bluuebunny/crossref_metadata_2025_split huggingface dataset:
Plasma Circulating Ki-67 Index as a Biomarker and Prognostic Indicator in Patients with Chronic Lymphocytic Leukemia.
Authors: Bruey Jean-Marie , Estrov Zeev , Kantarjian Hagop M. , Ma Wanlong , Yeh Chen-Hsiung , Weirda William , Donahue Amber , O'Brien Susan , Keating Michael , Albitar Maher 
Published: October 2019

Abstract
Abstract 1261
Poster Board I-283
Ki-67 is a nuclear antigen that is expressed in all stages of the cell cycle except G0 and is widely used as a marker of cellular proliferation in human tumors. We recently demonstrated that levels of plasma circulating Ki-67 (cKi-67) are significantly higher in patients with newly diagnosed acute lymphoblastic leukemia (ALL) than in healthy control subjects, and that elevated levels of cKi-67 are associated with a shorter survival in ALL patients. Here we examined the associations of cKi-67 levels with laboratory and clinical variables in patients with chronic lymphocytic leukemia (CLL). The study included 194 patients with CLL and 96 healthy control subjects. The cKi-67 levels in plasma were determined using electro-chemiluminescence-based immunoassay using the Mesoscale Discovery platform. Since usually Ki-67 is used as an index of tumor cell proliferation, we took into account the lymphocyte count of the CLL patients in peripheral blood and normalized the levels of the cKi67 to the absolute number of lymphocytes in the peripheral blood establishing plasma cKI-67 index (cKi-67 level ng/1000 circulating lymphocytes/μL plasma). Median (range) levels of absolute cKi-67 were significantly higher in patients with CLL than in control subjects (914.65 [102.0-4975.12] ng/mL vs 353 [35.76-2830.65] ng/mL; P<0.0001). However, absolute levels did not correlate with clinical or other laboratory variables (white cell count, hemoglobin, platelets, beta-2 microglobulin, or Rai stage, performance status). In contrast, the cKI-67 index correlated significantly with bone marrow involvement (p<0.001), number of lymph node sites involved (p<0.001), and Rai stage (p=0.05), but not with IgVH mutation (P=0.62) or performance status (p=0.71) The cK-I67 index was significantly associated with survival when used as either as a continuous variable (P=0.002) or as a dichotomous variable (P=0.005). Multivariate Cox proportional hazards analysis incorporating cKi67 index with IgVH mutation status and B2M, demonstrated that only cKi-67 and B2M were independent predictors of survival.
This data shows that there variability in proliferation between patients with CLL and those patients high relative proliferation (index) have more aggressive disease. Furthermore, plasma cKi-67 index and B2M levels are strong predictors of clinical behavior in CLL.

Disclosures
No relevant conflicts of interest to declare.
Functional Characterization of Three VWF- A1 Domain Mutations Causing Type 2 Von Willebrand Disease.
Authors: Othman Maha , Chegeni Rouzbeh , Vickars Linda M , Favaloro Emmanuel J , Lillicrap David 
Published: October 2019

Abstract
Abstract 1305
Poster Board I-327
Through the Canadian Platelet-Type VWD Project (www.pt-vwd.org), we have identified 3 novel candidate mutations within the A1 domain of VWF gene in 5 patients that were provisionally diagnosed as type 2 VWD. These mutations are: L1460F (2 related patients), Y1363C (1 patient), E1389K (2 related patients). The VWF:Ag ranged from 0.19 – 0.54 IU/mL, VWF:RCo ranged from 0.19- 0.21 IU/mL and platelet count ranged from 206-254 × 103/ mm3. High Molecular weight multimers of VWF were absent in the plasma of the patient with the 1460 aa change. These substitutions have not been previously reported in the literature or in the International VWF Mutation Database. All three mutations were created by site-directed mutagenesis in the full-length huVWF cDNA and transiently transfected in the HEK293T/17 cell line. The VWF protein secreted in the media was collected and quantified by ELISA before proceeding with Functional VWF-platelet binding studies. Quantification of the mutant rVWF proteins in the media and cell lysates and multimer analysis of the proteins showed normal secretion and multimer distributions. To determine the GpIb binding properties of the putative mutants, the VWF recombinant protein (0.25 ug) from each of the WT and three mutants was incubated with lyophilized platelets (1 × 108) for 10 minutes at 25°C with different concentrations of ristocetin between 0-1 mg/mL. The platelets were then pelleted by centrifugation at 1200 g and the resulting supernatant was tested for the remaining unbound VWF by ELISA. Compared to the WT VWF protein, only the L1460F mutant showed increased platelet binding under lower concentration of ristocetin. Ristocetin-induced platelet agglutination (RIPA) assays performed using these recombinant proteins confirmed enhanced ristocetin responsiveness for L1460F, and that Y1363C and E1389K alternatively showed reduced responsiveness. Based on these data, a type 2B VWD diagnosis can only be made with respect the L1460F and not the other two A1 domain missense mutations. These data confirm once again that the assignment of the phenotype on clinical and laboratory basis is useful prior to genetic analysis and mutation identification. However, in some situations both will be ultimately required to understand the clinical presentation and to assign a disease subtype.

Disclosures
Vickars: Novartis Canada: Honoraria, Research Funding.
Upregulation of TRAIL in Myeloma Cells Correlates with Skeletal-Related Events.
Authors: Kawano Yawara , Ueno Shikiko , Okuno Yutaka , Kikukawa Yoshitaka , Mitsuya Hiroaki , Hata Hiroyuki 
Published: October 2019

Abstract
Abstract 2844
Poster Board II-820

Introduction:
Skeletal complications including bone fracture, bone pain and hypercalcemia are major clinical events in patients of multiple myeloma (MM). Osteoclastgenesis is known to be induced by free receptor activator of nuclear factor kappa β ligand (RANKL) and inhibited by dimerization of RANKL and osteoprotegerin (OPG). OPG is also known as a soluble inhibitor of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL); therefore, a possible role of TRAIL as an osteoclast inducer is suggested, although the association of TRAIL with bone lesions in MM is a matter of debate. We thus investigated the expression of TRAIL mRNA in purified MM cells and analyzed its association with skeletal-related events.

Patients and Methods:
MM cells were purified from bone marrow samples from 40 MM patients by CD138-immunomagnetic beads (Miltenyi Biotech, Paris, France). TRAIL mRNA expression in purified MM cells was analyzed by real time PCR(ABI PRISM 7700 Sequence Detector, Applied Biosystems). Simultaneous analysis of serum TRAIL concentrations, analyzed by ELISA (Diaclone, Cedex, France), and TRAIL mRNA-expression levels was also performed in 23 cases. Each of these patients was given a score called skeletal-related event score (SRE score) according to the skeletal complications (pathological fracture, bone-associated plasmacytoma, >12mg/dL hypercalcemia, and receiving an pathological fracture-related operation or radiation therapy).

Results:
Significant association (p=0.0006) was seen between TRAIL mRNA expression levels and the SRE score (Fig.1). Serum calcium levels also had significant association to TRAIL mRNA expression levels (p=0.0050). On the other hand, no association of TRAIL mRNA with hemoglobin (p=0.3970) and platelets (p=0.9401) was seen. Serum TRAIL concentrations in MM cases, which were equivalent to those in healthy individuals, did not correlate to TRAIL mRNA expression levels in purified MM cells (p=0.4094).

Conclusions:
The data suggest that MM patients with high TRAIL expression in MM cells tend to have more skeletal complications, which may be mediated by increased osteoclastgenesis. Since serum TRAIL concentrations did not correlate with TRAIL mRNA levels in MM cells, increased TRAIL expression in bone marrow microenvironment could be important. Despite of previous reports suggesting TRAIL-induced apoptosis of hematopoietic cells, the observed high TRAIL expression did not correlate with anemia or thrombocytopenia in our cases. Although mechanisms regulating TRAIL expression in MM cells and protection from TRAIL-induced apoptosis remain to be determined, our findings may introduce a new strategy targeting TRAIL to reduce skeletal events in MM.

Disclosures:
No relevant conflicts of interest to declare.
Thrombosis in Primary Myelofibrosis: Incidence and Risk Factors.
Authors: Barbui Tiziano , Carobbio Alessandra , Cervantes Francisco , Vannucchi Alessandro M. , Guglielmelli Paola , Antonioli Elisabetta , Alvarez-Larràn Alberto , Rambaldi Alessandro , Finazzi Guido , Barosi Giovanni 
Published: October 2019

Abstract
Abstract 2915
Poster Board II-891
Few epidemiologic studies have been conducted to evaluate at which extent vascular complications can influence the prognosis of patients with primary myelofibrosis (PMF). We assessed the cumulative rate and predictive factors of major cardiovascular vascular (CV) events in 707 patients (M/F 465/242, median age 62, range 11-90 years) with PMF from 4 European institutions. Patients were diagnosed during the period February 1973 - April 2008 and followed for a total of 2,909 patient-years (pt-yr) of observations (median, 2.92 years; 25th percentile, 1.14 years; 75th percentile, 5.92 years). A total of 236 deaths (33%) were recorded for an overall mortality of 7.7% pt-yr. Of them, only 12 well-documented deaths (2%, 0.39% pt-yr) were attributable to cardiovascular causes, whereas the majority of fatalities were due to non-CV events. Fatal and non-fatal thrombosis (myocardial infarction, stroke, peripheral arterial thrombosis, venous thromboembolisms (including splanchnic, cerebral and legs) was registered in 51 (7.2%) patients, with a rate of 1.75% pt-yr. If deaths from non-CV causes were considered as competing events, we estimated that the adjusted rate of major thrombotic events at 10 years would have been 2.2% pt-yr. In a multivariable model, age >60 yrs (HR 2.34, 95%CI 1.24-4.39, p=0.01) and JAK2 mutational status (HR 1.92, 95%CI 1.10-3.34, p=0.02) were significantly associated with thrombosis while the strength of the association between leukocyte count > 15 ×109/L and CV events was of borderline significance (HR 1.72, 95%CI 0.97-2.72, P= 0.06). The highest incidence of fatal and non-fatal thrombosis was observed when the mutation was present along with leukocytosis (3.9% pt-yr, HR 3.13, CI 1.26-7.81). This study is the largest hitherto carried out in this setting and shows that the rate of major CV events in PMF is comparable to that reported in essential thrombocythemia and increases in aged patients as well as in those with the JAK2 V617F mutation and leukocytosis.

Disclosures:
No relevant conflicts of interest to declare.
The Type of Enteropathy Is a Prognostic Factor in Enteropathy-Associated T-Cell Lymphoma.
Authors: Chandesris Marie-Olivia , Malamut Georgia , Verkarre Virginie , Meresse Bertrand , Rahmi Gabriel , Macintyre Elizabeth , Brousse Nicole , Cerf-Bensussan Nadine , Cellier Christophe , Hermine Olivier 
Published: October 2019

Abstract
Abstract 2937
Poster Board II-913
Enteropathy-associated T-cell Lymphoma (EATL) may complicate celiac disease (CD), refractory celiac disease type I (RCD I) characterized by normal intraepithelial lymphocytes (IEL) or refractory celiac disease type II (RCD II) defined by abnormal IEL (CD3s-,CD3i+ and CD8-) and a clonal rearrangement of the gamma or delta chain of the T cell receptor (TCRγ/δ). It remains unknown whether the type of the associated enteropathy, non clonal (CD or RCD I) or clonal (RCD II), may be a prognostic factor in EATL.
We aimed to assess the prognosis of EATL according to the type of the associated enteropathy. Medical files of 29 patients with EATL were retrospectively studied. The type of associated enteropathy was confirmed by immunohistochemistry analysis in all cases and by flow cytometry phenotyping analysis of freshly isolated IEL and search for a TCR γ or δ clonal rearrangement by Multiplex PCR whenever possible. Kaplan-Meier curves and Logrank test were used to compare survival of EATL in the 2 groups of enteropathy (clonal or not).
Mean age at EATL (13 women / 16 men) onset was 57 years. The associated enteropathy was CD (n=10) or RCD I (n=2) in 12 patients and RCD II in 17 patients. No statistical difference was found in lymphoma staging with localized (IE and IIE) versus disseminated stages (IV) found in 42% and 58% of patients with CD or RCD I and in 53% and 47% of patients with RCD II, respectively. Diagnostic or therapeutic surgery was practiced in 83% and 47% of patients with CD or RCD I and with RCD II, respectively and chemotherapy in 92% and 82% of the same groups of patients, respectively (n.s). Considering all the EATL, the two-year and five-year survival rates of EATL were 34.1% and 20.2%, respectively. In subgroup analysis, the two-year and five-year survival rates of EATL were 66.7% and 53.3% in case of CD and/or RCD I and 11.8% and 0% in case of RCD II, respectively (p=0.0007).
In conclusion, the type of enteropathy significantly impacts the prognosis of EATL with a particular short survival in case of associated RCD II enteropathy.

Disclosures:
No relevant conflicts of interest to declare.
Identification of RHAMM-Derived CD8+ Restricted, Heteroclitical, Cryptic Epitope R9Y as a Promising Target for Immunotherapy of Chronic Lymphocytic Leukemia.
Authors: Giannopoulos Krzysztof , Dmoszynska Anna , Rolinski Jacek , Greiner Jochen , Stilgenbauer Stephan , Schmitt Michael 
Published: October 2019

Abstract
Abstract 3034
Poster Board II-1010
The receptor for hyaluronic acid mediated motility (RHAMM) is leukemia associated antigen (LAA) strongly correlated with proliferation and bearing negative prognosis for chronic lymphocytic leukemia (CLL). Recently, we completed a peptide vaccination clinical trial with dominant RHAMM-derived epitope (R3) for patients with CLL. To our surprise, R3 vaccination induced regulatory T cells (Treg) in 66% vaccinated patients which might limit the clinical efficacy of peptide immunotherapy. Obtained results also pointed to a pre-existing tolerance to LAA-derived peptides. While the tolerance is limited to dominant epitopes we aimed to identify a crypitic epitopes that might circumvent tolerance mechanisms and in order to increase affinity modified them heteroclitically.
We screened in silico 50 peptides with different heteroclitical modifications using SYFPEITHI algorithm and calculate affinity to MHC-I complex. Further, we choose 10 peptide pairs (cryptic – modified) which affinity increased to the highest extent and screened them in vitro assay for the affinity as well as the peptide-MHC-I complex stability in T2 binding test. In functional studies we screened 5 peptide pairs by ELISPOT analysis using CD8+ T cells isolated from peripheral blood (PB) of CLL patients and healthy donors. We found that CD8+ cells from CLL patients presensitized with peptides: R9Y (YLQLDAFEV); R13YL (YLVQSLEDV); R15Y (YLKQTLDEL); R17YL (YLQEQLNKI) as well as R19YL (YLIKHVVKL) specifically recognized T2 cells pulsed with respective peptides with the highest frequency for R9Y (90%). In chromium-51 release assays, R9Y-primed CD8+ T cells from CLL patients were able to effectively lyse R9Y-peptide pulsed T2 cells as well as CLL leukemic cells presenting RHAMM in a HLA-A2 restricted manner; in contrast, K562 cells and T2 cells lacking either RHAMM or HLA-A2 expression were not lysed above background levels. In addition, mAb-based MHC I blockade demonstrated that the observed peptide-specific lysis was MHC I-restricted.
In conclusion, we defined a novel RHAMM-derived CD8+ restricted, heteroclitical, cryptic epitope R9Y which might represent an interesting target for immunotherapy of CLL. In future clinical administration the R9Y peptide might circumvent tolerance to dominant epitopes and thereby increase efficacy of peptide vaccination.

Disclosures
Stilgenbauer: GSK: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.
Rapid Expansion of Naive CD4+ Cord Blood Lymphocytes Restores Adaptive Immunity within 2 Months After Unrelated Cord Blood Transplantation
Authors: Chiesa Robert , Hiwarkar Prashant , Rao Kanchan , Qasim Waseem , Gilmour Kimberly , Goulden Nicholas , Amrolia Persis J , Veys Paul 
Published: October 2019

Abstract
Abstract 2337
Viral infections remain a significant problem following haematopoietic cell transplantation (HCT). Recovery of CD4+ T-cells only occurs with return of thymopoiesis 6–12 months after HCT. In an attempt to improve immune reconstitution and reduce viral infections following unrelated cord blood transplantation (CBT), serotherapy was omitted from conditioning.
26 children, median age 19 months (range 1–147) underwent unrelated CBT for immunodeficiency (13) or high risk/relapsed leukemia (13). Pre-transplant co-morbidities included lung aspergillosis (2), adenoviraemia (1), Paraflu3 respiratory tract infection (3). Conditioning was myeloablative in 25 patients. HLA matching was 6/6, 5/6 and 4/6 in 13, 14 and 3 cases respectively (4 double cords). GvHD prophylaxis was mostly cyclosporine/mycophenolate mofetil (24). The median number of nucleated, CD34+ and CD3+ cells infused was 8.12 × 107/kg (range, 4.7–17.72), 3.5 × 105/kg (range, 1.23–11.95) and 5.63 × 106/kg (range, 1.9–24.45).
Median time to myeloid and platelet engraftment was 22 (range, 13–38) and 39 (range, 21–110) days. One patient died from conditioning related toxicity and 3 patients died after leukaemia relapse. 22 patients are alive and in remission with a median follow up of 12 months (range 3–66). 18 patients have full donor engraftment while 4 patients are mixed chimeras.
Acute GvHD > grade I occurred in 16 patients (grade II n=11; grade III-IV n=5) but in general this responded to steroids and only 1 child developed cGvHD. There was no infection-related mortality, despite early adenoviraemia (4), CMV viraemia (4), HHV6 encephalitis (1) and RSV/Paraflu3 respiratory tract infections (3).
The most striking finding was a rapid and sustained CD4+ T-cell expansion with conversion from naive to memory/effector phenotype. The median CD3+, CD4+ and CD8+ counts at 2 months post CBT was 0.85 ×109cells/L (range, 0.44–2.44), 0.55 ×109cells/L (range, 0.06–1.89), and 0.21 ×109cells/L (range, 0.01–1.21) respectively. As a comparison, the median CD4+ T-cell count at 2 months post a HCT from a matched sibling donor was 0.24 ×109cells/L in 23 children evaluated in our institution.
In 3 patients who developed adenoviral/CMV reactivation virus-specific T cells were detected by ELISPOT/IFN-g assays as early as 45 days post transplant, and viral infections resolved rapidly.
Unrelated CBT without serotherapy results in rapid “peripheral” expansion of naive/memory CD4+ T-cells, and restores cell-mediated immunity in children faster than any other HCT donor source.

Disclosures:
No relevant conflicts of interest to declare.
Dominant Negative Form of PAX5 Is Generated by Multimerization of Its DNA Binding Domain
Authors: Kawamata Norihiko , Pennella Mario , Woo Jennifer , Berk Arnold , Koeffler H. Phillip 
Published: October 2019

Abstract
Abstract 25
We have previously cloned a number of fusion genes involving PAX5 in acute lymphoblastic leukemia (ALL) (Kawamata N. et al. PNAS, 2008). All of these fusion products exerted a dominant negative effect over the wild-type PAX5. One of these fusion PAX5 proteins, PAX5-C20orf112, was generated by the fusion between the DNA binding domain of PAX5 (PAX5DB) and the C-terminal end of C20orf112. To find the mechanism of the dominant negative effect of the PAX5-C20 fusion, we performed Fluorescence Recovery After Photobleaching (FRAP) assay using PAX5-C20 and PAX5wt constructs connected with Yellow Fluorescence Proteins (YFP). Results showed extremely strong DNA binding affinity of PAX5-C20 compared to PAX5wt. FRAP experiments using deletion mutants of PAX5-C20 showed that both the DNA binding domain and C-terminal alpha-helix region of C20 were indispensable for this strong binding to DNA. Fluorescence Resonance Energy Transfer (FRET) assay, Bi-molecule Fluorescence Complementation (BiFC) assay, and co-immunoprecipitation assay showed that C-terminal end of C20 containing an alpha-helix region encodes a homo-multimerization domain. To confirm that homo-multimerization of PAX5DB increases DNA binding affinity, PAX5DB was fused to the inducible dimerization motif of FKBP (PAX5DB-FK). PAX5DB-FK increased its DNA binding affinity with addition of FKBP ligand inducing homo-dimerization. We also fused PAX5DB to homo-dimerization of MAX (bHLH domain), or tetramerization domain of TP53. FRAP assays showed that homo-dimerization increased its DNA binding activity, and homo-tetramerization further increased its DNA binding and its dominant negative effect over PAX5wt. PAX5-ETV6, also a common fusion protein in ALL, exerts a dominant negative effect over PAX5wt. The ETV6 region of this fusion protein has a multimerization (SAM) domain and the PAX5DB-ETV6SAM mutant protein also showed a dominant negative effect and strong binding to DNA. Importantly, in further studies, co-expression of PAX5-C20 and the YFP-C20-alpha-helix-region diminished the strong DNA binding and the dominant negative activity of the fusion protein. Our data show that multimerization of the DNA binding domain of PAX5 induces strong DNA binding activity, leading to its dominant negative effect over the wild type transcription factor. We believe this represents a new paradigm explaining how a number of fusion genes containing a DB motif from one protein and a multimerization motif from the other partner, can behave in a dominant negative fashion. These observations suggest that peptides/ small molecules inhibiting the multimerization of these oncogenic fusion transcription factors can be promising reagents for treating cancers.

Disclosures:
No relevant conflicts of interest to declare.
New Regulatory Mechanisms of Bone Marrow Mesenchymal Stem Cell Differentiation Involving An Adenosine Receptor.
Authors: Carroll Shannon H , Wigner Nathan A , Kulkarni Nitin , Gerstenfeld Louis C , Ravid Katya 
Published: October 2019

Abstract
Abstract 2584
The bone marrow cellular environment, including existing and newly formed bone cells, are important for the process of hematopoiesis at baseline and under stress, such as bone fracture. The A2b adenosine receptor (A2bAR) has a protective role in various models of chronic injury and in inflammation, particularly through its expression in macrophages. Bone fracture healing is a complex process composed of an initial inflammatory response, followed by differentiation of mesenchymal stem cells (MSC) into chondrocytes and osteoblasts to form bone, and finally resorption by osteoclasts to remodel the structure. Here we find A2bAR expression in bone marrow MSC cultures, and A2bAR knockout (KO) mice have lower bone density than wildtype counterparts. In vitro, induced differentiation of bone marrow derived MSCs from A2bAR KO mice resulted in lower expression of osteoblast differentiation genes and formed fewer mineralized nodules, as compared to wildtype mice. Furthermore, concurrent treatment of bone marrow derived MSCs with A2bAR agonists, and consequently an increase in cAMP levels, during differentiation resulted in elevated expression of the osteoblast differentiation gene Osterix. Examination of an in vivo model of bone fracture and showed a significant decrease in bone formation in the A2bAR KO mice, compensated for by upregulation of chondrocyte differentiation markers and cartilage formation. Delayed fracture healing in the A2bAR KO mice was associated with lower expression of genes involved in osteoblast differentiation. These results demonstrate a new role for the A2bAR and its ligand in promoting differentiation of MSCs into osteoblasts, which would affect the integrity of the bone marrow niche under stress.

Disclosures:
No relevant conflicts of interest to declare.