bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effect of B-Cell Depletion On Inhibitor Antibody Formation and Immune Tolerance Induction to Human Factor VIII in Hemophilia A Mice.
Authors: Zhang Ai-Hong Allan , Skupsky Jonathan , Scott David W. 
Published: October 2019

Abstract
Abstract 2137
Poster Board II-114
B-cell depletion using anti-human CD20 monoclonal antibodies has been reported to be effective in autoimmunity and in temporarily eliminating inhibitory antibodies in hemophilia A patients. In the current study, we examined the effect of anti-murine CD20 (αCD20) depletion on the immune response to factor VIII (FVIII) and its influence on an immune tolerance induction (ITI) protocol. Previous studies have shown that IgG subclasses of anti-murine CD20 monoclonal antibody (αCD20) have differential effects on B-cell depletion in the mouse. Thus, IgG1 αCD20 selectively depletes follicular B cells, while sparing marginal zone (MZ) B cells. Combined with evidence that MZ B cells may be tolerogenic antigen-presenting cells, we tested the hypothesis that follicular B-cell depletion using αCD20 IgG1 might favor tolerance induction to human FVIII. Hemophilic (FVIII knockout) mice were primed with physiological doses of recombinant human FVIII by weekly IV injection, followed by αCD20 IgG1 or control IgG1 treatment. Ten days after the αCD20 treatment, the mice were treated with daily high dose (2μg) FVIII IV injections to model ITI in hemophilia A patients. After 4 weekly injections, 70% of the mice developed titers of anti-FVIII IgG as high as 1:12,800. Unlike whole B-cell depletion, subsequent follicular B-cell depletion did not significantly decrease the anti-FVIII IgG titer, compared with mice receiving control IgG1. Repeated high dose FVIII injections to mimic ITI significantly increased the anti-FVIII IgG titer in both groups. However, in the mice that received αCD20 IgG1 treatment, the increase of anti-FVIII IgG levels were significantly lower than that in control IgG1 treated mice. In conclusion, we found that follicular B-cell depletion by αCD20 IgG1 antibody in hemophilia A mice did not switch the immune response to tolerance, but it diminished the immunogenicity of human FVIII in vivo in hemophilic mice. (Supported by NIH R01 HL061883)

Disclosures:
No relevant conflicts of interest to declare.
Ex Vivo Expansion of Highly Purified Human NK Cells..
Authors: Gao Chunji , Li Xiaohong , Ma Jian , Wu Xiaoxiong , Wang Feifei , Li Meng , Yu Li , Da Wanming 
Published: October 2019

Abstract
Abstract 2157
Poster Board II-134

Object
To optimize the expansion of high purity NK cells from human peripheral blood and explore the changes in biological functions of NK cells after Ex vivo expansion.

Methods
NK cells were isolated from PBMNC by using miniMACS (Magnetic cell-selection) and NK Cell Isolation Kit II(Miltenyi Biotec, Germany), then they were cultured in SCEM (Stemline Hematopoietic Stem Cell Expansion Medium, Sigma) supplemented with 10% human AB serum and different combinations of interleukin (IL)-2 and/or IL-12, IL-15 for 15 days. Cultures were fed with fresh media and cytokines every 3 days, and were evaluated for cell expansion, phenotype, perforin and granzyme B mRNA expressions, and IFN-γ secretion at the end of the culture period.

Results
In group IL2+IL15 and IL2+IL15+IL12, cells were expanded 50.46±4.31 and 52.35±6.72 fold, respectively, much more higher than others(P<0.01), but no significant difference between them (P>0.05). And the purity of CD3−CD56+NK cells was over 94% in all groups except the control. The expressions of perforin and granzyme B mRNA of expanded NK cells cultured with cytokines was significantly higher than the starting population(P<0.01), although IL2+IL15+IL12 group was slightly higher than that of IL2+IL15 group, without significant difference (P>0.05). There was great increase in IFN-γ levels in the supernatants of NK cells culture in the presence of cytokines; IL2+IL15+IL12 group and IL2+IL12 group was significantly higher than others(P<0.01).

Conclusion
High purity NK cells could be efficiently expanded in culture with IL2+IL15, and its biological functions were enhanced in this condition.

Disclosures:
No relevant conflicts of interest to declare.
Splice Variant JAK2 Transcript Deleting Exon 14 in Patients with Chronic Myeloproliferative Neoplasms.
Authors: Ma Wanlong , Kantarjian Hagop , Zhang XI , Wang Xiuqiang , Zhang Zhong , Yeh Chen-Hsiung , Sferruzza Anthony , O'Brien Susan , Albitar Maher 
Published: October 2019

Abstract
Abstract 2161
Poster Board II-138
The JAK2 V617F mutation in exon 14 is the most common mutation in chronic myeloproliferative neoplasms (MPNs). While other point mutations and small deletions and insertions in exons 12, 13, and 14 have been reported in the JAK2 JH2 domain, deletion of the entire exon 14 is rarely detected in patients with MPNs. In a series of >10,000 samples from patients with suspected MPNs tested for JAK2 mutations by direct sequencing of mRNA, we detected a complete exon 14 (88 bp) deletion mutation in <1% of those with JAK2 mutation. This appears to be an alternative splicing mutation, not detectable with DNA-based testing, leading to a frameshift deletion in the JAK2 JH2 domain and the expression of a truncated protein (S593I fsX8). Although this mutation was present at detectable levels (>15% of total JAK2 transcript) in a small proportion of MPN cases, we decided to investigate the possibility it may be expressed at low levels (<15% of JAK2 transcript) in patients with MPNs. Using a sensitive reverse transcription-PCR—based fluorescent fragment analysis method to quantify the percentage of truncated (minus exon 14) JAK2 mRNA as compared to wild-type, we tested 61 patients with confirmed MPN; 183 patients with suspected MPNs (93 V617F positive, 90 V617F negative); and 46 healthy normal control subjects. The wild-type PCR product was 273 bp, while the exon 14 splice mutant displayed a 185-bp peak. All samples with a splicing mutant expression to wild-type ratio >15% were confirmed by direct sequencing. The JAK2 exon 14 splice mutation was detected at low levels in 9 of the 61 MPN patients (15%), accounting for 3.96% to 33.85% (mean=12.04%) of JAK2 transcript in these individuals. Among the 183 suspected MPN patients, the exon 14 splice variant was detected in 20 of the 93 (21.5%) with V617F (mean expression level relative to wild type=5.41%, range=2.13%-26.22%) and 31 of the 90 (34.4%) without V617F (mean expression=3.88%; range=2.08%-12.22%). All 46 normal individuals were considered negative for the alternatively spliced transcript. In conclusion, the expression of an alternatively spliced JAK2 mRNA with deletion of exon 14, leading to a truncated JAK2 protein, is a common abnormality in patients with MPNs. This alternatively spliced transcript is more common in MPN patients without V617F mutation and might contribute to the leukemogenesis in these patients. Low levels of JAK2 exon 14 splice mutation may also contribute to the effects of the V617F mutation in patients with MPNs. However, this mutation is missed if DNA and not the RNA is used for testing for JAK2 mutations.

Disclosures:
No relevant conflicts of interest to declare.
Tyrosine Kinase Proteins profiling of Nilotinib Resistant Chronic Myelogenous Leukemia Cells Unravels a Tyrosine Kinase-Mediated Bypass.
Authors: Pasquet Jean-Max , Gioia Romain , Drullion Claire , Lagarde Valerie , Leroy Cedric , Roche Serge , Cardinaud Bruno , Mahon Francois-Xavier 
Published: October 2019

Abstract
Abstract 2175
Poster Board II-152
Targeting the tyrosine kinase activity of Bcr-Abl is an attractive therapeutic strategy in Chronic Myeloid Leukemia (CML) and in Bcr-Abl positive Acute Lymphoblastic Leukemia. Whereas imatinib, a selective inhibitor of Bcr-Abl tyrosine kinase, is now used in frontline therapy for CML, second generation inhibitors such as nilotinib or dasatinib have been developed for the treatment of imatinib-resistant or –intolerant disease. We have shown that one of the mechanisms of resistance to nilotinib is an increasing expression of the p53/56 Lyn kinase, both at mRNA and protein level in cell lines. This result was confirmed in vivo in nilotinib-resistant CML patients (Mahon et al. Cancer Res., 2008, 68(23):9809-16.). To elucidate Lyn mediated-nilotinib resistance, a phosphoproteomic study was performed by Stable Isotope Labelling with Amino acid in Cell culture (SILAC) which highlights the potential role of downstream tyrosine kinases. Among different candidate proteinsThe Spleen tyrosine kinase Syk and the UFO family receptor tyrosine kinase Axl were the most relevant in the nilotinib resistant cell line as compared to the sensitive counterpart. Syk hyperphosphorylation was confirmed in the nilotinib resistant cell line using western blot at least on tyrosine residues Y323 and Y525/526, two critical tyrosine residues respectively involved in Lyn-mediated Syk phosphorylation and autophosphorylation-associated Syk activation. Lyn interacts with Syk as detected in Syk immunoprecipitates in nilotinib resistant cells. Furthermore, Syk-Lyn interaction is inhibited by dasatinib suggesting the requirement of Lyn kinase activity and Syk phosphorylation. Targeting Syk expression in nilotinib resistant cells by siRNA or tyrosine kinase activity by pharmacological inhibitors leads respectively to a partial (35%) or to a full restoration of nilotinib sensitivity. Moreover, the identification of Axl by SILAC is correlated to a 9 fold increase of its level of expression in the resistant cell line and the inhibition of Axl tyrosine kinase activity decreases proliferation of both nilotinib sensitive and resistant CML cells. All together these results disclose a new pathway for tyrosine kinase inhibitors resistance in CML involving at least the two Lyn downstream tyrosine kinases Syk and Axl.

Disclosures:
Mahon: Amgen: Honoraria; Novartis Pharma: Consultancy, Honoraria, Research Funding; Alexion: Consultancy, Honoraria.
Small Interfering RNA against Bcr-Abl Transcripts Sensitized Mutated T315I Cells to Nilotinib.
Authors: Koldehoff Michael , Elmaagacli Ahmet H 
Published: October 2019

Abstract 2188
Poster Board II-165

Background:
Selective inhibition of the BCR-ABL tyrosine kinase by RNA interference has been demonstrated in leukemic cells. Therefore, we evaluated the specific bcr-abl small interfering RNA (siRNA) silencing in BCR-ABL positive cell lines, including those resistant to imatinib (IM) and particularly those with the T315I mutation.

Design and Methods:
The factor-independent 32Dp210 bcr-abl oligoclonal cell lines and in human IM-resistant bcr-abl positive cells from different patients with leukemia disorders were investigated. The effects of bcr-abl siRNA or the combination of bcr-abl siRNA with both IM and nilotinib were compared with those of the ABL inhibitors IM and nilotinib.

Results:
Coadministration of bcr-abl siRNA with IM or nilotinib dramatically reduced the .{/MAIN;133}BCR-ABL expression in wild-type (wt) and mutated bcr-abl cells and increased the lethal capacity. The bcr-abl siRNA significantly induced apoptosis and inhibited proliferation in wt (p<0.0001) and mutated cells (H396P, T315I, p<0.0001) versus controls. Cotreatment of bcr-abl siRNA with IM or nilotinib resulted in an increased inhibition of proliferation and induction of apoptosis as compared to IM or nilotinib (p<0.0001) in T315I cells. Furthermore, the combination of bcr-abl siRNA with IM or nilotinib significantly (p<0.01) reversed multidrug resistance gene 1-dependent resistance of mutated cells. In T315I cells bcr-abl siRNA with nilotinib has shown powerful effect on the cell-cycle distribution.

Conclusions:
Our data suggest that silencing by bcr-abl siRNA with IM or nilotinib may be associated with an additive antileukemic activity against tyrosine kinase inhibitor-sensitive and –resistant BCR-ABL cells, and might be an alternative approaches to overcome BCR-ABL mutations.

Disclosures:
No relevant conflicts of interest to declare.
Chronic Myelogenous Leukemia Cells Contribute to a Bone Marrow-Stroma in In Vivo NOD/SCID Mouse System.
Authors: Shirasaki Ryosuke , Tashiro Haruko , Oka Yoko , Sugao Toshihiko , Akiyama Nobu , Kawasugi Kazuo , Shirafuji Naoki 
Published: October 2019

Abstract
Abstract 2191
Poster Board II-168
Aims: The stroma-forming cells in a bone marrow are derived from hematopoietic stem cells. We reported previously that non-adherent leukemia blast cells converted into myofibroblasts to create a microenvironment for proliferation of leukemia blasts in vitro. In this report we demonstrate that with severe combined immunodeficiency (SCID) mouse system chronic myelogenous leukemia (CML) cells are also differentiated into myofibroblasts to contribute to a bone marrow-stroma in vivo. Materials and Methods: Bone marrow cells were collected from informed CML patients, from which mononuclear cells were separated with density-gradient sedimentation method. After discarded an adherent cell-fraction, non-adherent mononuclear cells were injected to the priory 2.5 Gray-irradiated non-obese diabetes (NOD)/SCID mice intravenously. For the inactivation of NK cells, anti-Asialo GM1 antibody was injected intra-peritoneally prior to the transplantation, and on each 11th day thereafter. Blood was collected to monitor Bcr-Abl transcript, and mice were sacrificed after chimeric mRNA was demonstrated. Bone marrow cells were obtained, and sorted with anti-human CD133 antibody and -CD106 to select CML-derived human stromal myofibroblasts referred to the in vitro data. The isolated positive fraction was further cultured, and the biological and the molecular characteristics were analyzed. Results and Discussion: When non-adherent CML cells were transplanted to NOD/SCID mice, CML cells were engrafted after 2 months. In the murine bone marrow human stromal cells were identified, in which BCR and ABL gene was fused with FISH analysis. When the parental CML cells were cultured on the CML-derived myofibroblasts, CML cells grew extensively in a vascular endothelial growth factor-A-dependent fashion. These results indicate that CML cells can create their own microenvironment for proliferation in vivo.

Disclosures:
No relevant conflicts of interest to declare.
Sirolimus Yields High Response Rates in Hematopoietic Allograft Recipients with Steroid-Refractory Acute Graft-Versus-Host Disease.
Authors: Pidala Joseph A. , Hoda Daanish , Salgado-Vila Norma , Kim Jongphil , Perkins Janelle , Bookout Ryan , Field Teresa , Perez Lia , Ayala Ernesto , Ochoa-Bayona J. Leonel , Raychaudhuri Jyotishankar , Alsina Melissa , Greene John N , Janssen William E. , Fernandez Hugo F. , Anasetti Claudio , Kharfan-Dabaja Mohamed A 
Published: October 2019

Abstract
Abstract 2235
Poster Board II-212
Acute graft-versus-host disease (aGVHD) remains the major cause of morbidity and mortality in allogeneic hematopoietic cell transplant recipients. There is no consensus in regards to the best therapy for patients who fail to respond to, or do not tolerate, systemic glucocorticoids. We evaluate the efficacy of sirolimus in 34 pts, median age of 49 (23-67) years, with steroid-refractory (N=31) or steroid-intolerant (N=3) aGVHD. The diagnosis of aGVHD was established at a median of 34 (7-1042) days after transplantation, and confirmed by biopsy in all cases. Initial treatment of aGVHD consisted of prednisone up to 2 mg/kg. Sirolimus was initiated at a median of 9 (1-255) days after glucocorticoid initiation. A loading dose of sirolimus was administered to 19 (56%) of 34 pts, median 6 (3-8) mg, followed by maintenance therapy of 1-2 mg per day with doses adjusted to target therapeutic trough levels between 4 and 12 ng/ml; therapeutic levels were achieved in all cases. The overall response rate was 76%. Fifteen (44%) of the 34 pts achieved a CR after sirolimus initiation and without requiring additional immune suppressive agents. CR was achieved in 11/31 (42%) steroid-refractory patients, and in 2/3 (67%) steroid-intolerant patients. The median overall survival (OS) after initiation of sirolimus was 5.6 months, and one year OS was 44% (95% C.I. 27%-60%). Maximum decrease in dose of steroids over 12 weeks following sirolimus was a median of 50% (range 0-100%). Fourteen (42%) of 33 pts reached a dose < 20 mg of prednisone at a median of 4 months (95% CI: 3.5-13.5) after initiation of sirolimus. Seven (21%) of 34 pts were free from steroids after a median of 20 months (95% CI: 9.2-20.0) after initiation of sirolimus. Two pts were shown to be successfully liberated from all immunosuppressive agents without recurrent GVHD at 2.7 and 7.1 months after initiation of sirolimus, respectively.
These data indicate the sirolimus is effective in controlling steroid-refractory aGVHD. Further studies are needed to determine the most appropriate timing for sirolimus after transplantation, whether prophylaxis, primary or secondary GVHD therapy.

Disclosures:
Off Label Use: Sirolimus for graft-versus-host disease.
Long-Term Results of Donor Lymphocyte Transfusions for the Treatment of Relapse After Allogeneic Transplantation for Chronic Myelogenous Leukemia - Is There a Role for Immunotherapy in CML?.
Authors: Kolb Hans-Jochem , Bund Dagmar , Schmetzer Helga , Schmid Christoph , Buhmann Raymund , Tischer Johanna , Ledderose Georg 
Published: October 2019

Abstract
Abstract 2258
Poster Board II-235
The long-term outcome of donor lymphocyte infusions (DLI) as first line treatment of relapse of chronic myeloid leukemia (CML) after allogeneic stem cell transplantation was studied and compared to other treatment modalities. Forty five patients were treated with DLI and 36 without DLI. Patients given DLI first did not differ significantly from those given other treatments in gender, age, donor, gender and histocompatibility of the donor, source of stem cells (blood vs marrow), depletion of T cells, stage of the disease, time from diagnosis and year at the time of transplant, acute and chronic graft-versus-host disease (GVHD) and remission duration. The survival of the DLI group was 66.4 percent at 10 and 15 years after transplantation, it was 42.2 and 23.8 percent respectively in the non-DLI group (p=0.019, log rank). Excluding patients with early relapse in the first 6 months the survival of the DLI group was 78 percent at 15 and 20 years, 70 and 26 percent respectively of the non-DLI group (p=0.009). Recurrent leukemia was the predominant cause of death in both groups (11 of 14 patients of the DLI-, 21 of 23 patients of the non-DLI group). Three patients in the DLI group died of recurrent infections, bronchiolitis obliterans (BO) and heart failure respectively, and 2 patients of BO in the non-DLI group. The proportion of surviving patients with positive PCR for bcr/abl was not different in both groups (2 of 13 patients of the non-DLI and 7 of 30 patients in the DLI group). The better survival of the DLI group indicates a better control of residual leukemia by a persistent immune effect. The response to DLI was improved by simultaneous treatment with low doses of interferon-a and GM-CSF. The combination of these cytokines without DLI and transplantation remains to be defined in patients that do not tolerate or respond incompletely to Imatinib. The role of immunotherapy for induction and maintenance of remission is well established in transplant patients.

Disclosures:
No relevant conflicts of interest to declare.
Potential Eradication of Myeloma Stem Cells by Combining ATRA with WNT and HH Signaling Pathways Inhibitors.
Authors: Tricot Guido J. , Wang Siqing , Shi Lei , Xu Hongwei , Xu Chunjiao , Zangari Maurizio , Zhan Fenghuang 
Published: October 2019

Abstract
Abstract 2839
Poster Board II-815
Multiple myeloma (MM) remains largely incurable. The existence of the putative myeloma stem cell population may account for drug-resistance and relapse. Previous investigation showed that the CD138-negative (CD138-) MM cell fraction contains the putative MM stem cells. However, little is known about the molecular characteristics of MM stem cells, which makes it difficult to specifically target such cells. In this study, by comparing gene expression profiles (GEP) of CD138+ and CD138- cells isolated from 10 MM cell lines, we discovered that RARα is the top one over-expressed gene in CD138- MM stem cells. RARα has two major isoforms, RARα1 and RARalpha2. Real-time PCR detected significantly higher expression of RARalpha2 but not of RARalpha1 in CD138- MM cells compared with CD138+ cells. We recently reported that increased RARalpha2 expression at diagnosis resulted in a significantly shorter overall-survival (P = 0.003); importantly, ATRA selectively killed RARalpha2-positive myeloma cells, but not RARalpha2-negative cells. These results suggested that ATRA could be used to eradicate specifically RARalpha2-over-expressing MM stem cells. Indeed, we found that ATRA selectively killed CD138- MM stem cells (P < 0.01) from ARP-1 and OCI-MY5 human myeloma cell lines and 5T33 murine myeloma cells but spared the CD138+ tumor cells from these cell lines. WNT and Hedgehog (HH) signaling pathways were activated in CD138- stem cells. To our surprise, ATRA treatment (10-6M) further increased WNT and HH signaling pathways in CD138- myeloma cells, based on increased protein levels of β-catenin and cleaved Shh/Ihh in ATRA-treated cells. Stimulation of these signaling pathways by LiCl (5 mM) and/or SHH(20 μg/mL) partially abrogated ATRA-induced cytotoxities on CD138- MM cells, demonstrating that stimulation of WNT and HH signaling induced partial ATRA-resistance in CD138- cells. A combinational treatment of ATRA (1 μM), a COX-2 inhibitor celecoxib (shown to inhibit WNT signaling, 50 μM) and a HH signaling inhibitor cyclopamine (10 μM) induced synergistic effects on cell death and growth inhibition of CD138- and RARalpha2-over-expressing MM cells in-vitro. In the 5T33 murine myeloma model, the combination of ATRA (20mg/Kg), celecoxib (10mg/Kg) and cyclopamine (20mg/Kg) induced synergistic inhibition of tumor growth in-vivo. Thus, our study provides novel approaches to specifically target MM stem cells.

Disclosures:
No relevant conflicts of interest to declare.