bluuebunny/crossref_metadata_2025 huggingface dataset:
The Role of Hematopoietic Cell Transplantation (HCT) for Burkitt Lymphoma: a Report From the Center for International Blood and Marrow Transplant Research (CIBMTR)
Authors: Gajewski James L. , Carreras Jeanette , Lazarus Hillard M. , Laport Ginna G. , Montoto Silvia , Maloney David G. , Hari Parameswaran
Published: October 2019

Abstract 2390

Burkitt lymphoma (BL) is an aggressive B cell lymphoma primarily affecting children and young adults and is characterized by the highest doubling time of any tumor. Cyclical intensive chemotherapy and rituximab confer high complete remission (CR) rates and 80% long term disease free survival in chemotherapy sensitive disease. The role of autologous (autoHCT) or allogeneic (alloHCT) transplant is not well described in BL. We report the outcomes of 241 recipients of HCT for BL between 1985 and 2007 reported to the CIBMTR. Five patients (pts) received syngeneic twin grafts in addition to autoHCT in 113 pts, HLA identical sibling alloHCT (SIB) in 80 pts and mismatched related or unrelated donor (UNR/MM) alloHCT in 48 pts. Baseline patient and disease related risk factors varied significantly between cohorts (table1). The autoHCT cohort had a higher proportion of pts with chemotherapy sensitive disease (86%), peripheral blood grafts (73%) and HCT in first CR (42%). In the UNR/MM cohort, 25% pts were chemotherapy resistant and only 6% were in CR1. The use of autoHCT has declined in recent years with the majority (81%) performed before 2001. Conditioning regimen for alloHCT was myeloablative in 88% (86% and 92% in SIB and UNR/MM respectively). Treatment related mortality (TRM) was higher in alloHCT recipients (table1). Cumulative incidence of relapse/progression at 5 yrs (95% CI) was 44 (35-53)% for autoHCT, 42(31-53)% for SIB and 48 (34-62)% for UNR/MM. For autoHCT, 5-yr progression free survival (PFS) was 48(39-58)%, 78% for those in first CR versus 27% for disease beyond CR1 (p<0.001). For alloHCT, 5-yr PFS was 50% for those in first CR versus 19% for disease beyond CR1 (p=0.001) (figure 1). 5-yr PFS was 30 (20-41)% for SIB and 22 (12-35)% for UNR/MM. Progressive BL was the commonest cause of death.

Conclusion:

While autoHCT and alloHCT are both feasible in patients with BL, the use of autoHCT appears to be declining in recent years concomitant with the advent of modern chemotherapy. AlloHCT was performed in those with considerably higher risk disease. Approximately one fifth of advanced BL pts receiving alloHCT beyond CR1 had long term disease free survival.

Disclosures:

No relevant conflicts of interest to declare.
Low-Dose Fludarabine and Cyclophosphamide Combined with Rituximab In the Treatment of Elderly/Comorbid Patients with chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma (CLL/SLL): Preliminary Results of Project Q-Lite by Czech CLL Study Group
Authors: Smolej Lukas , Spacek Martin , Brychtova Yvona , Belada David , Schwarz Jiri , Doubek Michael , Motyckova Monika , Cmunt Eduard , Rohon Peter , Klaskova Katerina , Kozak Tomas
Published: October 2019

Abstract 2466

Background:

Combination of fludarabine, cyclophosphamide and rituximab (FCR) is currently considered the treatment of choice in physically fit patients (pts) with chronic lymphocytic leukemia (CLL). However, many patients cannot tolerate this aggresive treatment because of advanced age and/or serious comorbid conditions. For these patients, chlorambucil has remained so far the standard of treatment. Low-dose fludarabine-based regimens have recently demonstrated promising results in small studies.

Aims:

to assess efficacy and safety of low-dose FC or FCR regimen used in elderly/comorbid patients with CLL.

Patients and Methods:

Between March 2009 and June 2010, we treated 74 pts with active disease (CLL, n=70, SLL, n=4, 57% males, median age, 70 years [range, 58–83], median Cumulative Illness Rating Score 4 [range, 0–10]) by low-dose FC/FCR at fourteen centers cooperating within Czech CLL Study Group. Dose reduction of chemotherapy was as follows: fludarabine to 50% (12 mg/m2 i.v. or 20 mg/m2 orally on Days 1–3), cyclophosphamide to 60% (150mg/m2 i.v./p.o. D1-3). The dose of rituximab was standard (375mg/m2 in 1st cycle, 500mg/m2 from 2nd cycle). The choice of regimen (FC vs FCR) was at the discretion of the attending physician. Treatment was repeated every 4 weeks. Antimicrobial prophylaxis with cotrimoxazole and aciclovir or equivalents was recommended. Fifty per cent of pts were treated in first line, the remaining half had relapsed/refractory CLL. Rai stage III/IV was present in 57% pts; 39% had bulky disease. IgVH genes were unmutated in 74%; according to hierarchical model, del 11q was present in 32% and del 17p in 8%.

Results:

FCR was used in 72 pts, FC in 2. Based on intention-to-treat principle, the overall response/complete response rate (including clinical CR and CR with incomplete blood count recovery) was 70/35%; 34 pts are still on treatment. No data on PFS/OS are available yet. Serious (CTC grade III/IV) neutropenia occurred in 51%, thrombocytopenia in 13% and anemia in 10% of pts. Serious infections were diagnosed in 13% of pts. Three pts (4%) died, all of them after failure of treatment (pneumonia, n=2, pulmonary embolism, n=1).

Conclusions:

Treatment of elderly/comorbid CLL patients with low-dose FC/FCR demonstrated very promising results. Toxicity was acceptable and manageable. Longer follow-up is needed for the assessment of PFS and OS. Supported by research project MZO 00179906 from Ministry of Health, Czech Republic.

Disclosures:

Smolej: Roche: Honoraria; Bayer-Schering: Honoraria; Genzyme: Honoraria. Off Label Use: Low-dose FCR in elderly/comorbid patients with CLL. Belada: Roche: Consultancy, Honoraria. Motyckova: Roche: Honoraria.
Recurrent Mutations Found In Acute Monocytic Leukemia by Exomes Sequencing
Authors: Yan Xiao-Jing , Xu Jie , Gu Zhao-Hui , Pan Chun-Ming , Lv Gang , Li Keqin Kathy , Mi Jian-Qing , Yang Shen , Song Huai-Dong , Chen Sai-Juan , Chen Zhu
Published: October 2019

Abstract 2490

Acute monocytic leukemia, the M5 subtype of acute myeloid leukemia (AML-M5), is a distinct group with characteristic clinical features and has been reported to have a poor prognosis. A subset of AML-M5 is associated with various chromosomal translocations involving the MLL locus at 11q23, while mutations are also reported in genes such as NPM1, FLT3, NRAS at different frequencies in this disease. However, these genetic changes occur only in a part of AML-M5 patients and some of them lack specificity due to the presence in other AML subtypes. To address the important genetic and molecular factors for the pathogenesis of M5 in a comprehensive manner, we sequenced exomes from nine AML-M5 initial bone marrow (BM) samples and matched control samples. We identified 64 somatic mutations within the coding sequences of 61 genes including 57 point mutations and 7 insertions or deletions (indels), among which 17 genes had at least 2 cases of mutations in 100 validated AML-M5 BM samples. We tested a part of these gene mutations in different subtypes of AML and found that some mutations are restricted to AML-M5 and AML-M4 (acute myelomonocytic leukemia) with more than 15% and 10% frequency, respectively. Thus, by systemic sequencing of exomes from a group of AML-M5 cases, we discovered recurring mutations that may play an essential role in the pathogenesis of AML with monocytic features.The prognostic significance of the mutations will be present in details.

Disclosures:

No relevant conflicts of interest to declare.
Macrothrombocytopenia and Hemolysis Associated with Phytosterolemia
Authors: Wang Gaifeng , Wang Zhaoyue , Cao Lijuan , Jiang Minghua , Bai Xia , Ruan Changgeng
Published: October 2019

Abstract 2515

Objective:

Phytosterolemia is a rare autosomal recessive sterol storage disease, characterized by increased serum concentrations of plant sterols (phytosterols). It is caused by gene mutations of ABCG5 and ABCG8. The patients suffer from xanthomas and premature atherosclerosis. In this study we demonstrate that macrothrombocytopenia and hemolysis are intrinsic features in four unrelated patients with phytosterolemia, and their clinical and pathogenesis are investigated.

Methods:

Erythrocyte and platelet morphology was examined under light microscope. Platelet aggregation was measured turbidometrically. Platelet glycoproteins were analyzed using flow cytometry. Biochemistry was analyzed by routine methods. The plasma sterol levels were measured by HPLC method. ABCG5 and ABCG8 genes were directly sequenced to identify mutations, Restriction endonuclease analysis was used to exclude the possibility of gene polymorphism.

Result:

All the four patients presented xanthomas, macrothrombocytopenia, hemolysis and splenomegaly. The blood smears showed large platelets, some as large as erythrocytes and abnormal erythrocyte shapes, such as stomatocytes. All the patients had bleeding tendency. There were mild defect in aggregation induced by ADP and ristocetin. The red cells showed an increased osmotic fragility. Plasma concentrations of phytosterols including sitosterol, stigmasterol and cholestanol were markedly elevated (30 fold) in the affected patients. Patient 1 was found to be homozygous for C→T mutation at nucleotide 64 in exon 1 of the ABCG5 gene, which results in the premature termination of the ABCG5 protein at amino acid 22 (Gln22X). Patient 2 was homozygous for C→T mutation at nucleotide 1336 in exon 10 of the ABCG5 gene, which results in the premature termination of the ABCG5 protein at amino acid 446 (Arg446X). Patient 3 was compound heterozygous for a large fragment deletion at nucleotide 1842 to 1883 in exon 12, and a complex mutation of del 1938C–1939G/ins1938T in exon 13 of ABCG8. Patient 4 was also compound heterozygous for C→T mutation at nucleotide 1336 in exon 10 and A→G mutation at classical cut locus in intron 9.

Conclusion:

In this study, we identified four patients with phytosterolemia who had macrothrombocytopenia and stomatocytic hemolysis in addition to common increased plasma plant sterols and xanthomas. Gene analysis confirmed the diagnosis. We suggest that the coexisting macrothrombocytopenia and stomatocytic hemolysis represent a new subtype of phytosterolemia, and plasma phytosterols should be measured in patients who have stomatocytic hemolysis and macrothrombocytopenia (so called“Mediteranean macrothrombocytopenia/ stomatocytosis”).

Disclosures:

No relevant conflicts of interest to declare.
Important Role for flt3 Ligand In the Generation of Early B and T Cell Precursors From Human Primitive Hematopoietic Progenitors, Revealed by Telomerized Human Stromal Cells.
Authors: Liu Bing , Ohishi Kohshi , Masuya Masahiro , Hamada Hirofumi , Katayama Naoyuki
Published: October 2019

Abstract 2582

The regulatory mechanism of human early T and B lymphopoiesis has not been well defined. It has been mostly studied, using mouse stromal cell lines, but either T or B lineage cells were developed. We first tested if human telomerase catalytic subunit (hTERT)-transfected telomerized human stromal cells, which have been shown to efficiently support myelopoiesis in serum-containing and serum-free cultures, are able to support the generation of T and B lineage cells from human primitive hematopoietic progenitors.

When we examined the relationship of the expression of lymphoid markers CD7, CD19, CD10, and cytoplasmic CD79a (cyCD79a) with CD38 on cord blood CD34+ cells, these antigens were mainly expressed on CD38high population. The CD19+ or CD10+ cell population mostly overlapped with the cyCD79a+ population. The CD19+ or CD10+ population was largely mutually exclusive with the CD7+ population. We therefore isolated CD34+CD38low/-CD7−CD19−CD10− cells to obtain immature hematopoietic progenitors and incubated the cells with the hTERT-stromal cells in the presence of 20%FCSaMEM. After three weeks, CD19−CD10−CD20−VpreB−cyCD79a+ early B, CD19+CD10+CD20+/−VpreB−cyCD79a+ proB, and CD7+CD56−CD2+/−CD1a−cyCD3− proT stage of cells were detected, in addition to myeloid cells.

We then examined the effect of cytokines on the generation of the proB and proT cells. Addition of flt3 ligand (flt3L) to the culture led to an increase in the numbers of the early B, proB, and proT cells by 8-, 13- and 12-fold, respectively, while flt3L did not significantly affect their differentiation. On the other hand, SCF or TPO alone did not significantly affect the generation of early B, proB, and proT cells, but, in combination with flt3L, enhanced the generation of these cells. No effects were seen with IL-7. In serum-free cultures without cytokines, considerable number of CD7+ cells was detected, but few or no B lineage cells including cyCD79a+CD19− early B cells were not observed. Nevertheless, flt3L not only enhanced the generation of CD7+ cells but also induced the development of cyCD79a+CD19− early B cells.

Our data show the usefulness of telomerized human stromal cells to assess human lymphopoiesis in vitro and suggest an important role for flt3L in human early B and T lymphopoiesis in the human bone marrow environment.

Disclosures:

No relevant conflicts of interest to declare.
Neuropsychology and MRI Observational Study In An Adult Sickle Cell Disease Population In South East London
Authors: Rawle Heather , Holmes Paul , Thomas Veronica J , Cartwright Ronwyn , Howard Jo
Published: October 2019

Abstract 2642

We studied 36 patients attending the sickle cell clinic in our large adult population of over 600 patients. These patients presented to the sickle psychology service because they had a past history of a stroke, or presented with concerns about memory. An MRI brain scan and neuropsychological testing (including tests for IQ, processing speed, executive function and memory) were performed on each patient. The data were organised into four groups in terms of severity of MRI abnormalities: normal MRI n=13; silent cerebral infarcts (subcortical punctate small vessel cerebrovascular disease) n= 11; severe infarcts as an adult n=8; and severe infarcts as a child n=4. The majority of patients with a history of stroke had evidence of large vessel infarcts as well as features of deep watershed ischaemia seen in large vessel (distal internal carotid artery) disease. These groups did not differ significantly in terms of gender, phenotype, age and mood. Cognitive impairments were more prevalent in the severe infarct groups but were also found in patients with silent cerebral infarcts and normal MRIs. Executive functioning and processing speed deficits were evident in all groups but were more severe in the silent cerebral infarct and severe infarct groups. Chi-squared tests for trend showed that the following test scores tended to reduce as MRI abnormalities increased: Full-Scale IQ (p=0.016), Processing Speed Index (p=0.015), Trail Making Test A (p=0.014), Trail Making Test B (p=0.018), and FAS Verbal Fluency Test (p=0.006). This suggests that executive functioning, processing speed and full-scale IQ are particularly vulnerable to the effects of MRI abnormalities in this patient population. Although the cognitive impairments were more severe in the groups with abnormal MRIs, there was significant cognitive impairment in some patients with normal MRIs, suggesting that other factors are also causative of cognitive impairments. These factors may include physiological causes such as impaired perfusion, and psychosocial factors such as disruption to education. These results agree with a recent US study (Vichinsky et al, 2010, JAMA, 303, 1823–1831) showing cognitive impairment in patients with normal MRI scans which implies that MRI is not an adequate screening tool to identify patients with cognitive impairment. This study has important clinical implications in terms of how cognitive deficits can affect the effectiveness of patient – health care professional consultations, patients' ability to manage their SCD and adhere to medication and health care advice. It is important therefore to identify patients with SCD who have such cognitive impairments so appropriate support can be offered.

Disclosures:

No relevant conflicts of interest to declare.
Clinicopathological and Cytogenetic Comparison of Patients with Acute T Lymphoblastic Leukemia and Lymphoblastic Lymphoma
Authors: Raza Sania , Patnaik Mrinal M. , Feldman Andrew L , Ansell Stephen M.
Published: October 2019

Abstract 2705

Background:

Due to similar cytomorphological features and treatment strategies, T lineage lymphomas (T-LBL) and lymphoblastic leukemias (T-ALL) are usually grouped together as a single entity. Traditionally, the difference between T-LBL and T-ALL has been based on percentage of blasts found in the bone marrow (BM): >25% BM blasts and/or presence of peripheral blood (PB) blasts consistent with T-ALL. Recent studies, based on immunophenotypic, cytogenetic and molecular differences, emphasize the clinical uniqueness between T-LBL and T-ALL and argue against grouping them as a single entity.

Objectives:

To perform a clinicopathologic and cytogenetic comparison between patients with T-LBL and T-ALL in order to identify differences between these disease groups.

Methods:

We utilized the Mayo Clinic Lymphoma database to identify consecutive adult patients (>18 years) diagnosed with T-cell leukemia and lymphoma from 1980 to 2010. The patients were sub-divided into T-LBL and T-ALL based on BM and PB blasts (absence of BM and PB blasts consistent with T-LBL). Clinicopathological and cytogenetic characteristics were retrospectively reviewed.

Result:

50 patients met the criteria for T-cell leukemia/lymphoma (29-T-ALL, 21-T-LBL). 50% of patients in each group were females with median age at diagnosis of 32 years for T-LBL and 29 years for T-ALL. Characteristics associated with a statistically significant survival difference for the cohort included: white blood cell count at diagnosis (>100,000/cmm), platelet count (<50,000/cmm), LDH (>500 U/L), presence of BM blasts and an adverse karyotype (hypodiploidy and MLL rearrangements). ECOG performance status, number of lymph node groups involved, extranodal disease, CNS involvement and constitutional symptoms had no impact on survival. Furthermore, no overall survival difference between patients with T-LBL and T-ALL was identified (figure 1).

Among patients with T-LBL, 11 (51%) patients with extranodal disease had shortened survival (p=0.02). CNS involvement was seen in 4 (19%) of T-LBL patients and 5 (17%) of T-ALL patients, and did not affect overall survival (p=0.8). 4 (19%) T-LBL patients and 1 (3%) T-ALL patient had pleural involvement at diagnosis. There were no morphologic, flow cytometric, or immunohistochemical differences noted between the two groups.

Conclusion:

In a large study from a single institution, we confirm no survival difference between T-LBL and T-ALL, which further provides ongoing support that from a therapeutic standpoint these groups can be viewed as a single entity. However, we hope to further clarify clinical differences by performing microarray and chemokine analysis on available tissue specimens.

Disclosures:

No relevant conflicts of interest to declare.
Long-Term Experience with Allogeneic Bone Marrow Transplant (BMT) in Chronic Myeloid Leukemia
Authors: Enrico Alicia Inés , Basquiera Ana , Napal Juan Jose , Sturich Ana Gabriela , Prates Maria Virginia , Garcia Juan , Yantorno Sebastian , Milone Santiago , Navickas Alicia , Milone Jorge
Published: October 2019

Abstract 4582

Introduction:

Introduction of imatinib mesylate has changed the approach towards bone marrow transplantation in Chronic Myeloid Leukemia (CML). Historically considered as a curative therapeutic option, with a probability of disease free survival ranging from 40 to 60%. Its indicated use has been restricted to patients with treatment failure to prior tyrosin-kinase inhibitors or blast crisis.

Objective:

To assess the outcome of patients with CML who undergo BMT

Materials and methods:

66 patients who underwent allogeneic BMT between the years 1994 and 2011 in two health care facilities were analyzed. 63 (93%) were performed between 1994 and 2005. At the time of transplantation, patient status was: 56 patients in 1st Chronic Phase and 10 in advanced phases. Fourteen patients received Unrelated Donor BMT and 37 had Related Donor BMT. Transplant risk was assessed, based on the European Bone Marrow Transplantation (EBMT) score (sc). The conditioning regimens which were used included: for patients in chronic phase, cyclophosphamide (Cy) and busulfan (Bu) and for patients in accelerated phase etoposide (VP 16) was added to the previous regimen, and for unrelated donors BuCy + ATG or alentuzumab was used. Prophylaxis for Graft vs Host Disease (GVHD) was performed with a two-drug regimen (cyclosporine, methotrexate), with or without methylprednisone.

Result:

66 patients were recorded (28 female/38 male), with a mean age of 35 years at the time of transplantation (range 8–55). The source of Stem Cell was bone marrow in 60 patients and peripheral blood in 6 patients. 43% of patients developed GVHD: (29 patients) according to clinical criteria established by the European Cooperative Group. Overall Survival (OS) was 50% with a mean follow-up of 48 months. Analyzed according to the EBMT risk score, OS was 64% in the 0–2 score group and 31% in patients with a 3–5 score. Procedure-related mortality was 16%, and the most frequent causes included: hepatic veno-occlusive disease, infections, GVHD, and graft failure.

Conclusions:

BMT in patients with CML has been substantially reduced since the introduction of tyrosin-kinase inhibitors. In our experience, this shows its healing potential with an extended follow-up and a prolonged survival.

Disclosures:

No relevant conflicts of interest to declare.
Polymorphisms of Mir-34b/c, Mir-146a and Mir-196a-2 and Predisposition to Chronic Lymphocytic Leukemia and Monoclonal B-Cell Lymphocytosis
Authors: Jamroziak Krzysztof , Szemraj Janusz , Rawstron Andy , Szemraj-Rogucka Zofia , Grzybowska-Izydorczyk Olga , Giannopoulos Krzysztof , Buda Gabriele , Martino Alessandro , Jesionek-Kupnicka Dorota , Franiak-Pietryga Ida , Piotrowska Magdalena , Robak Tadeusz
Published: October 2019

Abstract 4585

Since microRNAs control expression of protein-coding oncogenes and tumor suppressor genes, functional microRNAs single nucleotide polymorphisms (SNPs) may modulate the risk of tumorigenesis including susceptibility to chronic lymphocytic leukemia (CLL). In this case-control study we investigated whether pri-miR-34b/c rs4938723 (T-to-C), miR-196a2 rs11614913 (C-to-T) and miR-146a rs2910164 (G-to-C) SNPs influence predisposition to CLL or monoclonal B-cell lymphocytosis (MBL). miR-34 family members are direct transcriptional targets of tumor suppressor p53, and miR-34b/miR-34c have been recently proposed as regulators of TCL1 (T-cell leukaemia/lymphoma 1) expression in CLL. A rs4938723 SNP in the promoter region of pri-miR-34b/c might affect transcription factor GATA binding and pri-miR-34b/c expression. Alterations in miR-196a2 and miR-196a2 may represent common cancer predisposition pathways as miR-196a2 rs11614913 and miR-146a rs2910164 have been recently associated with altered risk of different solid tumors including breast, lung, prostate and liver cancers. Additionally, we verified the impact of these microRNA SNPs on prognostic factors and clinical course of CLL. Genotyping was performed using PCR-based assays in a total of 561 Caucasians including 195 patients with CLL, 166 patients with MBL and 200 healthy control individuals. The assessed minor allele frequencies (MAFs) of the investigated SNPs were as follows: for rs4938723 SNP C allele frequency was 0.37 in CLL, 0.36 in MBL and 0.30 in controls, for rs11614913 SNP T allele frequency reached 0.43 in CLL, 0.42 in MBL and 0.39 in controls, and for rs2910164 SNP C allele frequency was 0.29 in CLL, 0.30 in MBL and 0.26 in controls. Logistic regression analysis did not detect significant associations of CLL or MBL with studied genotypes or alleles (p>0.05). Moreover, none of the tested genetic variants was found to influence CLL patients’ progression-free survival (PFS) or overall survival (OS) with median follow-up time from diagnosis of 3.0 (0–13.9) years. In conclusion, our data suggest that investigated SNPs in pri-miR-34b/c, miR-146a and miR-196a-2 genes are not likely to play a major role in the susceptibility to CLL and MBL or in clinical outcome of CLL.

Disclosures:

No relevant conflicts of interest to declare.
FLT3 Internal Tandem Duplication and Kinase Domain Mutations Remain Poor Prognostic Markers in Intermediate Cytogenetic AML After Treatment with Transplant in First Remission
Authors: Aljurf Mahmoud , Abalkhail Hala , Almhareb Fahed , Chaudhri Naeem A. , Walter Claudia Ulrike , Toulimat Mohammed , Al Zahrani Hazzaa , Khalil Salem , Mohamed Said , Rasheed Walid , Alsharif Fahad , Albitar Maher
Published: October 2019

Abstract 4627

The internal tandem duplication (ITD) and tyrosin kinase domain (TKD) D835 mutation in the Fms-like tyrosine kinase receptor-3 gene (FLT3) are perhaps the most studied prognostic molecular markers in acute myeloid leukemia (AML). However most of the studies have been in patients treated with standard intensive chemotherapy. The prognostic significance of FLT3, especially the TKD D835, in AML patients treated with intensive chemotherapy followed by allogeneic hematopoietic stem cell transplantation (HSCT) is not well studied.We studied the prognostic value of both FLT3-ITD and FLT3-D535 mutations in patients with AML treated with HSCT.

Methods:

Samples from 87 patients with AML were analyzed for FLT3 ITD and D835 mutations using fragment length analysis for ITD and restriction enzyme digestion followed by fragment length analysis for D835. All patients were diagnosed with AML, treated with intensive chemotherapy, then HSCT. They included 66 (76%) patients with intermediate cytogenetic abnormalities and 21 (24%) patients with adverse cytogenetics.

Results:

ITD mutation was detected in 18 patients (21%) patients, while the D835 mutation detected in 6 patients (7%). FLT3-ITD was associated with significantly shorter overall survival (OS) when all patients are considered (P=0.006). When only patients with intermediate cytogenetic abnormalities treated with HSCT in their first complete response (CR1) are considered, FLT3-ITD was significantly associated with poor OS (P=0.001). Event free survival (EFS) was also significantly shorter in patients with the FLT3-ITD (P=0.005). Similarly, D835 mutation was associated with significantly shorter OS (P=0.002) in this group of patients. Surprisingly, in patients with poor cytogenetic abnormalities, patients with FLT3-ITD had significantly longer survival (P=0.05), although the number is too small.

Conclusion:

Our data suggests that FLT3 mutations, both ITD and D835, remain poor prognostic markers in patients with AML treated with intensive chemotherapy followed by HSCT, but only in patients with intermediate risk cytogenetic abnormalities and this may not be relevant in patients with high-risk AML.

Disclosures:

No relevant conflicts of interest to declare.
4Ts HIT Scoring in the Medical-Surgical ICU
Authors: Crowther Mark A. , Heels-Ansdell Diane , Williamson David , Granton John , Burry Lisa , Lamontagne Francois , Finfer Simon , McDonald Ellen , Vallance Shirley , Klinger James , Friedrich Jan , Paunovic Bojan , Walter Stephen , Zytaruk Nicole , Cook Deborah J , Warkentin Theodore E.
Published: October 2019

Abstract 4669

Background:

Thrombocytopenia occurs in 20–45% of critically-ill medical-surgical patients. The ‘4Ts’ HIT score (with 4 domains: Thrombocytopenia, Timing of thrombocytopenia, Thrombosis and oTher reason for thrombocytopenia) might reliably identify patients at low risk of HIT. Agreement on 4Ts scoring is uncertain in this setting.

Objective:

To compare 4Ts HIT scores among research coordinators (who scored real-time), and 2 adjudicators (who scored retrospectively, independently) during an international heparin thromboprophylaxis trial.

Methods:

763 of 3746 medical-surgical ICU patients in PROTECT met enrolment criteria in this HIT substudy, if any of the following occurred: platelets <50×109/L, platelets decreased to 50% of ICU admission value, venous thrombosis, or if HIT was otherwise clinically suspected. Persons independently completed 4Ts scores blinded to all laboratory HIT results and each others' scores. 475 of these patients had a central or local laboratory HIT test performed and had 4Ts scoring by adjudicators; 432 were scored by research coordinators.

Results:

Most patients (410, 86.3%) had a 4Ts score ≤3 conferring a low pretest probability (PTP) of HIT whereas 57 (12.0%) had a moderate PTP, and 8 (1.7%) had a high PTP, as determined by adjudicator consensus. Raw agreement on the 4Ts score was good for central adjudicators overall; however, agreement between adjudicated consensus scores and real time research coordinator scores was not as high. 4Ts agreement (raw and chance-corrected) is presented below.

Conclusions:

Real time 4Ts scoring by research coordinators was reasonably close to that obtained by calibrated central adjudicators, suggesting the 4Ts score is somewhat reliable at the bedside when performed by trained personnel. The fourth domain of 4Ts (oTher causes of thrombocytopenia) generated the most disagreement. Further studies of 4Ts scoring by bedside clinicians is needed.

Funding:

Heart and Stroke Foundation of Canada

Disclosures:

Crowther: Pfizer: Consultancy, Honoraria; Leo Pharma: Consultancy, Honoraria; Bayer: Consultancy, Honoraria; BI: Honoraria; CSL Behring: Consultancy; Octaphram: Consultancy; Artisan: Consultancy. Off Label Use: Dalterparin is not indicated for prolonged prophylaxis in critically ill medical patients. Zytaruk:Pfizer: donated study drug dalteparin for PROTECT. Cook:Pfizer: donated study drug dalteparin for PROTECT. Warkentin:Sanofi-Aventis: Speakers Bureau; Pfizer Canada: Speakers Bureau; GlaxoSmithKline: Consultancy, Research Funding; GTI Diagnostics: Consultancy, Research Funding; Canyon Pharma: Consultancy, Speakers Bureau; Informa: Patents & Royalties.
Heparin-Induced Thrombocytopenia Antibody Is More Common in Patients with Active Bacterial Infections
Authors: Smith Melody , Patel Prapti A. , Dowell Jonathan E , Frenkel Eugene P. , Sarode Ravindra , Shen Yu-Min P
Published: October 2019

Abstract 4678

Heparin mobilizes and binds to platelet factor 4 (PF4), forming a heparin-PF4 complex on the platelet surface. Rarely, IgG antibodies recognize and bind to the immune complex, causing platelet activation and thrombin generation, resulting in heparin induced thrombocytopenia (HIT). There is evidence that PF4 has a role in antibacterial host defense and can concurrently prime the immune system with IgG antibodies that can cross-react with the heparin-PF4 complex (Krauel et al, Blood 2011). In the setting of a bacterial infection, the PF4 molecules interact with the bacterial cell wall and induce antibody production. These antibodies are able to recognize any bacteria coated in PF4, resulting in phagocytosis. In the setting of heparin, this antibody can cross react with the heparin-PF4 complex and result in HIT. In vitro studies suggest that E. coli, S. pneumo, and S. aureus coated in PF4 cross-react with human heparin induced anti-PF4/heparin antibodies.

We have collected a database of patients being tested for HIT at our institution and performed a retrospective analysis of the incidence of bacterial infections. We hypothesize that patients who have a bacterial infection will be more likely to develop HIT due to anti-bacterial antibodies that cross-react with the heparin-PF4 complex. We collected culture data from 54 patients with positive HIT antibody by ELISA and 178 patients with negative HIT antibody. The incidence of positive cultures and active infections in described in table 1. In the HIT negative patients, 61/178 (34.3%) had positive cultures, compared to 28/54 (51.9%) in the HIT positive patients (p=0.0198). Given that there is data to support E. coli, S. pneumo, and S. aureus-induced antibody cross-reactivity, we performed a subgroup analysis of the incidence of these specific bacterial infection in HIT positive and negative patients (33.3% versus 19.7%, p=0.0357). There is a statistically significant difference noted in the active infections, subgroup analysis, as well presence of any positive cultures in the HIT positive patients as compared to the HIT negative patients.Table 1Colonized Infections (%)Active Infections (%)E. coli, S. pneumo, S. aureus (%)Any + culture (%)HIT negative (n=178)15 (8.4)61 (34.3)35 (19.7)66 (37.1)HIT positive (n=54)2 (3.7)28 (51.9)18 (33.3)29 (53.7)p value0.2420.01980.03570.0293

This data supports that in the setting of active infection, particularly with E. coli, S. pneumo, and S. aureus, HIT antibody results should be carefully evaluated. Patients with positive cultures are likely to have thrombocytopenia from infection or sepsis, regardless of the presence of a HIT antibody. Thus, additional factors, such as the patient's 4T score and the presence of thrombosis should be considered prior to initiating treatment for HIT.

Disclosures:

No relevant conflicts of interest to declare.
Initial Management of Suspected Heparin-Induced Thrombocytopenia: Opportunities for Education
Authors: Smith Melody , Dowell Jonathan E , Frenkel Eugene P. , Sarode Ravindra , Shen Yu-Min P
Published: October 2019

Abstract 4686

According to the current recommendations for patients being evaluated for heparin-induced thrombocytopenia (HIT), a pretest assessment for the probability of HIT should be performed to evaluate the need for laboratory testing and the treatment modality that the patient should receive. The 4T score is an accepted clinical tool used for this purpose. For patients with a 4T score <4 discontinuation of heparin products is not indicated. However, in those patients who have a 4T ≥4, heparin should be discontinued and treatment with a direct thrombin inhibitor (DTI) should be initiated while performing the immunologic assay for HIT antibody.

In this study, a database of patients being tested for HIT at our institution was compiled and retrospective analysis was performed to determine whether our treatment population was being managed appropriately. The treatment of 159 patients in whom HIT antibody had been sent over a 2- year period, from 5/2008 to 5/2010, was assessed. Of the sample population, 32/159 (20%) had not been exposed to heparin prior to the HIT antibody being sent. Furthermore, the majority of the patients, 104/159 (65%) had a 4T score <4. Thus fully 85% of the patients tested in this cohort did not need to be tested. Of the latter group with heparin exposure but low clinical probability for HIT, 57/104 (54%) had heparin unnecessarily discontinued once HIT was suspected.

Most notably, 23/159 (14%) of the patients had a 4T greater than 4. The data for these patients indicates that the majority of them were not treated as per the current guidelines. As noted in the Table, 69% of these patients had either no treatment, meaning that heparin was not discontinued, or simply had heparin stopped with no DTI initiated. Only 5/23 (22%) of the patients in this cohort were placed on a DTI. Of these patients, 2 were also placed on warfarin, as they were noted to have active thromboses.

This data suggests that the management of patients suspected of having HIT at our institution is not consistent with the current recommendations with wasteful practices and inappropriate therapy. These results highlight opportunities for further education of clinicians regarding HIT.No treatmentHeparin stopped onlyDTIWarfarinNot exposed to heparin (n=32)324T less than 4 (n=104)4757004T greater than 4 (n=23)41452

Disclosures:

No relevant conflicts of interest to declare.
Rituximab May Be a Useful Option in Children with Severe Chronic Immunthrombocytopenia (ITP)
Authors: Halimeh Susan , Schubert Erdmuth , Paulus George , Rott Hannelore , Schaefers Michael , Quabeck Klaus
Published: October 2019

Abstract 4692

This otherwise healthy 12-year-old girl presented in March, 2011 with a history of severe ITP that had been diagnosed in November, 2007. Since then, she had suffered from recurrent bruises, petechiae, epistaxis, persistent bleending after tooth-loss and during menstruation (menarche: April, 2010), and subsequent iron-deficiency. Severe headache of sudden onset turned out to be caused by intracranial hemorrhage (ICH) in December, 2009, and in September, 2010, respectively. CT-scans in September 2010 revealed 2 additional ICH which had occured without symptomps. Fortunately, she recovered without sequelae from these complications. The initial and the subsequent treatment consisted of immunoglobulie (IG) and prednisone (IV or orally) given in various doses and for various time-intervals, always followed by a rise in platelet count for short periods of time, and a subsequent decline, when the drugs were withdrawn. Both ICH occured in phases of watchful waiting, while platelet count were below 1000. Splenectomy was offered and repeatedly denied by the patient and her parents.

After the second ICH (Sept 2010) the patient received continuous weekly IG as prophylaxis of further ICH. Serious side-effects of prednisone in this patient (Cushing’s habitus, abdominal striae, insomnia and secondary depression) led to withdrawal of this drug. Since February 2011, the efficacy of IG was decreasing significantly, and the patient was referred to hematological practice for further treatment. We obtained the patient’s and her parents’ informed consent for a trial of 4 weekly doses rituximab (375mg/ square meter body surface after premedication with an antihistamine and 50mg prednisone). The infusions were given over a time-period of 5 weeks (May – June 2011) and were well tolerated. Platelet counts began to rise significantly after the 2. course and have remained above 200 000 since the beginning of July, 2011 without any other medication.

Through the long-term course in this patient yet has to be followed, we feel that Rituximab (though not licensed for ITP in Germany) has been a useful option in this heavily pretreated patient. Because of good clinical results from US trials, like cases confirm previous reports of B.U. Mueller and C.M. Benett (Pediatr. Blood Cancer 2009; 52:259 and Blood 2006; 107:2639) we are now able to perform this treatment.

Disclosures:

No relevant conflicts of interest to declare.
Neutrophil Volume, Conductivity and Scatter Parameters to Diagnose Neonatal Sepsis
Authors: Celik Istemi Han , Demirel Gamze , Sukhachev Dmitry , Erdeve Omer , Dilmen Ugur
Published: October 2019

Abstract 4726

INTRODUCTION:

Neonatal sepsis remains an important clinical syndrome despite advances in neonatology. Early diagnosis and adequate antibiotic treatment are required because of high rates of mortality and morbidity. Current hematology analysers can determine cell volume (V), conductivity for internal composition of cell (C) and light scatter for cytoplasmic granularity and nuclear structure (S) and standart deviations. This VCS measurements were used to diagnose sepsis particularly in adults and higher levels of V and lower levels of S were seen in septic patients. We investigated these parameters in diagnosis of neonatal sepsis.

METHOD:

We investigated these parameters in secreening of neonatal sepsis, defined as a systemic infection, validated by a positive blood culture, diagnosed beyond the first day of life. We used LH780 hematological analyzer (Beckman Coulter, Fullerton, CA). We combined these parameters with interleukin-6 (IL-6) and C-reactive protein (CRP), and developed models to diagnose sepsis by Effective Modelling of Moleculer Activity (EMMA). It uses combinatorial algorithm of the selection parameters for regression equation based on modified stepwise procedure. It allows compute a number of “best” regression equations with different combinations of parameters.

RESULTS:

A total of 237 newborn, 61 proven sepsis, 108 clinical sepsis and 68 control, were enrolled the study. Gram positive and negative bacteria including 12 types of microorganism were isolated from 34 and 27 patients, respectively. Mean neutrophil volume (MNV) and volume distribution width (VDW) were found to be statistically increased both in proven and clinical sepsis groups. We developed models using MNV, VDV, IL-6 and CRP (Table 1). We used these models both for proven sepsis and sepsis (proven and clinical sepsis) groups. Table 2 and 3 summarize the cut-off levels of MNV, VDV, IL-6, CRP and models' performance of proven sepsis and sepsis groups. These models gave more sensitivity and specificity than usage of MNV, VDW, IL-6 and CRP alone.

CONCLUSION:

We suggest to use combination of MNV and VDW with markers such as CRP and IL-6, and use diagnostic models created by using EMMA including these markers.

Disclosures:

No relevant conflicts of interest to declare.
Inhibition of STAT5 Phosphorylation by Anti-GM-CSF Monoclonal Antibodies: Validation of a New Whole Blood Assay
Authors: Grivel Karine , Magnenat Laurent , Moulard Maxime
Published: October 2019

Abstract 4728

Introduction.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) regulates survival, proliferation, and differentiation of monocytes/macrophages. The GM-CSF receptor (CD116) is expressed on primary monocytes and mediates cell signaling through STAT5 phosphorylation. Therefore STAT5 is central to GM-CSF action. BioCytex has developed a new semi-quantitative flow cytometry (FC) assay to measure the phosphorylation status of STAT5 in human primary monocytes and lymphocytes in whole blood samples. The assay was used to investigate the potency of recombinant monoclonal antibodies (MAbs) for the neutralization of human GM-CSF.

Methods.

Engineered anti-GM-CSF MAbs were generated and characterized by Merck Serono. Several clones and preparation were tested, including MAbs 343 (low and high LPS), 467; 340; 339; 366, and MAb 285 as isotype control. Their different in vitro potencies for blocking recombinant GM-CSF activity in stable cell lines was determined using the TF-1 proliferation and IL-8 secretion in U937 cell assays. Whole blood FC assay was designed and validated at BioCytex using either a rabbit MAb or a rabbit polyclonal antibody specific for phospho-STAT5 (Tyr694). Monocytes were gated using a PE-labeled anti-CD14 probe. Inhibition of STAT5 phosphorylation by anti-GM-CSF MAbs was performed using recombinant GM-CSF at EC90.

Results.

EC90 of GM-CSF was determined at 2.3 pM from n=5 healthy volunteers. All GM-CSF specific MAbs inhibited STAT5 phosphorylation in monocytes, whereas the control (MAb 285) did not. More interestingly, there was a marked effect of LPS content since inhibition of STAT5 phosphorylation was not completed in presence of high LPS load (MAb 343). IC50 and IC90 for MAbs 339 and 366 were 10-fold increased as compared to MAbs 467 and 340. IC50 were ranging from 0.12 to 0.19 nM for clones 343 (low LPS), 467 and 340. IC50 and IC90 for clone 339 were 0.016 and 0.034 nM, respectively.

Conclusion.

Inhibition of GM-CSF mediated monocyte activation by neutralizing MAbs can be monitored in a whole blood assay. STAT5 phophorylation could be viewed as a potential biomarker for GM-CSF receptor activation. The cell-signaling inhibition potency in primary cells correlated with in vitro potency using stable cell lines. In summary, the new validated FC could be used for research and clinical development purposes.

Disclosures:

Grivel: LFB Biotechnologies: Consultancy. Magnenat:Merck Serono S.A.: Employment. Moulard:BioCytex: Consultancy, Employment.
mTOR Signaling Is Required for the Survival and Proliferation of Hematopoietic Stem/Progenitor Cells
Authors: Xiao Xia , Luo Hongmei , LaRue Amanda C. , Schulte Bradley A. , Wang Yong
Published: October 2019

Abstract 4787

The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that regulates protein synthesis, gene transcription, cell growth and cell proliferation. Previous studies have demonstrated that abnormalities in the Pten-mTOR pathway may contribute to the development of leukemia and lead to premature exhaustion of hematopoietic stem cells (HSCs). These findings suggest a role for mTOR in the regulation of HSC self-renewal and cell transformation. The aim of this study was to investigate the involvement of mTOR signaling in the survival and proliferation of HSCs and hematopoietic progenitor cells (HPCs). Immunofluorescent staining with antibodies against phosphorylated mTOR and S6 kinase revealed that Thrombopoietin (Tpo) activates mTOR in cultured mouse bone marrow (BM) lineage negative cells, suggesting that Tpo may promote the survival and proliferation of HSCs/HPCs via activation of the mTOR signaling pathway. Further studies revealed that treatment with an mTOR specific small molecule inhibitor (Ku-69734) significantly suppressed the colony-forming ability of HPCs as evidenced by a dose-dependent decrease in the production of CFU-GM, BFU-E and CFU-GEMM. We also examined the clonogenic function of HSCs using cobblestone-area forming cell (CAFC) assays and found that Ku-69734 treatment markedly reduced the number of CAFCs in long-term BM culture. Moreover, immunophenotyping and flow cytometric analyses showed that inhibition of mTOR induced apoptosis primarily in HSCs and to a lesser degree in HPCs, indicating that mTOR inhibition may suppress the clonogenic function of HSCs and HPCs via the induction of apoptosis. Together, these data demonstrate that mTOR signaling is required for the survival and proliferation of HSCs and HPCs. Given that many mTOR inhibitors are currently in clinical trials for the treatment of cancers, our findings provide the rationale to further evaluate the potential toxicity of mTOR inhibitors in HSCs/HPCs in vivo.

Disclosures:

No relevant conflicts of interest to declare.
A Wharton's Jelly Mesenchymal Stromal Cell Derived 3D Osteogenic Niche Allows for Cord Blood Stem Cell Attachment
Authors: Aljitawi Omar S. , Keefe Peggy , Ott Lindsey , Li Dandan , Zhang Da , Abhyankar Sunil , Garimella Rama , McGuirk Joseph , Detamore Michael
Published: October 2019

Abstract 4813

Background:

Most of umbilical cord blood (UCB) stem cell ex vivo expansion methods have led to UCB stem cell differentiation instead of their self-renewal. Evidence suggests that bone osteoblastic cells are responsible, through physical contact with hematopoietic stem cells (HSCs), for the maintenance of long-term HSCs.

Aims:

To develop a 3 dimensional (3D) osteogenic structure that provides a niche for UCB stem cells and secondarily, assess the ability of this structure, in addition, to a cocktail of cytokines to expand UCB stem cells.>

Methods:

Mesenchymal stromal cells (MSCs) isolated form Wharton's Jelly (WJ) were seeded into biodegradable scaffolds followed by osteogenic differentiation induction using osteogenic differentiation media for up to 4 and 6 weeks to develop a 4-week and a 6-week osteogenic scaffolds, respectively. CD34+ selected UCB stem cells were expanded on a monolayer of WJMSCs using a cocktail of cytokines for one week, following which the monolayer and expanded CD34+ UCB stem cells were trypsinized and added to the 4-week and 6-week 3D osteogenic scaffolds (3D conditions), or plated again in culture flask (2D conditions). Pre- and post-expansion total nucleated cell counts were determined and flow cytometry was used to assess the phenotype of the expanded population.

Results:

Osteogenic differentiation was successfully induced in 3D scaffolds as evident by Alizarin-red staining, scanning electron microscopy (SEM) and molecular testing. UCB stem cell attachment to the osteogenic scaffold was verified by SEM. TNCs were expanded 10X in 2D and 200X in 3D conditions. However, the percentage of CD34+ cells was 5.31% and 4.91% in 2D, and 2.98% and 1.14% in 4-week and 6-week 3D conditions, respectively.

Conclusion:

Attachment of CD34+ UCB stem cells to 3D osteogenic scaffold allowed for their expansion, though the majority of the expanded cells lost their CD34 expression possibly secondary to their differentiation.

Disclosures:

No relevant conflicts of interest to declare.