bluuebunny/crossref_metadata_2025 huggingface dataset:
Significance of Telomere Length Measurement in the Diagnosis of Dyskeratosis Congenita.
Authors: Du Hong-Yan , Pumbo Elena , Shimamura Akiko , Vlachos Adrianna , Lipton Jeffrey M. , Goldman Fred , Wilson David B. , Mason Philip , Bessler Monica
Published: October 2019

Dyskeratosis congenita (DC) is a rare inherited bone marrow failure (BMF) syndrome. The classical features of DC include nail dystrophy, abnormal skin pigmentation, and mucosal leukoplakia. The diagnosis of DC can be difficult. Originally, the diagnosis was based on the presence of the classical mucocutaneous features. However, the identification of four genes responsible for DC (DKC1, TERC, TERT, and NOP10) showed that these mucocutaneous features are only present in a proportion of patients with DC. Additionally, screening for mutations in the affected genes is expensive and is negative in about 50% of patients with classical features of DC. The products of the genes mutated in DC are the components of the telomerase ribonucleoprotein complex, which is essential for telomere maintenance. Therefore it has been postulated that DC is a disease arising from excessive telomere shortening. Here we examined whether the measurement of telomeres could be used as a screening test to identify individuals with DC. For this purpose we examined telomere length in peripheral blood mononuclear cells from 169 patients who presented with bone marrow failure including 17 patients with DC, diagnosed by the presence of classical cutaneous features or the identification of mutations in DKC1, TERC or TERT, 28 patients with paroxysmal nocturnal hemoglobinuria, 25 patients with Diamond Blackfan anemia, 5 patients with Shwachman-Diamond syndrome, 8 patients with myelodysplastic syndrome, and 74 patients with aplastic anemia of unknown cause classified as idiopathic aplastic anemia. In addition we measured telomere length in 12 patients with idiopathic pulmonary fibrosis and in 45 individuals with a de novo deletion of chromosome 5p including the TERT gene. Their telomere lengths were compared with those of 202 age-matched healthy controls. Moreover, mutations were screened in the genes associated with DC. In cases where a mutation was identified, telomere length and mutations were also examined in all the family members. Our results show that all patients with DC and bone marrow failure have very short telomeres far below the first percentile of healthy controls. Not all mutation carriers, including some carriers of apparently dominant mutations, have very short telomeres. What is more, very short telomeres could be found in healthy individuals in these families, some of whom were not mutation carriers. These findings indicate that in patients with BMF the measurement of telomere length is a sensitive screening method for DC, whether very short telomeres in this setting are also specific for DC remains to be determined. However, in contrast to a previous study, we find that telomere length does not always identify mutation carriers in the families of DC.
Retrospective Comparison of Transplant Outcomes in Patients with Multiple Myeloma According to Induction Therapy with Thalidomide/Dexamethasone (TD) with or without Bortezomib (VTD).
Authors: Giralt Sergio , Thandi Rupi , Qazilbash Muzaffar , Mendoza Floralyn , Han Eric , Hosing Chitra , Wang Michael , Thomas Sheeba , Alexanian Raymond , Champlin Richard , Weber Donna
Published: October 2019

Background: Thalidomide/Dexamethasone (TD) has become one of the most commonly used induction therapies for patients with symptomatic multiple myeloma (MM) eligible for high dose therapy (HDT) intensification with autologous stem cell transplant (ASCT). Bortezomib (Velcade) has been added to the combination of TD (VTD) in an effort to reduce MM tumor burden further prior to HDT.The impact of this addition on HDT outcomes has not been fully explored.

Purpose: To determine the impact of the addition of bortezomib to TD induction therapy in patients with MM undergoing HDT and ASCT consolidation.

Patients and Methods: Patients were eligible for this analysis if they had undergone HDT with ASCT for first remission consolidation or primary refractory disease within 12 months of diagnosis between 9/03 and 12/05 and had received either TD or VTD as induction therapy. Patients receiving VTD after TD were excluded. Patients receiving more than 1 chemo regimen other than TD or VTD were excluded. Chemomobilization was NOT considered an exclusion criteria. Results A total of 78 patients qualified for the analysis (27 VTD; 51 TD). Patient and treatment characteristics are summarized in table 1. In brief, the patients receiving VTD had a higher rate of cytogenetic abnormalities and received less cycles of chemotherapy prior to SCT. Although pre-SCT response rates were similar between patients receiving VTD or TD (95% vs 92%) there was a trend for a higher CR rate in the VTD group (15% vs 6%). Post transplants response rates assessed between 3–6 months demonstrated that 28% and 38% of VTD patients achieved near CR and CR respectively while 19% and 23% had these responses post TD induction. There was no difference in 2 year OS and PFS among patients receiving VTD or TD (91% vs 81% and 35% and 56% respectively).

Conclusion: Both VTD and TD as induction treatment are associated with high response rates prior to SCT as well as 6 months post SCT. In this retrospective analysis no survival benefit was seen for induction therapy with VTD over TD, despite higher near CR and CR rates. However randomized trials need to be performed addressing type of induction as well as duration of induction therapy prior to high dose therapy consolidation.

Patient and Treatment Characteristics Variables VTD TD N 27 51 Median Age 54 (34–71) 56 (34–71) %ISS> 1 76% 65% % CG Abnormal 37% 19% p=.009 B2M @ Dx 2.99 3.19 Cycles Prior to SCT 2 4 p=.00009 % Mel 200 74% 69% Post SCT Maintenance 15/27 23/51
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 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 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 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 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 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 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 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 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 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.
Superior Survival of Combined Gastrointestinal and Skin GvHD Compared to Isolated Gastrointestinal GvHD in Children Post Allogeneic Stem Cell Transplantation
Authors: Gassas Adam , Ali Muhammad , Dupuis Annie , Doyle John J , Schechter Tal
Published: October 2019

Abstract 4535

Objective:

To compare the prognosis of isolated gastrointestinal (GI) graft versus host disease (GvHD) vs combined (GI and skin) GvHD in children post allogeneic stem cell transplantation (SCT).

Methods:

Allogeneic SCT recipients from January 2000-Dec 2009 were included and patients who were diagnosed with skin and GI GVHD were identified. Demographics and outcome data were collected and analyzed.

Results:

Four hundred and fifty children (0–18 years) underwent allogeneic SCT in the study period. Seventy-nine (17.5%) patient underwent endoscopy and GI biopsy for GI GvHD. Of the seventy-nine patients, 49 had isolated GI GvHD and 30 had combined GvHD. Fifty-one patients received SCT for a malignant indication and 28 for a non-malignant cause. Donor sources were; isolated GI group, related 10, unrelated 20: combined group, related 20, unrelated 29). All patients received myeloablative conditioning regimens and the majority received cyclosporine A and methotrexate for GvHD prophylaxis. Sixty-seven percent of patients with combined GvHD had grade III-IV while only 31% had grade III-IV in patients with isolated GI GvHD. To ensure there was no bias in a quicker initiation of anti GvHD therapy in children with skin involvement, we included only children who had a GI scope and GI biopsy looking for GI GvHD and also we compared if GvHD therapy initiation was different in patients with or without skin involvement [In the combined group 16 patients (53%), therapy was started prior to GI biopsy versus 16 patients (33%) in the isolated GIT group P=0.2]. Interestingly, 40% of children with combined GvHD had also liver GvHD while only 6% of patients with isolated GI GvHD had liver involvement (p≤0.0001). Overall response to GvHD therapy was similar in both groups (83% vs 87%). However, despite having less patients in the isolated GI group with severe (grade III-IV GvHD), there was a significant increase in non-relapse mortality (NRM) in the isolated GI group compared to the combined group (37% vs 16%) resulting in a statistically significant superior overall survival for those children with combined GvHD (79% vs 49% P= 0.02). Disease relapse was similar in both groups (8% and 6% for combined and isolated GIT group, respectively).

Conclusion:

Children with GI GvHD has better prognosis when there is skin involvement. Further research is warranted to examine if there is certain biological features that make GI GvHD extends to skin and other organs and why isolated GI GVHD is associated with worse prognosis?

Disclosures:

No relevant conflicts of interest to declare.
First Reported Use of a Biosimilar G-CSF in Allogeneic Stem Cell Mobilisation
Authors: Azar Nabih , Choquet Sylvain , Garnier Alice , Roos-Weil Damien , Leblond Véronique
Published: October 2019

Abstract 4571

OBJECTIVES:

Biosimilar granulocyte colony-stimulating factor (G-CSF) has been approved on the basis of comparable quality, safety and efficacy as the originator product. Approval of biosimilar G-CSF is in the same indications as the originator, including autologous and allogeneic peripheral blood stem cell (PBSC) mobilisation, for which it is being used throughout our large hospital group in Paris, France. Concerns have been raised by professional bodies over use of biosimilar G-CSF in allogeneic transplants. To our knowledge, this is the first reported use of biosimilar G-CSF in healthy donors for allogeneic transplantation.

METHODS:

Healthy related donors received biosimilar G-CSF (EP-2006, Sandoz Biopharmaceuticals) 10 μg/kg/day for PBSC mobilisation. G-CSF was administered on days 1–4 with the first leukapheresis performed on day 5. If the required number of CD34+ cells per recipient body weight (4 × 106 CD34+ cells/kg) was not collected, G-CSF administration was continued and a second PBSC collection was performed on day 6. Further G-CSF administration and a third leukapheresis was done on day 7 if required.

RESULTS:

Twelve healthy donors (7 male, 5 female; mean ± SD age 61.0 ± 7.8 years, range 51–73) received biosimilar G-CSF for PBSC mobilisation. Median donor body weight was 82 kg (range 55–115 kg). Median CD34+ cell count on day 5 was 75/ml (range 23–140). A single leukapheresis after 4 injections of biosimilar G-CSF was sufficient to collect the CD34+ cell dose required in six donors with peripheral blood (PB) CD34+ counts ranging from 80–140 ml. Six donors underwent a second leukapheresis after 5 days of biosimilar G-CSF, one of whom (a 73-year old female) underwent a third leukaphereses on day 7 (PB CD34+ at first apheresis 23 ml). Median number of CD34+ cells per recipient bodyweight was 5.5 × 106 cells/kg (range 2.6–8.9). Five donors reported bone pain (4 mild, 1 moderate severity) which was effectively treated with paracetamol. No other adverse events were reported. Blood measurements were normal in all donors when assessed one week after the end of mobilisation. These findings are consistent with our previous use of originator G-CSF (Neupogen®) in allogeneic stem cell mobilisation.

CONCLUSION:

These preliminary findings suggest biosimilar G-CSF is effective and well-tolerated in allogeneic stem cell mobilisation. Further studies are required to assess longer-term safety outcomes. The use of biosimilar G-CSF may provide important cost-savings when compared with the originator product.

Disclosures:

No relevant conflicts of interest to declare.
PRMT1 Interacts with AML1-ETO to Promote Its Transcriptional Activation and Progenitor Cell Proliferative Potential
Authors: Shia Wei-Jong , Okumura Akiko J. , Yan Ming , Sarkeshik Ali , Lo Miao-Chia , Matsuura Shinobu , Komeno Yukiko , Zhao Xinyang , Nimer Stephen D. , Yates John R. , Zhang Dong-Er
Published: October 2019

Abstract 4621

Fusion protein AML1-ETO resulting from t(8;21) translocation is highly related to leukemia development. It has been demonstrated that full-length AML1-ETO blocks AML1 function and requires additional mutagenic events to promote leukemia. We have previously shown that the expression of AE9a, a splice isoform of AML1-ETO, can rapidly cause leukemia in mice. To understand how AML1-ETO is involved in leukemia development, we took advantage of our AE9a leukemia model and sought to identify its interacting proteins from primary leukemic cells. Here we report the discovery of a novel AE9a binding partner PRMT1 (protein arginine methyltransferase 1). PRMT1 not only interacts with but also weakly methylates Arginine 142 of AE9a. Knockdown of PRMT1 affects expression of a specific group of AE9a-activated genes. We also show that AE9a recruits PRMT1 to promoters of AE9a activated genes, resulting in enrichment of H4 Arg3 methylation, H3 Lys9/14 acetylation, and transcription activation. More importantly, knockdown of PRMT1 suppresses AE9a’s self-renewal capability, suggesting a potential role of PRMT1 in regulating leukemia development.

Disclosures:

No relevant conflicts of interest to declare.
Monitoring the Chimerism Status of Subpopulation in HLA Haplo-Identical Stem Cell Transplantation Using the Anti HLA Antibodies: Based on the Study of HLA Distribution in Korean Population
Authors: Choe Won Ho , Jang Seongsoo , Chi Hyun-Sook , Park Chan-Jeoung , Im Ho Joon
Published: October 2019

Abstract 4706

Background:

Determining the chimerism in stem cell transplantation (SCT) is important in the monitoring of engraftment. Conventional monitoring methods such as short tandem repeat (STR) PCR are labor intensive and difficult in showing the changes of cell subpopulations. Nowadays, HLA-haploidentical SCT is performed actively and flow cytometric analysis using anti HLA antibody targeted for the different HLA between donor and recipient in HLA-haploidentical SCT can be useful by observing changes of cell subpopulations and determining the chimerism.

Methods:

Based on the study of HLA distribution in Korean population, we designed the panel of HLA monoclonal antibody that can detect and differentiate major HLA of Korean population. This panel was verified against 23 cases of HLA-haploidentical SCT. Flow cytometric analysis was performed using anti-HLA-antibodies on one pediatric patient with aplastic anemia who underwent HLA-haploidentical SCT. Tests were performed every day since SCT. The appearance or disappearance of donor and/or recipient HLA was observed using antibodies against mismatched HLA. Donor and/or recipient T cells, B cells, NK cells, and monocytes were observed using CD3, CD19, CD56, and CD13 respectively. The flow cytometric results were interpreted by observing the changes in subpopulations of detected cells and determining the engraftment status of patients.

Results:

A total of 11 anti-HLA-A-antibodies and anti-HLA-B-antibodies were selected. They cover 97.9% of HLA-A and 8.6% of HLA-B in Korean population. In the verification of our panel of HLA monoclonal antibody, we distinguished donor and recipient cells in 21 of 23 HLA-haploidentical SCT cases. In our case, the patient had HLA-A2/A24 while the donor had HLA-A2/A33. Recipient type of CD3(+) and HLA-A33(-) T cells appeared in first few days but donor type CD3(+) and HLA-A33(+) T cells appeared at day 20 and were increased. Donor type CD56(+) and HLA-A33(+) cells were seen at analysis done in day 8 and continued to appear. HLA-A33(+) cells were consistently observed and increased throughout the follow up period, showing the process of engraftment of donor stem cells.

Conclusion:

We were able to apply our flow cytometric analysis design using HLA monoclonal antibody for the detection of chimerism in HLA-haploidentical SCT. This method was more simple and sensitive than conventional monitoring techniques. Moreover, this method allowed us to observe the dynamics of changes in cell subpopulations after HLA-haploidentical SCT. Flow cytometric analysis can be considered as a strong tool for observing the changes in cell subpopulations and the monitoring the engraftment of HLA-haploidentical SCT.

Disclosures:

No relevant conflicts of interest to declare.
Upregulation of TCRζ Chain Overcome T Cell Immunodeficiency in Patients with Chronic Myeloid Leukemia
Authors: Zha Xianfeng , Chen Shaohua , Yang Lijian , Li Bo , Chen Yu , Yan Xiaojuan , Li Yangqiu
Published: October 2019

Abstract 4719

TCRζ chain is the key molecular of TCR signaling, the defective TCRζ chain not only decreases the expression of TCR on the T cell surface and the quantity of circulation T cells, but also influences T cell activation and proliferation. Previous studies indicated that the obvious downregulation of TCRζ chain was one of main factors which caused T cell immunodeficiency in patients with chronic myeloid leukemia (CML), which lead to dysfunction of immune supervision to tumor. This study was undertaken to explore the possibility that forced expression of TCRζ chain may restored the T cell immune function. In present study, the freshly CD3 + T cells were isolated by MACS from de novo CML patients; freshly T cells were transfected with TCRζ chain recombinant vector (TCRζ-IRES-EGFP) and control vector (pIRES2-EGFP) by nucleoporation technique. The transfection efficiency was detected by FCM at 18 hours post-transfection, and TCRζ chain protein and its phosphorylation were detected by Western blotting after activation with OKT3 antibody for 1 minute. The supernatants and RNA were collected from transfected cells stimulated with OKT3 and anti-CD28 antibody, for analysis of IL-2 levels and the mRNA expression of ZAP70 and NF-κB. The results showed the transfection efficiency of TCRζ chain vector and control vector construct was 72.16±6.95% and 73.4±7.90% in CML T cells from different patients respectively. In T cells transfected with TCRζ chain, the expression of TCR chain was increased, the IL-2 production induced by OKT3 and anti-CD28 antibody in TCRζ chain transfected T cells (175.1±66.3pg/mL) were higher than that from control group (107.6±65.5pg/mL) (n=6, p=0.039). The bother expression levels of ZAP-70 and NF-κ B in the experimental group was higher than the control group (n = 4, p < 0.05), moreover the expression level between ZAP-70 and NF-κB showed linear correlation (n = 4, p = 0.013, r = 0.98). In conclusion, the results indicate that upregulation of deficient TCRζ chain may reverse the TCR/CD3-mediated signaling abnormalities, which may improve the T cell immune function in patients with CML.

This study was supported by grant from the Key project of Natural Science Foundation of Guangdong Province, China (No. 9251063201000001).

Disclosures:

Zha: This study was supported by grant from the Key project of Natural Science Foundation of Guangdong Province, China (No. 9251063201000001): Research Funding. Li:the Key project of Natural Science Foundation of Guangdong Province: Research Funding.
The Feature of SALL4 and BMI-1 Expression in Placenta and Umbilical Cord Blood
Authors: Chen Shaohua , Shen Qi , Yang Lijian , Li Bo , Ma Yupo , Li Yangqiu
Published: October 2019

Abstract 4800

The SALL4 is a newly discovered pluripotency stem cell factor involved in the self-renewing, hematopoietic stem cells (HSCs) and leukemic stem cells. Its important role in normal HSCs and leukemic stem cells is supported by its interactions with several key players, like BMI-1, a putative oncogene of the Polycomb group family, controlling specific target genes involved in development, cell differentiation, proliferation, and senescence. Little is known the expression feature of SALL4 and BMI-1 mRNA in placenta and umbilical cord blood (UCB). In this study, the mRNA expression level of SALL4 and BMI-1 in 10 cases placenta tissues and the mononuclear cells from the corresponding UCB (CBMCs) were measured by real-time PCR with SYBR Green I technique, β2-microglobulin gene (β2M) was used as an endogenous reference, mononuclear cells from 10 healthy adults peripheral blood (PBMCs) and 2 cases of adult thymic cells served as control. The result showed that the expression level of both SALL4 and BMI-1 in placenta (Median: 34.36 and 17.55 respectively) were significantly higher than those from CBMCs (Median: 11.3 and 2.07 respectively) (p=0.007, p=0.001) and PBMCs (Median: 0.64 and 0.03 respectively) (P<0.001, P<0.001). Moreover, both SALL4 and BMI-1 expression levels in CBMCs were significant higher than those from PBMCs (P=0.002, P=0.001). And the expression of SALL4 could be detected in thymic cells. Correlation analysis was performed among the relative expression levels of SALL4 and BMI-1. The results showed that significant positive correlation between the expression levels of SALL4 and BMI-1 was observed in placenta tissues and CBMCs, as well as in PBMCs (rs=0.648, P=0.043, rs=0.721, P=0.019, rs=-0.697, P=0.025). In conclusions, the expression level of SALL4 and BMI-1 might be related to the numbers of hematopoietic stem cells, placenta tissues may be as an appropriate model for investigating gene regulation of SALL4 and BMI-1, while the significance of expression both genes in PBMCs and thymic cells is needed further investigation.

Disclosures:

Chen: National Natural Science Foundation of China(no. 30871091): Research Funding; the Fundamental Research Funds for the Central Universities (No. 21610603): Research Funding.
Hypoxic Induction of Vascular Endothelial Growth Factor Regulates Fetal Erythropoiesis but Not Stem Cell Function
Authors: Rehn Matilda , Cammenga Jorg
Published: October 2019

Abstract 4823

It has recently become apparent that hypoxia is an important factor in the hematopoietic stem cell (HSC) niche in the bone marrow (BM) but whether hypoxic niches also plays a role in regulation of fetal liver (FL) HSCs is not clear. Vascular endothelial growth factor A (VEGFA) is a key molecule in angiogenesis but is also known to be essential for adult HSC survival, mediated by a cell-intrinsic, autocrine mechanism. Vegfa expression can be induced by hypoxia-inducible factors (HIF), transcription factors that are stabilized in low-oxygen conditions and that activate transcription by binding to hypoxia-response elements (HRE). We have previously shown that hypoxic induction of Vegfa in adult HSCs is needed for proper function, using the mouse model Vegfaδ/δ, where the HRE in the Vegfa promoter is deleted. Here we have investigated the role of hypoxic Vegfa in fetal hematopoiesis. The Vegfaδ/δ genotype is partly embryonic lethal and we established that the major part of lethality in utero takes place between day 15.5 of development and birth. However, the exact reason for the lethality is not known. We speculated that the function of FL HSC might be reduced, and therefore BM seeding is inefficient and a cause of prenatal deaths. In contrary to our hypothesis, we now show that HSCs from Vegfaδ/δ FL have a normal capacity to competitively repopulate adult recipients. On the other hand, fetal erythropoiesis is severely impaired in absence of hypoxia-induced Vegfa, possibly explaining the embryonic lethality of Vegfaδ/δ mice. Fetuses at day 15.5 are pale and have significantly lower amounts of Ter119+ CD71low erythrocytes in the FL compared to wild type controls. Our findings indicate that hypoxic regulation of Vegfa expression is important in fetal erythropoiesis, suggesting that hypoxic areas exist within the FL. However, HSCs are affected by such hypoxic areas only to a small extent and hypoxic Vegfa expression is not highly important in FL HSCs.

Disclosures:

No relevant conflicts of interest to declare.