed separation device for clinical cell purification. In addition, high throughput cell selection for diagnostic or basic research applications can be achieved by embedding the matrix into pipette tips and the use of suitable pipetting robots.

Disclosures:

No relevant conflicts of interest to declare.
Non-Irradiation Approach To Study Hematopoiesis and Leukemogenesis In a De Novo Murine Model Of Hematopoietic/Leukemia Stem Cell Transplant
Authors: Liu Xiaofei , Yang Dan , Fang Shumin , Geng Yang , Zhao Qianhao , Yang Pengjie , Peng Siying , Shen Yuelei , Pan Guangjin , Wang Jinyong
Published: October 2019

Traditional mouse models for hematopoietic stem cell transplant (HSCT) require whole body irradiation to ablate the hematopoietic cells in recipients, with the defect of disturbing non-hematopoietic cells and introducing potential tumorgenesis in a nonautonomous manner. Here, we use a novel approach to produce mice that whole hematopoietic-specific ablation can be conditionally achieved without global body irradiation. Briefly, a Diphtheria toxin receptor (DTR)-GFP reporter element was targeted into the ROSA26 locus to produce DTR-GFP reporter mice, with a loxp-stop-loxp cassette. Then the DTR-GFP reporter mice were crossed to Vav-Cre mice to produce double transgenic mice (DTR-GFP mice). We injected DT to ablate the bone marrow cells from DTR-GFP mice, and transplant WT bone marrow cells into them. Our data showed that all hematopoietic cells including hematopoietic stem cells, Myeloid, lymphoid lineages are GFP positive in DTR-GFP mice. A single dose of DT can kill all the hematopoietic cells from DTR-GFP mice. One month later, WT bone marrow cells were successfully engrafted into the DTR-GFP recipients without irradiation. We are using this model to re-evaluate some leukemia models that irradiated bone marrow niches might be involved in the tumorigenesis.Thus, we establish a de novel HSCT approach without irradiated myeloablation, which will benefit studies of hematopoiesis, leukemogenesis, hematopoietic stem cell niche, as well as other types of tissue transplants that need ablation of recipient hematopoietic system or immune system.

Disclosures:

Peng: Biocytogen: Employment, Membership on an entity’s Board of Directors or advisory committees. Shen:Biocytogen: Employment, Membership on an entity’s Board of Directors or advisory committees.
GPI-80 Defines Self-Renewal Ability In Hematopoietic Stem Cells During Human Development
Authors: Prashad Sacha L. , Calvanese Vincenzo , Yao Catherine , Kaiser Joshua , Sasidharan Rajkumar , Magnusson Mattias , Mikkola Hanna K.A
Published: October 2019

Advances in pluripotent stem cell and reprogramming technologies have provided hope of generating transplantable hematopoietic stem cells (HSC) in culture. However, better understanding of the identity and regulatory mechanisms that define the self-renewing HSC during human development is required. We discovered that the glycophosphatidylinositol-anchored surface protein GPI-80 (Vanin-2), previously implicated in neutrophil diapedesis, distinguishes a functionally distinct subpopulation of human fetal hematopoietic stem and progenitor cells (HSPC) that possess self-renewal ability. CD34+CD90+CD38-GPI80+ HSPCs were the only population that could maintain proliferative potential and undifferentiated state in co-culture on supportive stroma, and displayed engraftment potential in sublethally irradiated NSG mice. GPI-80 expression also enabled tracking of human HSC during development as they migrate across fetal hematopoietic niches, including early fetal liver and bone marrow. Microarray analysis comparing CD34+CD90+CD38-GPI80+ HSPC to their immediate progeny (CD34+CD90+CD38-GPI80-) identified novel candidate self-renewal regulators. Knockdown of GPI80, or the top enriched transcripts encoding surface proteins (ITGAM) or transcription factors (HIF3a) documented the necessity of all three molecules in sustaining human fetal HSC self-renewal. These findings provide new insights to the poorly understood regulation of human HSC development and suggest that human fetal HSCs utilize common mechanisms with leukocytes to enable cell-cell interactions critical for HSC self-renewal.

Disclosures:

No relevant conflicts of interest to declare.
Characteristics Of Paroxysmal Nocturnal Hemoglobinuria Clones In Colombian Patients
Authors: Londoño Mónica , Arenas-Mantilla Mario , Henao-Uribe Alicia Maria , Novoa Ricardo , Naranjo Andrea , Bedón Fernanda
Published: October 2019

Introduction

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired disorder, characterized by the clonal expansion of hematopoietic stem cells lacking glycosylphosphatidylinositol-anchored proteins. PNH clones are frequently found in patients with aplastic anemia (AA), myelodysplastic syndrome (MDS), unusual site thrombosis and non-inmume hemolysis. In Colombia, the DECF laboratory analyses blood samples from all over the country, summited by the APEC (association of patients with complement diseases) for PNH screening.

Methods

We reviewed the results of flow cytometry (FC) analyses from 1448 patients screened for PNH in DECF laboratory, between 2010 and 2013, and evaluated the association between clinical characteristics and distribution of PNH clone sizes. Clinical characteristics were considered as referred by treating physicians in the FC analysis request forms. All patients included gave written informed consent.

Results

Mean age of the study population was 44.6±18.5 years and 60.5% were female patients. The most frequent indications for screening were thrombosis (23.5%) and unexplained cytopenias (21.8%). Only 14% of the samples were PNH positive. Table 1 shows the results of FC analysis according to the indications for screening. Median clone sizes were 0.1% (interquartile range (IQR): 0-5.8%) in erythrocytes, 4.2% (IQR: 1.1-53.9%) in granulocytes and 3.8% (IQR: 0.7-64.1%) in monocytes. PNH clone size in granulocytes showed strong correlation with clone size in monocytes (r=0.86, p<0.001) and moderate correlation with clone size in erythrocytes (r=0.65, p=0.001). Figure 1 depicts the distribution of PNH granulocyte clone sizes among indications for screening. In the group of patients with PNH granulocyte clone sizes >5%, those with AA had smaller clone sizes than the rest (Figure 2), while patients with hemolysis showed larger clone sizes than the rest (Figure 3). No significant differences in clone size distribution were found within patients with PNH granulocyte clone sizes ≤5%.

Conclusions

Our findings are comparable to those reported in other countries. PNH granulocyte clone sizes had stronger correlation with PNH monocyte clones than with PNH erythrocyte clones. About one third of the patients with AA and one fourth of those with hemolysis were PNH positive. Over 50% of patients with AA showed PNH clones sizes <5%. Patients with hemolysis have larger clones than the rest. Further studies are needed to establish the association between PNH clone sizes and clinical outcomes of related disorders.

Disclosures:

No relevant conflicts of interest to declare.
Defects In DNA Double-Strand Break Repair and Increased Alternative Non-Homologous End Joining Pathways In Multiple Myeloma: Implications For Genome Stability
Authors: Herrero Ana B , San Miguel Jesus F , Gutierrez Norma
Published: October 2019

Multiple myeloma (MM) is a hematological malignancy characterized by frequent chromosome abnormalities. However, the molecular basis for this genome instability remains unknown. Since DNA rearrangements can be generated thought an improper repair of double strand breaks (DSBs), we investigated the functionality of DSB repair in MM cells. We found that four out of seven MM cell lines analyzed exhibited clear defects in the repair of ionizing radiation (IR)-induced DSBs, revealed by a slow kinetics of g-H2AX disappearance, a prolonged G2/M DNA damage checkpoint activation and an increased sensitivity to IR. An analysis of the proteins that participate in DSB repair revealed no lowered amounts of proteins of the classical NHEJ pathway. However, increased levels of proteins involved in homologous recombination (HR), and in an alternative NHEJ subpathway (Alt-NHEJ) were found in all MM cell lines compared to controls. Interestingly, the Alt-NHEJ protein DNA ligase III was also overexpressed in three out of five samples of plasmatic cells isolated from patients with MM. In vivo assays using a digested plasmid as a substrate revealed slight or non-significant defects in extrachomosomal NHEJ in MM repair-deficient cells, however increased HR was detected by a high percentage of cells with endogenous and IR-induced Rad51 foci. Importantly, the Alt-NHEJ pathway, known to play a role in the generation of deletions and translocations leading to cancer progression, was also found upregulated in MM cells, as revealed by larger deletions and higher sequence microhomology at repair junctions compared to control cells. Taken together, our results uncover aberrant DSB repair in MM. Consequences for genome instability, progression and treatment of the disease will be discussed.

Disclosures:

No relevant conflicts of interest to declare.
NPM-RAR Binding To Tradd Selectively Blocks TNF-Induced Apoptosis While Allowing TNF Activation Of JNK
Authors: Redner Robert L. , Chattopadhyay Anuja , Hood Brian L. , Conrads Thomas P.
Published: October 2019

The t(5;17)(q35;q21) variant of acute promyelocytic leukemia fuses nucleophosmin (NPM) to the retinoic acid receptor alpha (RARA). Expression of the NPM-RAR fusion protein leads to a leukemic phenotype similar to that caused by the t(15;17)(q22;q21) PML-RAR fusion. Using a proteomic approach to identify proteins that bind NPM-RAR, we previously demonstrated that TRADD, amongst other proteins, is a distinct binding partner for NPM-RAR. TRADD serves as an adapter protein that mediates TNF-receptor-mediated activation of the caspase, NFkB, and JNK pathways. We reported that NPM-RAR inhibits TNF-activation of caspase 8 and caspase 3, with subsequent impairment of TNF-induced apoptosis. Extending these findings, we assayed activation of JNK kinase in NPM-RAR-expressing subclones of HELA and U937 cells. We find that TNF activated JNK kinase in NPM-RAR HELA and U937 cells to a similar extent as nontransfected parental controls. This indicates that NPM-RAR binding to TRADD selectively inhibits TNF’s ability to activate caspases, without impairing its ability to activate JNK. In this regard, NPM-RAR effects on TRADD is similar to the EBV oncogenic viral protein LMP1, which binds to the death domain of TRADD, to selectively inhibit caspase activity, while still allowing TNF-mediated activation of survival pathways. Our studies identify a novel mechanism through which NPM-RAR may impact leukemogenesis.

Disclosures:

No relevant conflicts of interest to declare.
The Glutaminase Activity Of L-Asparaginase Is Not Required For Anticancer Activity Against Asns-Negative Cell Lines
Authors: Lorenzi Philip L , Chan Wai Kin , Anishkin Andriy , Purwaha Preeti , Rogers David M , Sukharev Sergei , Rempe Susan B , Weinstein John N
Published: October 2019

L-Asparaginase (L-ASP) is a key component of acute lymphoblastic leukemia therapy. Its mechanism of action, however, is still poorly understood, in part because of its dual asparaginase and glutaminase activities. In the present study, we tested the hypothesis that L-ASP glutaminase activity is required for anticancer activity. We first used molecular dynamics simulations of the clinically used E. coli L-ASP enzyme to guide engineering of mutants that lack glutaminase activity. Dynamic mapping of enzyme-substrate contacts identified the backbone amine of residue Q59 as having frequent contact with glutamine but not asparagine substrate. That difference identified Q59 as a promising mutagenesis target for modifying substrate selectivity. Saturation mutagenesis and screening of the resulting Q59 mutants identified Q59L as retaining asparaginase activity yet exhibiting undetectable glutaminase activity. Using Q59L to test the glutaminase-anticancer hypothesis, we observed no anticancer activity by Q59L against cell lines that do express asparagine synthetase (ASNS), including six leukemia lines—CCRF-CEM, SR, MOLT-4, K562, NALM-6, and REH—and two ovarian cancer lines—OVCAR-8 and SK-OV-3. Wild-type (WT) L-ASP, on the other hand, effected a dose-response in all of those cell lines, suggesting that glutaminase activity is required to kill cancer cells that express ASNS.  Unexpectedly, Q59L exhibited potent anticancer activity against cell lines that do not express detectable ASNS, including the leukemia cell lines Sup-B15 and RS4;11 and ASNS siRNA-treated OVCAR-8 cells. We conclude that the glutaminase activity of L-ASP is not necessary for anticancer activity against cell types that do not express ASNS. Since Q59L is expected to exhibit reduced toxicity relative to wild-type L-ASP because of its reduced glutaminase activity, these findings provide rationale for clinical assessment of Q59L L-ASP for the treatment of ASNS-deficient cancers.

Disclosures:

Lorenzi: ERYtech Pharma: Consultancy, Membership on an entity’s Board of Directors or advisory committees, US 7985548, US 7985548 Patents & Royalties. Off Label Use: L-asparaginase is an enzyme-drug approved for treatment of acute lymphoblastic leukemia.
Differential Response Of Leukemic Blasts To Dexamethasone and Prednisone Occurs At Sub-Saturating Doses
Authors: Pufall Miles A. , Yang Hongxing , Shelton Dawne N.
Published: October 2019

Dexamethasone (dex) and prednisone (pred) are highly related glucocorticoids that are used in the treatment of leukemias and lymphomas. Recently it was shown that dex produces better outcomes in children with high-risk B cell precursor acute lymphoblastic leukemia than pred. Although this result was consistent with the accepted potencies of the two drugs, the reason for this difference is not well understood. Glucocorticoids work primarily by binding and activating the glucocorticoid receptor, which, when bound, associates with genomic response elements to orchestrate gene expression programs. We hypothesized that dex regulates key genes that drive apoptosis in BCP-ALL more strongly. To test this we used microarrays to measure the differential expression of genes in response to saturating doses of dex and pred in three cell lines. Dex and pred induced very similar patterns of gene expression in each cell type, with an overlap of about 60%. Surprisingly, when we compared the 40% of genes that were regulated by dex or pred alone in each cell line, there were no commonly regulated genes among all cell lines. Further, for the 150 genes commonly regulated by both dex and pred in all three cell lines, there were no differences in the magnitude of response, suggested that the difference might lie at a lower, more functional doses. We tested this using RNA sequencing at five concentrations of both dex and pred in a primary patient sample. Consistent with their potency, the average regulated gene required eight times more pred than dex. Interestingly, when we performed unsupervised clustering of regulated genes by concentration, we found more modest differences in the concentration required for the onset of regulation, and that pred selectively suppressed expression of a block of genes at lower concentrations. These data suggest that rather than specific genes being regulated by dex, or simply a more potent response of the same genes, that the difference in apoptosis induction may be due to surprisingly different responses at sub-saturating concentrations.

Disclosures:

No relevant conflicts of interest to declare.
GLI1 Inhibitor GANT61 Deregulates stat3 Phosphorylation In Chronic Lymphocytic Leukemia Cells
Authors: Wang Cheng , MM Kang Lu , Wang Xin
Published: October 2019

Background

Hedgehog(Hh) family has come to be recognized as key fundamental mediators of many carcinomas, but conflicting results exist about its role in chronic lymphocytic leukemia (CLL). Here we examined the effect of GLI inhibitor GANT61 to investigate the role of the Hh-signaling pathway in CLL.

Methods and Results

We conduct real-time PCR for Hedgehog family members on isolated mononuclear cells from peripheral blood (n=35) and bone marrow cells(n=6) to evaluate the presence of the Hh-signaling pathway in CLL. There's no significant difference between peripheral blood and bone marrow cells in levels of Hh members. Profiling of cognate Hh pathway members revealed reduced expression of three key Hh signaling effectors, Patched, Smoothened (SMOH) and GLI, in peripheral blood mononuclear cells (PBMC), whereas transcription levels of other investigated members(SHH, IHH, DHH, GLI2, GLI3 etc.) resembled normal B-lymphocyte levels. However, we found a great heterogeneity for the expression levels of the Hh family with a subset of about 25% of CLL PBMC samples showing high transcript levels ( 1.5-fold than the median) for GLI1 and SMO. There is a direct positive correlation between GLI1 expression and SMO expression. We performed western-blot in CLL PBMC samples and found a positive correlation between phosphorylation of stat3 and GLI1 (figure 1). We examined the activity of GANT61 on viability of cell lines and primary CLL cells (N=3) in vitro by CCK8. GANT61 reduced the cell viability to 65% ± 14% after 24 hours of culture at concentration of 20uM (mean +/¨C SD, P < 0.05). We found that the capacity of GANT61 to inhibit CLL cell viability was associated with stat3 phosphorylation, which is time and dose dependent (figure 2).

Conclusion

These results suggest that in CLL Hh pathway is closely related to stat3 pathway. Moreover, these studies reveal a potential mechanism for the anti-leukemia activity of GANT61 which might inhibit viability of CLL cells by deregulating stat3 phosphorylation.

Disclosures:

No relevant conflicts of interest to declare.
Comparison Of Clinical Implications Of p16 Deletion In B-Lineage Acute Lymphoblastic Leukemia
Authors: Xu Na , Xiao Xiaozhen , Zhou Xuan , Gao Guanlun , Xiao Yajuan , Du Qingfeng , Xiaoli Liu
Published: October 2019

Objective

This paper probes into the impact of P16 gene deletion on the prognosis of children and adults B-ALL.

Method

With the adoption of the Flow Cytometry, G-banding Technique Karyotype Analysis and Fluorescence in situ hybridization (I-FISH) technology, this paper will retrospectively analyze the clinical data of the immunological and cytogenetic detection to the total 124 initial onset B-ALL patients, who were treated in our hospital from January 2006 to December 2008, including children (73 cases, median age 6 years old) and adults (51 cases, median age 29 years).

Result

With the adoption of the I-FISH technology, the result data of the survey are exemplified as follows: in the group of 73 cases of child B-ALL patients, the incidence of P16 gene homozygous deletion, loss of heterozygosity and non-deletion are 13.7% (7 cases), 5.9% (3 cases), 80.4% (41 cases); in the group of 51 cases of adults, they are 13.7% (7 cases), 5.9% (3 cases), 80.4% (41 cases). In this data,recurrence rate in patients with p16 gene deletion is 43.5% (10 cases) in group of children, which is lower than that 90.0% (9 cases) in group of adults. The difference is statistically significant (P = 0.013 ). Overall Survival (OS) is compared in patients of children and adults with and without p16 gene deletion. The result shows that children aged from 1 to 14 with p16 gene deletion have lower Overall Survival (OS) than that without p16 gene deletion (P = 0.013). And it is the same in adult patients older than or are 15 years old (P = 0.020). The long-term disease-free survival DFS is no significant difference (P = 0.154) between that with and without p16 gene deletion in child group. On the contrary, the P16 gene deletion DFS is significantly lower than those without it in the adult group (P <0.001).

Conclusion

p16 gene deletion in B-ALL patients of children and adults is equally poor prognostic factors. Consequently, it is significant for the assessing prognosis and guiding clinical treatment by making a definite status of the p16 gene deletion.

Disclosures:

No relevant conflicts of interest to declare.
Monoclonal Igκ and Igλ Antibodies Minimize Cross-Reactivity With NK Cells In a Multicolor Immunophenotyping Panel
Authors: Deal Emily M. , Inokuma Margaret S. , Yang Xiaoyun , Shye Shou , Franzblau Manuela , Gladding Daiva A. , Crespo Oliver , Wai Hobert
Published: October 2019

Immunophenotyping provides a valuable aid to leukemia and lymphoma diagnosis and classification. Comprehensive initial screening panels must include markers that identify all major cell populations, such as T cells, B cells, and NK cells. It also is useful to further phenotype the subsets of the major populations within the same tube. This can be achieved by using a single fluorochrome for two markers that are specific for subsets of different cell types (for example, CD8 FITC for NK cells and Igλ FITC for B cells). Such a cocktail minimizes the amount of patient sample required for classification while simultaneously decreasing workflow, reagent consumption, and time.

The current study examines the interaction between Igλ FITC, Igκ PE, CD8 FITC, and CD56 PE surface staining. We identified that Igλ and Igκ polyclonal antibodies cause an artificial dragup of the CD8 FITC NK population in a fraction of healthy donors. This dragup results in the majority of NK cells appearing to be CD8+, which is consistent with the phenotype of chronic lymphoproliferative disorders of NK cells [Am J Clin Pathol 127:881; Br J Haematol 120:1026].

Since this artificial dragup may indicate an inappropriate result for the patient specimen, we assessed the effects of different Igκ and Igλ antibodies on this stain. The highly conserved nature of Igλ and Igκ across species caused us to evaluate polyclonal antibodies column-adsorbed against cross-reactive antibodies in addition to total polyclonal and monoclonal antibodies. We conclude that monoclonal Igκ and Igλ antibodies minimize the phenotypic effects on the NK population when using the same fluorochromes, while providing sufficient separation to discriminate Igκ+ and Igλ+ B cells. These studies decrease the risk of errors in disease classification in assays that simultaneously evaluate subsets of multiple cell types.

Disclosures:

Deal: BD Biosciences: Employment. Inokuma:BD Biosciences: Employment. Yang:BD Biosciences: Employment. Shye:BD Biosciences: Employment. Franzblau:BD Biosciences: Employment. Gladding:BD Biosciences: Employment. Crespo:BD Biosciences: Employment. Wai:BD Biosciences: Employment.
Increased ADAM28 Levels In Plasma and Bone Marrow Cells of Patients With Adult Acute Leukemia
Authors: Zhang Xiaohui , Wang Qianming , Hanby Hayley A , Zhou Shiyuan , Zhou Yi , Wang Min , Zhao Jingzhong , Huang Xiao-Jun , Zheng Long
Published: October 2019

ADAM28, a member of the ADAM (A Disintegrin And Metalloproteinase) family, cleaves various substrates including von Willebrand factor. Two isoforms of ADAM28: the membrane associated (ADAM28m) and the secreted (ADAM28s) were identified. Both were overexpressed in solid tumors including breast carcinoma, non-small cell lung cancer, and bladder transitional cell carcinoma. Overexpression of ADAM28 has been shown to promote cell growth, invasion, and metastasis of solid tumors in humans. However, little is known about expression and potential role of ADAM28 in hematological malignancies. In this study, we determined ADAM28 protein and/or mRNA expression in plasma and/or bone marrow cells of patients with various acute and chronic leukemia. An informed consent was obtained from all participants. Plasma and bone marrow aspirates were obtained from a total of 65 patients including 24 patients with acute lymphoblastic leukemia (ALL), 23 patients acute myelocytic leukemia (AML), and 18 patients with other leukemia. Twenty one healthy volunteers were also included in the study (Table 1). By flow cytometry, we showed that mean fluorescent index ratios that are specific for ADAM28m were significantly <>

<>increased in the bone marrow malignant cells in patients with ALL (2.9±0.13) and AML (2.1±0.17) compared with those in the control (0.4±0.06). The p values were <0.01 and <0.05, respectively. In addition, plasma levels of ADAM28s antigen assessed by a specific sandwich ELISA were significantly higher in patients with ALL (386.1±44.6 pg/ml) (p<0.01) and AML (291.3±24.0 pg/ml) (p<0.05) than those in the control (120.7±12.1 pg/ml). Furthermore, the mRNA levels encoding ADAM28m (1.7±0.0) and ADAM28s (2.9±0.1) in the bone marrow cells were significantly higher than those in the control (p< 0.05) (Table 1). Interestingly, cellular ADAM28 overexpression was inversely associated with cellular apoptosis in ALL patients. In four ALL patients with an early relapse, ADAM28 levels (both protein and mRNA) in bone marrow cells were significantly higher than those in patients with complete remission. In conclusion, our data indicate for the first time that a measurement of plasma and bone marrow ADAM28 levels may help identify patients with high risk of early relapse. Whether a treatment that targets ADAM28 has an effect in adult acute leukemia remains to be determined.

Disclosures:

No relevant conflicts of interest to declare.
Genomic Analysis Of Notch Mutations In a Case Of Alagille Syndrome With Acute Lymphoblastic Leukemia
Authors: Bernardi Simona , Tononi Paola , Vargas Sergio Marin , Bassi Giulio , Ambrosetti Achille , Meneghini Vittorio , Scaffidi Luigi , Martinelli Giovanni , Iacobucci Ilaria , Pizzolo Giovanni , Delledonne Massimo , Krampera Mauro
Published: October 2019

Alagille syndrome (ALGS), or arteriohepatic dysplasia, is a congenital multisystem disease due to Notch signalling pathway mutations, most commonly affecting JAG1 (ALGS type 1), and more rarely NOTCH2 (ALGS type 2), leading to hepatic, lung, renal and ocular dysfunction (chronic cholestasis, peripheral pulmonary artery stenosis, dysplastic kidneys pigmentary retinopathy), and skeletal abnormalies (minor vertebral segmentation, characteristic facies, posterior embryotoxon/anterior segment defects). ALGS is an autosomal dominant disease, but it is characterized also by variable penetrance and clinical expression and somatic/germline mosaicism.

A 20-year-old man with ALGS was admitted to the University Hospital of Verona because of pancytopenia. Following analyses led to the diagnosis of Philadelphia chromosome/bcr-abl-negative, CD10-positive, B-lineage acute lymphoblastic leukemia (common B-ALL).

In order to identify the genetic components involved in this complex phenotype, we sequenced the exome of a bone marrow sample collected from the patient. By genome interpretation with Knome pipeline applied to the reference genome UCSC hg19, we found missense variants both in NOTCH2 (E38K) and JAG1 (P871R) genes that are mainly involved in the syndrome, although their effect on protein function was predicted not to be deleterious. However, we detected putative damaging mutations in genes such as PAX5 (R38H) and NOTCH1 (K1821N) which might be strongly related to the observed disease. In fact, PAX5 is a member of PAX protein family of transcription factors implicated into regulation of early development, that binds NOTCH2 and likely altering its functionality. On the other hand, NOTCH1 is involved in cell growth and proliferation and thus the predicted alteration of function of the corresponding protein may have an important role in neoplastic transformation.

Disclosures:

No relevant conflicts of interest to declare.
Body Mass Index and Uncontrolled DM Impact On Acute Myeloid Leukemia (AML) Outcomes
Authors: Abdelfatah Mohamed , Anand Ankit , Kanaan Zeyad , Al-Ameri Ali , Haller Nairmeen
Published: October 2019

Background

The incidence of obesity is increasing worldwide and is associated with numerous adverse health outcomes. Obesity was associated with more toxicity and worse prognosis in pediatric AML patients. On the other hand diabetic patients diagnosed with AML have multiple complications related to infection organ failure and chemotherapy side effects, Previous epidemiological studies in children have shown significant correlations in incidence between AML and DM in children.

Aim

To study Body-Mass Index (BMI) and uncontrolled DM impact on clinical outcome of adult AML pts.

Methods

A total of 187 adult pts with AML between 2002-2011 were enrolled. Retrospective data included demographics, labs, cytogenetics and outcome. LeukemiaNET Standardization (LNS), complete remission (CR), overall survival (OS) and relapse free survival (RFS) were obtained (Dohner E, Blood 2010). BMI of <18.5 defined as underweight, 25-30 was defined as overweight, while >30 as obesity. On the other hand Fasting blood glucose and Hemoglobin A1c were measured, DM severity was classified according to guidelines, as non diabetics (A1c)<5.7 and normal FBG<100, high risk for DM with (A1c) from (5.7 - 6.4) and uncontrolled DM (A1c) >6.5. Fischer's and Wilcoxon tests were used for comparatives between groups, cox proportional hazards and logistic regression for associations for OS/RFS and CR, Kaplan-Meier test for OS and RFS estimates via JMP software V9.0. IRB approval was obtained according to Helsinki declaration.

Results

The median age was 70 years, with 98 (52%) were men. 45 pts (24%) had complex cytogenetics, 28 pts (15%) had AML progressed from MDS. Median BMI was 28.7 (range 14.8-51.4). 10 (5%) were underweight, 34 (18%) had normal BMI, 59 (32%) were overweight, and 84 (45%) were obese. 149 pts (80%) were not Diabetic, 12 pts (6%) had high risk for DM, 26 pts (14%) uncontrolled DM. Overall Median OS was 23 weeks; 46 weeks in normal BMI, 30 weeks in overweight, 14 weeks in obese, 15 weeks in underweight, A according to DM subgroups; 24 weeks non-diabetics, 15 weeks had high risk for DM, 37 had uncontrolled DM. At diagnosis, BMI or Hemoglobin A1c classes were not associated with age, sex, white blood count (WBC), platelets, blasts, ECOG status, LNS; nor treatment toxicities. Even though median survival in weeks was lower in obese and underweight pts, Median OS and RFS were not different in univariate or across BMI subgroups (p=0.32 and 0.22) or Hemoglobin A1c subgroups (p=0.19 and 0.71)

Conclusions

BMI and uncontrolled DM did not show statistically significant correlation with LNS, relapse rates, OS or RFS.

Disclosures:

No relevant conflicts of interest to declare.
Recurrent STAT6 Mutations In Follicular Lymphoma
Authors: Malek Sami , Kaminski Mark S. , Li Hongxiu , Ouillette Peter , Jones Sian , Fox Heather , Jacobi Kathryn , Saiya-Cork Kamlai , Bixby Dale , Lebovic Daniel , Shedden Kerby , Sabel Michael , Marentette Lawrence , Cimmino Vincent , Chang Alfred , Yildiz Mehmet
Published: October 2019

Introduction

Follicular lymphoma (FL) constitutes the second most common non-Hodgkin’s lymphoma in the Western world. FL carries characteristic recurrent structural genomic aberrations. However despite recent advances, knowledge regarding the coding genome in FL is still evolving and is currently incomplete.

Methods

To further our understanding of the genetic basis of follicular lymphoma (FL), we used solution exon capture of sheared and processed genomic DNA isolated from FACS-sorted lymphomatous B-cells and paired CD3+ T-cells isolated from twenty three cases of FL and one case of DLBCL (which was transformed from prior FL), followed by paired-end (96-101 base pair read length per side) massively parallel sequencing. The sequence data were characterized by a mean depth of coverage of 41, and 90% of bases in the target region were covered by at least 10 reads. Bioinformatics pipelines developed by our bioinformatics core served as the primary data source to nominate candidate mutated genes in downstream data analysis.

Results

The bioinformatics pipeline nominated 711 distinct candidate mutations in 24 FL cases. Sanger sequence validation confirmed 39 recurrently (≥ 2 FL cases) mutated genes. Genes with confirmed mutations in ≥ 2 FL cases in the discovery cohort were subsequently selectively expanded into a combined FL validation cohort of 114 cases. In addition to frequent mutations in MLL2, CREBBP, BCL2, TNFRSF14, EZH2, OCT2, ARID1A, IRF8 and MEF2B, we here report novel mutations in STAT6 in FL. STAT6 mutations were identified in 11% (12/114) of FL and predominantly affected the DNA binding domain (DBD; comprising STAT6 amino acids 268-430). Two FL cases each carried two distinct STAT6 mutations, presumably targeting both alleles. Of interest, the majority of FL-associated STAT6 mutations affected a single amino acid codon (codon 419), resulting in the STAT6 mutants p.419D>D/G or p.419D>D/H. These FL-associated STAT6 mutations are distinct from mutations previously described in primary mediastinal B-cell lymphoma (PMBCL).

Given the involvement of STAT6 in signal transduction pathways activated by multiple cell surface receptors, as well as the recently described involvement of STAT6 in antiviral innate immunity (involving an interaction between the STAT6 DBD and the protein STING), we are currently exploring functional consequences of the novel STAT6 mutations in FL and cell line models.

Conclusion

We report identification of somatic mutations in STAT6 in 11% of FL. These mutations predominantly affected the STAT6 DNA binding domain. We identify a novel STAT6 mutation hotspot in STAT6 codon 419 (p.419D>D/G or p.419D>D/H).

Disclosures:

Lebovic: Genentech: Speakers Bureau; Allos/Spectrum: Speakers Bureau; Celgene: Speakers Bureau; Onyx: Speakers Bureau.
A Phase II Study Of Aprepitant As Anti-Emetic Prophylaxis In Patients Receiving Induction Chemotherapy For AML
Authors: Brandwein Joseph , Yee Karen W.L. WL , Schuh Andre C , Gupta Vikas , Seki Jack T
Published: October 2019

Background

Standard AML induction chemotherapy includes an anthracycline plus cytarabine (3+7); the former is considered moderately emetogenic. Such patients typically received anti-emetic prophylaxis using a 5-HT3 antagonist; however, many patients continue to experience chemotherapy-induced nausea and vomiting (CINV) requiring additional therapy. Aprepitant is a neurokinin inhibitor with documented efficacy against CINV, but has not been evaluated to date in the setting of AML induction therapy.

Patients and Methods

Patients with previously untreated AML, receiving induction chemotherapy with daunorubicin 60 mg/m2 IV daily on Days 1-3 plus cytarabine 200 mg/m2 IV daily (100 mg/m2 for patients age 60 and over) as a continuous IV infusion x 7 days, were enrolled in a Phase II single arm open label study. All patients received 5-HT3 antagonist prophylaxis using either granisetron 1 mg IV daily or ondansetron 8 mg IV q12h on the 3 daunorubicin dosing days. In addition, patients received aprepitant 125 mg PO on Day 1 followed by 80 mg PO daily on Days 2-5. No corticosteroids were used. Additional anti-emetic therapy was given as needed, at the discretion of the medical staff, for any nausea or vomiting. The primary endpoint was any vomiting or retching, while secondary endpoints included nausea requiring supplemental anti-emetics. Results were compared to a retrospective cohort of 40 patients who had received the same AML induction chemotherapy and 5-HT3 antagonist prophylaxis, but without aprepitant.

Results

At a pre-planned interim analysis, 27 patients receiving aprepitant on study were evaluated. By the end of Day 5, 3/27 patients (11%) had experienced at least one episode of vomiting or retching, while by the end of Day 8 four additional patients had experienced vomiting/retching, for a cumulative incidence of 26%. In comparison, the cumulative incidence of vomiting or retching by the end of Day 5 in the retrospective cohort was 50% (20/40), and 53% by Day 8. The proportion of patients experiencing vomiting was below 8% per day on aprepitant dosing days, but increased to11% on Day 6.  The cumulative incidence of nausea and/or vomiting in the aprepitant group was 52% by the end of Day 5 and 60% at the end of Day 8, as compared to 78% at both time points in the retrospective cohort. There were no toxicities attributable to aprepitant.

Conclusion

The results indicate that aprepitant is highly effective, when combined with a 5-HT3 antagonist, in preventing vomiting in patients receiving AML induction chemotherapy with 3+7. The combination appears less effective in controlling nausea. The results warrant further evaluation in a Phase III randomized placebo controlled study.

Disclosures:

Brandwein: Merck: Honoraria, Research Funding. Off Label Use: Aprepitant as anti-emetic prophylaxis for AML induction. Seki:Merck: Honoraria.