bluuebunny/crossref_metadata_2025_split huggingface dataset:
BPI A645G SNP but Not NOD2 Genotype Predicts for Acute Lung Injury After Allogeneic Stem Cell Transplantation
Authors: Wermke Martin , Maiwald Stefanie , Thiede Christian , Schetelig Johannes , Ehninger Gerhard , Platzbecker Uwe , Bornhäuser Martin , Wassmuth Ralf 
Published: October 2019

Abstract
Abstract 2324

Introduction:
Infectious as well as non-infectious pulmonary complications are key determinants of non-relapse mortality (NRM) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Single nucleotide polymorphisms (SNP) in two genes coding for nucleotide-binding oligomerization domain containing 2 (NOD2) and bactericidal/permeability-increasing protein (BPI) have been shown to predict for the occurrence of GvHD and chronic pulmonary complications after allo-HSCT. We aimed at determining the effect of SNPs in these genes on the occurrence of acute lung injury (ALI) after allo-HSCT.

Patients and Methods:
We retrospectively analyzed a cohort of 272 patients undergoing allo-HSCT and their respective related and unrelated donors. ALI was functionally defined as a drop of the oxygenation index (OI = partial pressure of oxygen in arterialized capillary blood/inhaled oxygen concentration) below 300. Genotyping for NOD2 SNP 8, 12 and 13 and BPI SNP A645G was done using Real Time-PCR. The incidence of ALI was assessed by univariate competing events statistics and by a multivariate model according to Fine and Gray.

Results:
Overall, 115 of 272 (42.3%) patients suffered from ALI at least once during the first 100 days post allo-HSCT. Median time to onset was 11 days. Neither NOD2 polymorphisms in the donor nor in the recipient correlated with the occurrence of ALI. Further, BPI recipient genotype was not associated with an increased incidence of this complication. On the other hand, ALI was more frequent in patients receiving a transplant from a donor homozygous for the G allele of the BPI A645G SNP (Figure 1). This association was confirmed in multivariate analysis (OR 1.46, p = 0.048). Other independent risk factors for the occurrence of ALI were age (as continuous variable, OR 1.03, p = 0.002) and transplantation from an unrelated vs. related donor (OR 1.66, p = 0.018) whereas the use of intensive conditioning was a risk factor of borderline significance (OR 1.59, p = 0.077). In spite of its association with ALI, BPI donor genotype did not correlate with cumulative incidence of NRM at 100 days (G/G: 6.7%, 95% CI: 1.0 – 12.4% vs. A/A or A/G: 10.2%, 95% CI: 6.0 – 14.4%; p = 0.380) or overall survival.

Conclusion:
In contrast to other investigators we found no correlation between NOD2 genotype and the occurrence of acute pulmonary complications after allo-HSCT. On the other hand, BPI A645G SNP in the donor was identified as an independent risk factor for ALI in these patients.

Disclosures:
No relevant conflicts of interest to declare.
HLA-Haploidentical Stem Cell Transplantation for Malignant Hematological Diseases Using G-CSF Mobilized Bone Marrow and Peripheral Blood Stem Cells
Authors: Luo Yi , Tan Yamin , Lai Xiaoyu , Shi Jimin , Zheng Weiyan , Huang He 
Published: October 2019

Abstract
Abstract 2384

Introduction:
Allogeneic haematopoietic stem cell transplantation (HSCT) has been used to treat malignant hematological diseases. For patients who do not have a matched sibling donor or a matched unrelated donor (MUD) for transplantation, an eligible HLA-haploidentical donor can be identified rapidly in nearly all cases. However, HLA-haploidentical BMT has been associated with significant risks of complication, such as graft rejection and severe GVHD and infection.

Materials and Methods:
Thirty eight consecutive patients with malignant hematological diseases received HLA-haploidentical stem cell transplant from April 2008 to June 2010. Eligible patients were 9– 48 years of age with acute myeloid leukemia (n=10),acute lymphocytic leukemia (n=17), myelodysplastic syndrome(n=5), chronic myeloid leukemia (n=4), non Hodgkin lymphoma (n=2). Transplantation conditioning consisted of Ara-C (4 g/m2/d, i.v.) on day -10 and -9, Bu (9.6mg/kg i.v. in 12 doses) on day -8, -7 and -6, Cy (1.8 g/m2/d, i.v.) on day -5 and -4, Me-CCNU (250 mg/kg, i.v.) on day -3, and ATG (2.5 mg/kg/d i.v.) on day -5 to -2. The prophylaxis of aGVHD consisted of cyclosporine A (CsA), mycophenolate mofetil (MMF), and short-term methotrexate. Donors were primed with rhG-CSF (7.5 mg/kg per day) injected subcutaneous (s.c.). Bone marrow cells and peripheral blood stem cells(PBSC) were harvested in nineteen donors, and only PBSC were harvested in the other 19 donors.

Results:
Primary engraftment was achieved in all patients. All the patients achieved complete donor chimerism in the peripheral blood before day +28. Seven patients (18.4%) had grade I aGVHD, ten (26.3%) had grade II aGVHD, four(10.5%) had grade III aGVHD and four (10.5%) had grade IV aGVHD. II-IV aGVHD occurred in nine patients treated with bone marrow cells and PBSCs, the same number patients suffered from II-IV aGVHD treated with only PBSCs. Transplant related mortality within 100 days after transplantation was 15.8%. At a median follow-up of 10 months (range 2–28months), the overall survival was 68.4%.

Conclusion:
Haploidentical stem cell transplantation is relatively safe and efficient for the patients who have no HLA matched donors.The results require to confirm and show that G-BM combined with PBSC or G-PBSC from haploidentical family donors have equal effect.

Disclosures:
No relevant conflicts of interest to declare.
The York Platelet Syndrome: A Third Case and New Findings
Authors: White James G , Professor Regents , Gunay-Aygun Merel 
Published: October 2019

Abstract
Abstract 2526
Studies published in 2003 described a mother and son with a mitochondrial myopathy and abnormal platelets containing giant electron opaque organelles and large, multilayered organelles resembling targets. The condition has remained a familial disorder until recently when a new patient, a four year old female, with the same myopathy and platelet ultrastructure was discovered. Now the disorder can be considered a syndrome named after the initial family, the York Platelet Syndrome (YPS). Whole mount preparations of unfixed, unstained YPS platelets revealed that the giant opaque organelles (OO) were as inherently electron dense as delta granules (dense bodies, DB) but several times larger. Analytical electron microscopy revealed both organelles were rich in phosphorous and calcium, suggesting they might have the same origin. However, measurements of serotonin and adenine nucleotides in YPS platelets revealed normal levels. Also, staining YPS platelets for acid phosphatase with cerium as the capture ion and using analytical electron microscopy to determine its location in whole mount preparations revealed the presence of cerium in the giant OO, but not in DB. The findings suggested the OO might be giant secretory organelles. However, exposure of YPS platelets to high dose thrombin caused complete secretion of α granules and DB, but OO and TO were not released through the open canalicular system or the surface membrane. Thus, OO and TO are not secretory organelles. Studies of YPS in the new patient have confirmed and extended these findings. Staining her cells with diaminobenzidine and H202 to detect platelet peroxidase has revealed enzyme reaction product in both OO and TO, as well as in channels of the dense tubular system. Thus, the giant OO and TO organelles contain proteins and elements usually separated into vesicles in the Golgi Complex and reassembled into alpha granules, dense bodies and lysosomes in megakaryocytes. Instead proteins and elements from the rough and smooth endoplasmic reticulum (SER) develop into giant OO and TO without passing through lamellae of the Golgi complex. Involvement of the SER and DTS in formation of the giant organelles is strongly supported by YPS platelets from the new patient. Large, flat sheets and coils of SER were a common finding in the new YPS patient platelets, and present, but less frequent in the original two YPS patients. The findings strongly support the concept that the YPS in a disorder of megakaryocyte endoplasmic reticulum allowing the formation OO and TO which continue to develop in the DTS of YPS circulating platelets. It is the first such disorder to be described in human megakaryocytes and platelets.

Disclosures:
No relevant conflicts of interest to declare.
Acetylation-Dependent Interaction of GATA-1 with the Potential Mitotic “Bookmarking” Protein Brd3.
Authors: Lamonica Janine M , Kadauke Stephan , Deng Wulan , Blobel Gerd 
Published: October 2019

Abstract
Abstract 2598
Acetylation of the transcription factor GATA-1 facilitates its ability to drive erythroid differentiation by enhancing its association with in vivo target sites. However, the underlying mechanism through which GATA-1 acetylation functions has remained elusive. To test whether GATA-1 acetylation serves to recruit essential cofactors, we performed a peptide affinity screen and identified Brd3 as an acetylated GATA-1 interacting partner. Brd3 belongs to the BET protein family that also includes Brd2, Brd4, and Brdt, and is characterized by tandem bromodomains (BD1 and BD2) and an extraterminal (ET) domain. We show that Brd3 and GATA-1 physically interact in an acetylation-dependent manner in vitro and in vivo. Mapping studies revealed that the interaction depends on BD1 of Brd3 and one of the two major acetylation sites that resides near the C terminal zinc finger of GATA-1. By ChIP-seq and ChIP-qPCR, endogenous Brd3 is recruited to virtually all GATA-1-occupied regulatory elements in erythroid cells, including both GATA-1activated and repressed genes. Although Brd3 has been reported to associate with acetylated histones along the entire length of transcribed genes, we found that Brd3 recruitment correlates poorly with histone acetylation along gene bodies. In agreement with our biochemical data, an intact BD1 is essential for the in vivo recruitment of Brd3 to GATA-1-occupied elements, further demonstrating that acetylation of GATA-1 is essential for Brd3 association in vivo. Notably, a pharmacological compound that targets acetyl lysine binding sites in BD1 and BD2 disrupts the Brd3/GATA-1 interaction in vitro, diminishes Brd3 and GATA-1 association at key erythroid genes in vivo, and impairs GATA-1 target gene expression and erythroid maturation. In concert, these findings suggest a mechanism by which the first bromodomain of Brd3 recognizes acetyl-lysines on GATA-1 to facilitate GATA-1 chromatin occupancy. These studies raise an interesting question: In contrast to the great majority of transcription factors, BET family proteins bind to chromatin during mitosis and might serve an epigenetic memory function to properly reactivate gene transcription upon exit of mitosis. We are currently investigating whether Brd3 functions by bookmarking GATA-1-bound sites throughout mitosis to aid in transcriptional memory and stability of lineage specific gene expression.

Disclosures:
No relevant conflicts of interest to declare.
Natalizumab Increases Circulating CD34+ Cells with An Impaired Functional Potential In Patients with Multiple Sclerosis without Affecting the Bone Marrow.
Authors: Saure Christian , Zohren Fabian , Schroeder Thomas , Bruns Ingmar , Cadeddu Ron Patrick , Fischer Ute , Kobbe Guido , Hartung Hans Peter , Kieseier Bernd C , Haas Rainer 
Published: October 2019

Abstract
Abstract 2620

Introduction:
In our previous report (Zohren et al., Blood 2008) we could show that the blockade of the heterodimer VLA-4 by the monoclonal IgG4 antibody natalizumab leads to a significant increase in circulating CD34+ cells in patients with multiple sclerosis (MS). We now extend our analysis on the influence of natalizumab on CD34+ cells comparing bone marrow (BM) and peripheral blood (PB) derived CD34+ cells of natalizumab patients with those from healthy donors.

Methods:
A total of 83 patients with MS receiving natalizumab were included. In vitro adhesion, migration and apoptosis assays as well as LTC-IC of immunomagnetically enriched CD34+ cells were conducted. Flow cytometric analyses were performed to assess phenotype and composition of the CD34+ subsets.

Results:
The median concentration of circulating CD34+ cells was significantly greater compared to normal donors (7.7/μL vs. 1.8/μ L; p= 0.0001) and remained relatively stable during a one year treatment with natalizumab. Leukocyte cell counts, the number of T cell subsets as well as the number of CD19+ B cells and CD56+ natural killer cells were in normal range in PB and BM after short- and long-term treatment with natalizumab. However, we found significantly reduced adhesion and migration abilities of circulating CD34+ cells under natalizumab treatment in comparison to G-CSF mobilized CD34+ cells of healthy donors. Moreover, the self-renewal capacity of these cells was poor. In contrast, no significant difference was seen between the BM of natalizumab patients and the BM of healthy donors with regard to cellularity and proportion of CD34+ cells. In addition, neither co-expression of CD49d nor the adhesion ability of the BM derived CD34+ cells revealed a significant difference between the two collective.

Conclusions:
Our data indicate that natalizumab mediates an increase in circulating CD34+ cells by impaired homing. These findings argue against the use of natalizumab-exposed PB CD34+ cells for transplantation.

Disclosures:
No relevant conflicts of interest to declare.
Distinct CD8+/CD57+ Large Granular Lymphocytic Populations Seen by Flow Cytometry Are Associated with Monoclonal T-Cell Populations, a Retrospective Case-Control Study of Large Granular Lymphocytic Leukemia
Authors: Ohgami Robert S. , Zehnder James L. , Arber Daniel A. 
Published: October 2019

Abstract
Abstract 2707
Large granular lymphocytic (LGL) leukemia is a disease with chronic elevations of large granular lymphocytes for > 6 months associated with a T-cell clone and often cytopenias. While a defined classification has been established in the 2008 WHO, the clinical and pathologic diagnosis often remains elusive, as patients can exhibit significant polyclonal or oligoclonal proliferations of LGLs in reactive conditions. Therefore predicting and defining patients with increased LGLs and significant monoclonal proliferations remains difficult. In an attempt to identify flow cytometry patterns which might be associated with monoclonality, we performed a retrospective review of patients at our institute between 2003–2010 with significant populations of peripheral blood LGLs in which T-cell clonality tests were also performed. 66 independent cases were identified and were divided impartially by time into a study set (cases from 2008–2010) and an experimental set (cases from 2003–2007). In the study set (2008-2010), 31 flow cytometry cases were identified, which stratified into 16 cases with monoclonal T-cell populations and 15 with oligoclonal or polyclonal populations. Comparing flow cytometry scatter-plots, we identified what appeared to be a discrete flow cytometry pattern. Specifically, those cases with distinct CD8+(dim)/CD57+ populations were significantly associated with T-cell monoclonality (p =< 0.0001) as well as lower neutrophil counts (average ANC= 1.78, p < 0.001) (See Figure 1). Such a preliminary analysis suggested that identification of patients with specific CD8+(dim)/CD57+ populations might define patients with not only monoclonal proliferations, but also with clinically and pathologically significant disease. To further evaluate this hypothesis, we used these criteria (discrete CD8+(dim)/CD57+ populations) to examine, in a blinded analysis, our experimental set of 35 consecutive cases of large granular lymphocytosis from 2003–2007. 11 cases were identified which demonstrated significant CD8+(dim)/CD57+ populations. Yet again, we observed that distinct populations of CD8+(dim)/CD57+ populations were associated with T-cell monoclonality (p =0.037), as well as neutropenia (average ANC=1.49; p=0.046). These findings demonstrate and define a major subset of clinically and pathologically significant patients through the use of flow cytometry immunophenotyping.

Disclosures:
No relevant conflicts of interest to declare.
The Kinase Inhibitor TKI258 Is Active Against the Novel CUX1-FGFR1 Fusion Detected In a Patient with T-Lymphoblastic Leukemia/Lymphoma and t(7;8)(q22;p11)
Authors: Wasag Bartosz , Lierman Els , Meeus Peter , Cools Jan , Vandenberghe Peter 
Published: October 2019

Abstract
Abstract 2715
The 8p11 myeloproliferative syndrome (EMS) is an aggressive, atypical stem cell disorder associated with chromosome translocations that constitutively activate FGFR1 by fusion to diverse partner genes. Here, we describe a case with a clinical and hematological diagnosis of T-lymphoblastic leukemia/lymphoma and with a t(7;8)(q22;p11) on cytogenetic analysis. We identified the fusion partner involved and characterized this translocation functionally in vitro using the interleukin 3 (IL3) dependent Ba/F3 cell line.
The translocation was analyzed in more detail by FISH using FGFR1 flanking probes. We could confirm the 8p11 breakpoint and 7q as the partner chromosome. Using 5'-RACE CUX1 (7q22) was identified as the fusion partner of FGFR1 in this patient with T-lymphoblastic leukemia/lymphoma. CUX1 is a homeobox family DNA binding protein not previously described as a fusion partner in hematological malignancies. To evaluate the transforming potential of this novel fusion, the CUX1-FGFR1 fusion was cloned and used to transform Ba/F3 cells. CUX1-FGFR1 expressing Ba/F3 cells displayed IL3 independent proliferation thus demonstrating the oncogenic character of this fusion protein. Western blotting of the transformed Ba/F3 cells showed activation of FGFR1 as well as its downstream target STAT5. Treatment of the CUX1-FGFR1 expressing Ba/F3 cells with the kinase inhibitors PKC412 and TKI258 significantly inhibited cell growth with an IC50 of 483 and 489 nM respectively. With western blotting a direct effect of both inhibitors on FGFR1 kinase activity as well as on different downstream effectors was proven. Furthermore using an annexinV/propidium iodide-based apoptosis assay, we could show that PKC412 and TKI258 both induced apoptosis followed by cell death in inhibitor treated CUX1-FGFR1 transformed Ba/F3 cells. The antiproliferative effect of the inhibitors could be rescued by addition of IL3 for the TKI258 treated but not for PKC412 treated CUX1-FGFR1 expressing cells. This observation indicates a selective action of TKI258 on FGFR1 signaling at the concentrations used. In contrast, for PKC412 non-specific cytotoxicity is also contributing to the antiproliferative effect. In summary, we identified a novel CUX1-FGFR1 fusion in a case with EMS and a novel t(7;8)(q22;p11), and demonstrated the oncogenic potential of CUX1-FGFR1 in the Ba/F3 cell line. This new fusion partner CUX1 contains a potential coiled coil domain that can explain the observed constitutive FGFR1 activation, as has been elaborately demonstrated for other oncogenic kinase fusions. The in vitro data presented here using the inhibitor TKI258 support the use of this compound for the treatment of the novel CUX1-FGFR1 fusion as well as other constitutively active FGFR1 fusion proteins.

Disclosures:
No relevant conflicts of interest to declare.
Detection, Comprehensive Analysis and Clinical Validation of Chromosomal Aberrations by Multiplex Ligation-Dependent Probe Amplification In Chronic Lymphocytic Leukemia
Authors: Abdool Adam , Donahue Amber C. , Wohlgemuth Jay G , Yeh Chen-Hsiung 
Published: October 2019

Abstract
Abstract 2716

Background:
Current strategies based on karyotyping or fluorescent in situ hybridization (FISH) for detection of chromosomal abnormalities in chronic lymphocytic leukemia (CLL) are laborious, time-consuming, and costly and have limitations in resolution. Multiplex ligation-dependent probe amplification (MLPA) can simultaneously detect copy number changes of multiple loci in a single PCR, making it an attractive alternative. We developed and validated an MLPA protocol for comprehensive, automated data analysis and interpretation of chromosome abnormalities associated with CLL.

Patients and Methods:
Reference ranges for individual MLPA probes were established from a group of 50 healthy control subjects. Using these ranges we built an automated spreadsheet-based analysis system that includes multiple quality checks; samples that fail these checks are flagged and not reported. Each target was given a call of “deletion,” “normal,” or “amplification,” depending on whether the normalized ratio fell within or outside of the established normal range (mean ± 2SD or mean ± 3SD). After establishing the normal references ranges for each probe, we used the MLPA assay to characterize chromosome abnormalities in blood samples from 100 patients with suspected CLL that had been previously tested with FISH.

Results:
The maximum normal ranges were distributed between 0.82 and 1.18 for the mean ± 2SD values (ie, 95% CI, P = 0.05), and between 0.73 and 1.27 for the mean ± 3SD values (ie, 99.7% CI, P = 0.01). MLPA showed good concordance with FISH results in the 100 clinically suspected CLL cases. In 6 of these cases, abnormalities detected by MLPA were in regions not covered by FISH, including additional copy number gains on chromosomes 18q21.1 and 19, and novel micro-deletions at 19q13.43 and 19p13.2 loci. Excluding these cases, MLPA showed 94% sensitivity, 94% concordance, and 93% specificity relative to FISH. MLPA detected abnormalities in 3 FISH-negative cases and failed to detect abnormalities in three 13q- cases with low percentages of leukemic cells (7%, 12% and 19%). The limit of detection of the CLL MLPA assay was about 20% leukemic clones.

Conclusions:
This MLPA-based assay for chromosome abnormalities in CLL showed excellent concordance with FISH. This multiplex assay represents a fast (roughly 2–3 days total process to report time vs 7–10 days for FISH), high-throughput, accurate, and user-friendly process for that has potential for use as a first-line screening test for detection of chromosome abnormalities associated with CLL in the clinical laboratory.

Disclosures:
No relevant conflicts of interest to declare.