bluuebunny/crossref_metadata_2025 huggingface dataset:
Dematin Is Essential for Calcium Mobilization in Platelets
Authors: Wieschhaus Adam Joseph , Breton Guy Le , Chishti Athar H
Published: October 2019

Abstract 365

Dematin is an actin binding protein that was originally identified in the human erythrocyte membranes; however, dematin polypeptides are detectable in many non-erythroid cells. In erythrocytes, dematin is located at the spectrin-actin junctions thus linking the skeleton to the plasma membrane. This junctional complex serves to maintain both erythrocyte shape and membrane stability. Dematin is phosphorylated by multiple protein kinases, including the cAMP-dependent protein kinase and protein kinase C, and its actin bundling activity is regulated by phosphorylation. Dematin is also an excellent substrate of calpain-1, a calcium-dependent cysteine protease. Because of the functional similarities between the core components of the membrane skeleton in erythrocytes and platelets, we sought to investigate the physiological function of dematin in platelets. The remodeling of the actin cytoskeleton is known to regulate platelet activation and secretion of platelet granule contents. Western blotting demonstrated abundant expression of the ∼52 kDa polypeptide of dematin in both human and mouse platelets. To evaluate the functional role of dematin in platelets, we utilized the dematin headpiece knockout (HPKO) mouse model previously generated in our laboratory. Headpiece domain of dematin is an actin binding module sharing sequence similarity with the villin-family of cytoskeletal proteins. Importantly, the in vivo deletion of the headpiece domain of dematin resulted in substantial diminution of calcium mobilization in response to multiple agonists of platelet activation (Fig. 1). The reduced calcium mobilization in HPKO platelets was associated with significant inhibition of the platelet aggregation and granule secretion pathways. The HPKO platelets exhibit decreased activation of both the integrin αIIbβ3 receptor and RhoA upon platelet stimulation. Moreover, the HPKO platelets display aberrant morphology upon spreading on the fibrinogen and vWF-coated surfaces. Consistent with these findings, the HPKO mice show a significant clot retraction defect associated with a general tail bleeding phenotype. Mechanistically, the basal level of cAMP remained unaltered in the HPKO platelets suggesting the independence of the observed phenotype upon changes in the cAMP concentration. Immunofluorescence analysis indicated that dematin is associated with two platelet membrane compartments known to be involved in calcium fluxes, i.e., the dense tubular system (DTS) and the plasma membrane. Studies are currently in progress to identify the dematin-associated complex in platelets by Western blotting and proteomics approaches. Together, these results unveil dematin as a novel regulator of calcium homeostasis in platelets with functional implications for the development of new anti-platelet therapies.

Disclosures:

No relevant conflicts of interest to declare.
Expression of Phospholipse C Gamma 2 in Diffuse Large B-Cell Lymphomas,
Authors: Huynh Minh Quang , Kaiser Ulrich , Goßmann Jennifer , Neubauer Andreas
Published: October 2019

Abstract 3667

Cell survival and proliferation of diffuse large B-cell lymphomas (DLBCL) are dependent on the NF-κB signaling pathway. Antigen binding of the B-cell receptor (BCR) leads to phosphorylation of Src tyrosine kinases, resulting in PLCg2 activation, which is important for regulation of NF-κB through the protein kinase C (PKC). The PLC-PKC cascade also activates the Ras signaling pathway. There are several clinical studies showing that inhibition of PKCβ with the specific inhibitor Enzastaurin could be a promising new therapy target for DLBCL. To our knowledge, there are little data about the role of PLCg2 in DLBCL. It has been shown that the activity of Syk and PLCg2 are well correlated with sensitivity of DLBCL to dasatinib.

Our study performed immunohistological staining of PLCg2 in 86 primary DLBCL. PLCg2 was strongly expressed in 54 out of 86 cases (63%) and weakly expressed in 28 cases (32%). Only 4 cases (5%) were negative for PLCg2. In three DLBCL cell lines (SuDHL-4, SuDHL-6 and U2932) we could show an inhibitory effect of the PLC-inhibitor U73122 on cell proliferation. Treatment in combination with Enzastaurin or the Src-inhibitor pp2 had an additive effect on cell proliferation compared to U73122 alone.

In conclusion, PLCg2 is strongly expressed in a great subset of DLBCL. Inhibition of PLCg2 could be, at least in these cases, a new target for lymphoma treatment.

Disclosures:

No relevant conflicts of interest to declare.
Phase I Trial Evaluating the Safety and Biological Activity of Iboctadekin (rhIL-18) in Combination with Rituximab in Patients with CD20+ B Cell Non-Hodgkin's Lymphoma,
Authors: Robertson Michael J. , Bauman John , Gardner Olivia , Jonak Zdenka , Struemper Herbert , Germaschewski Fiona , Koch Kevin M. , Murray Sharon , Weisenbach Jill , Toso John
Published: October 2019

Abstract 3697

Background:

Iboctadekin (recombinant human interleukin-18; rhIL-18) is an immunostimulatory cytokine with anti-tumor activity in preclinical animal models. Phase I clinical trials have demonstrated that rhIL-18 is well-tolerated as monotherapy for patients with advanced solid tumors. Preclinical and clinical data indicate that rhIL-18 acts a costimulatory cytokine that may be optimally used in combination with other agents such as monoclonal antibodies. Methods: Patients with CD20+ B-cell non-Hodgkin's lymphoma (NHL) were given rituximab 375 mg/m2 IV weekly for 4 consecutive weeks in combination with rhIL-18 (in six dose cohorts of 1, 3, 10, 20, 30, and 100 mcg/kg) IV weekly for 12 weeks. Eligible patients had disease which progressed after standard therapy or for which there was no effective standard treatment. Assessments included safety/tolerability, pharmacokinetics, pharmacodynamics (serum cytokines, peripheral blood phenotypic markers and tumor biomarkers), immunogenicity, and anti-tumor activity. Results: Nineteen patients were enrolled on study (10 follicular NHL, 5 mantle cell lymphoma, 2 diffuse large B-NHL, 2 other subtypes). The combination was well-tolerated with a safety profile similar to that observed with rituximab or rhIL-18 given as monotherapy. The pharmacodynamic response is as expected based on results of prior clinical trials of rhIL-18 as monotherapy for cancer. Using the IWC response criteria, two (11%) of 18 evaluable patients had complete responses (CR) and 3 (16%) had partial responses (PR). Six (33%) patients had stable disease. Overall response rate for follicular lymphoma was 33% (1 CR, 2 PR). Conclusions: The combination of rhIL-18 and rituximab is safe, well-tolerated, and induces potent biological activity. A maximum tolerated dose of rhIL-18 was not identified. Further study of rhIL-18 plus rituximab in patients with CD20+ B cell malignancies is warranted.

Disclosures:

Bauman: GlaxoSmithKline: Employment, Equity Ownership. Gardner:GlaxoSmithKline: Employment, Equity Ownership. Jonak:GlaxoSmithKline: Employment, Equity Ownership. Struemper:GlaxoSmithKline: Employment, Equity Ownership. Germaschewski:GlaxoSmithKline: Employment, Equity Ownership. Koch:GlaxoSmithKline: Employment, Equity Ownership. Murray:GlaxoSmithKline: Employment, Equity Ownership. Toso:GlaxoSmithKline: Employment, Equity Ownership.
The Role of Imatinib Plasma Level in the Achievment of Complete Cytogenetic Response (CCyR) in Chronic Myeloid Leukemia (CML) Therapy
Authors: Kutsev Sergey , Oxenjuk Oxana , Mordanov Sergey , Shatokhin Yuri , Pospelova Tatiana , Khoroshko Nina , Turkina Anna
Published: October 2019

Abstract 3789

Background.

The treatment of patients with chronic phase (CP) Ph-positive chronic myeloid leukemia (CML) with imatinib has resulted in high rates of cytogenetic and molecular responses. There are evidences that the achievement of CCyR and MMR to imatinib therapy is related with Imatinib plasma level (IPL) in some studies1,2 but not in others 3. This discrepancy may be possibly explained by the heterogeneity in the analysed cohort patients differing with respect to the phase of the disease and imatinib dose.

The Aim of our study was to elucidate the trough role of IPL in the achievement of CCyR in homogeneous cohort of CP CML patients.

Methods.

IPL were detected in 321 CP CML patients with Imatinib treatment duration more than 12 months (the median – 90,3). Imatinib doses was 400 mg QD. The age of patients was 54,6 (24–76). Male/female ratio was 157/164. All patients gave informed consent before blood sampling. Blood samples were collected in 21–27h after the last Imatinib dose intake. Imatinib concentrations (C trough) were determined by validated LC/MS/MS method.

Results.

The patients were subdivided in 4 quartiles (Q): Q1 (n=81) with IPL 0 – 670 ng/ml, Q2 (n=80) with IPL 671 – 1042ng/ml, Q3 (n=80) with IPL 1043 – 1362ng/ml, Q4 (n=80) with IPL 1363 – 3826/ml. The results of imatinib treatment in each quartile were estimated according ELN recommendation. The obtained findings have shown that 48,1% CP CML patients in Q1 have achieved CCyR whereas 77,5%, 81,3% and 85% - in Q2,Q3 and Q4 respectively (Fig.1.).

Conclusion.

Our findings show that the achievement of CCyR in large cohort of CP CML patients (n=321) on imatinib with 400 mg/QD depends on IPL. The low level of IPL may indicate the nonadherence of some CML patients as well as some intrinsic mechanisms of imatinib plasma concentration decrease.

Disclosures:

Kutsev: Novartis: Research Funding, Speakers Bureau. Pospelova:Novartis: Research Funding, Speakers Bureau; Bristol Myers Squibb: Speakers Bureau. Khoroshko:Novartis: Speakers Bureau.
Activation of Chromatin Structure Upstream PU.1 Gene and in Vitro Differentiation in High Risk Myelodysplastic Syndrome Following 5-Azacytidine,
Authors: Stopka Tomas , Curik Nikola , Burda Pavel , Vargova Karin , Pospisil Vit , Vlckova Petra , Savvulidi Filipp , Necas Emanuel , Hájková Hana , Haskovec Cedrik , Belickova Monika , Cermak Jaroslav , Trneny Marek , Jonasova Anna
Published: October 2019

Abstract 3791

Use of DNA demethylating agent 5-azacytidine (AZA) in high risk Myelodysplastic syndromes (MDS) represents an important strategy yet not fully understood at molecular levels. Previous work demonstrated that normal progenitors treated by DNA demethylation agent Decitabine upregulated a key hematopoietic transcription factor PU.1. This was potentiated in presence of granulocyte colony stimulating factor (G-CSF) prior the DNA demethylation (Hu et al. 2010). Similarly, Decitabine administered to multiple myeloma cells resulted in DNA demethylation and upregulation of PU.1 gene (Tatetsu et al. 2007). To advance our understanding of molecular events mediated by AZA in MDS we measured effect of AZA on transformed cell lines (OCI-M2, SKM-1, MOLM-13, HNT-34, F-36P) and monitored the hematopoietic transcriptional program. We herein present evidence that AZA, while inducing efficiently cell cycle arrest and apoptosis of MDS tumor cells, stimulates also the expression of PU.1 and its target C/EBPa that is followed by acquisition of the myeloid differentiation program. Gene derepression of PU.1 is marked by epigenetic modifications at the upstream regulatory element (URE) of PU.1. G-CSF potentiated effects of AZA in MOLM-13 and SKM-1 cells. The effects of G-CSF and other cytokines (such as M-CSF or GM-CSF) on monocytic versus granulocytic lineage commitment of MDS cells are currently tested. To advance our study on the patient level we used primary high risk MDS CD34+ cells (N=36). Tumor cells with the lowest PU.1 expression displayed partial methylation at URE and incomplete sensitivity to AZA/G-CSF-mediated in vitro differentiation. We observed that patients with permissive chromatin at URE and higher PU.1 (and C/EBPa) levels responded significantly better to AZA/G-CSF in vitro. Currently we enrolled more patients in order to ask whether permissive PU.1 locus predicts responsiveness to AZA and whether it associates with different clinical patient outcome. (Grants: IGA NT10310-3/2009, MPO FR-TI2/509, GAUK251135 82210 & 251070 45410, SVV-2011-262507).

Disclosures:

No relevant conflicts of interest to declare.
IER3 Expression in Childhood Myelodysplastic Syndrome,
Authors: de Vries Andrica , Zwaan Christian M. , van Oorschot Astrid Danen- , de Haas Valerie , Hasle Henrik , Locatelli Franco , De Moerloose Barbara , Polychronopoulou Sophia , Stary Jan , Schmugge Markus , Dworzak Michael , Zecca Marco , Beverloo H. Berna , Niemeyer Charlotte M , Pieters Rob , van den Heuvel Marry M.
Published: October 2019

Abstract 3817

Background:

Childhood myelodysplastic syndrome (MDS) is a rare disease accounting for less than 5% of all hematological malignancies. In about 50% of the MDS cases an abnormal karyotype is found by conventional karyotyping, of which chromosome 6 is involved in 10%. The immediate-early-response 3 (IER3) gene, which is located on chromosome 6p21, encodes for a glycoprotein that plays a role in the regulation of apoptosis and cell cycle progression. Recently, it was shown that IER3 gene aberrations frequently occur in adult MDS patients, which are not restricted to patients with chromosome 6 aberrations and that low IER3 expression was associated with a worse outcome. Therefore, we investigated the frequency and prognostic impact of IER3 expression in childhood MDS.

Methods:

IER3 mRNA expression was determined by quantitative real-time PCR in 58 childhood MDS patients of which 17 carried a chromosome 6 aberration, and in normal bone marrow (n=8). In addition, methylation-specific-PCR (MSP) was performed to investigate the methylation status of the promoter region of the IER3 gene, as a plausible cause for the downregulation of IER3.

Results:

Median IER3 mRNA expression was 0.9% in MDS (range 0.01–73.3% relative to GAPDH expression) and 3.3% in normal bone marrow (range 0.81–85.5% relative to GAPDH expression) (p=0.05). A more than 4-fold decrease in IER3 expression below the mean of healthy controls was found in 74% (43/58) of the childhood MDS patients. There was no difference in IER3 mRNA expression between MDS patients with or without chromosome 6 aberrations (MWU p= 0.89). Also, no difference in IER3 mRNA expression was found between the different WHO-subtypes or between primary versus secondary MDS. Three patients with a chromosome 6p21 rearrangements in the IER3 region based on genomic profiling (array-CGH) showed expression-levels in the range of normal bone marrow. Low IER3 mRNA expression was associated with adverse outcome in childhood MDS patients (Cox regression analysis estimated hazard ratio 0.73, p=0.027, 95% CI-interval 0.55–0.97). The low IER3 expression appeared not to be due to hypermethylation of its promoter region.

Conclusion:

IER3 expression is low in childhood MDS patients with and without chromosome 6 aberrations and this low expression is associated with poor outcome. However this seems to be due to therapy related mortality, rather than by the increased risk of relapse. The IER3 downregulation is not regulated by hypermethylation of the IER3 promoter region.

Disclosures:

Hasle: Pfizer: Research Funding.
Frequent Clonal Evolution in Multiple Myeloma,
Authors: Hwang Sang Mee , Choi Jungeun , Yim Sunhee , Kim Tae Young , See Chaja , Kim Miyoung , Yoon Sung-Soo , Lee Dong Soon
Published: October 2019

Abstract 3919

Background:

Multiple myeloma is a clonal bone marrow disease characterized by the neoplastic transformation of differentiated B cells. Various complex cytogenetic and molecular genetic aberrations are present that are important for prognostication and follow up investigation. We investigated the clonal evolution of multiple myeloma patients at relapse or at progression compared from the diagnosis by conventional cytogenetics, fluorescence in situ hybridization (FISH) and cytoplasmic immunoglobulin fluroscence in situ hybridization (cIg FISH). Methods: 35 patients diagnosed as multiple myeloma by bone marrow examination from January 2003 to March 2011 were included. Conventional cytogenetics were performed in all patients at diagnosis and at relapse or progression. FISH was performed in 24 patients with available specimen for at least 3 items including −13/del(13q), p53 deletion/del(17p), 1q21 gain, p16 deletion, IgH rearrangement, t(4;14) and t(14;16). The FISH results were confirmed with cytoplasmic immunoglobulin FISH specifically staining plasma cells. Results: Forty-nine percent of the patients had relapsed or progressed with additional clonal evolutions and they were detected by conventional cytogenetics. Numerical abnormalities were more frequent than structural abnormalities and structural abnormalities involving chromosome 1 was frequent. Thirty-five percent had developed −13/13q loss which is considered a poor prognostic factor. cIg-FISH found additional aberrations in 20% of the patients such as RB1 deletion, del(17p) and t(14;16). Conclusion: Conventional cytogenetics and cIG-FISH are both necessary in relapsed patient since clonal evolutions develop in many patients which may only be detected by one method. Full evaluation of cIg-FISH including non-poor prognostic factors may be considered since new clones evolve that can be a candidate of follow-up marker and since prognostic factors can change as treatment modality changes.

Disclosures:

No relevant conflicts of interest to declare.
Treatment Patterns and Outcomes in Elderly Patients with Multiple Myeloma,
Authors: Bang Soo-Mee , Kyle Robert A , Lee Jae Hoon , Dispenzieri Angela , Larson Dirk , Colby Colin L. , Rajkumar S. Vincent , Kumar Shaji
Published: October 2019

Abstract 3980

Background:

Nearly half of patients with multiple myeloma (MM) are over 70 years at the time of diagnosis. While the survival of patients with myeloma has improved in the last decade, several studies have suggested no significant change for the elderly patients. These patients often have other comorbidities and are less likely to tolerate standard treatment approaches. We retrospectively studied the treatment patterns, outcomes, and prognostic factors in patients over 75 years of age.

PATIENTS AND METHODS:

We identified 212 patients who were older than 75 years from among 1545 patients seen at the Mayo Clinic within 90 days of diagnosis during 1999 to 2008. We only included 139 patients in whom complete treatment details were available.

RESULTS:

The median age was 80 years (range: 76–94) with 20 (14%) patients older than 85 years; 79 (57%) were male. Thirty-five patients (25%) had a history or coexistence of one or more malignancies. Chromosome analysis and FISH were performed in 65% and 22% respectively. Abnormal cytogenetics were seen in 30 (33%) of the 90 tested patients. The median survival of the 139 elderly patients was 27 months (95% CI: 20∼35) compared to 62 months (56∼68) for 1,290 patients ≤ 75 years of age seen during the same period. Sixty-nine (50%) patients received one or more novel agents (thalidomide, lenalidomide, pomalidomide, bortezomib) and showed longer median survival (40 months vs 13 months) compared with those not receiving these agents (p=0.0008). Age over 85, initial poor performance score > 1, thrombocytopenia, elevated serum LDH or beta2-microglobulin, response more than stable disease to first therapy resulted in inferior survival. Among them, only performance status had the persistent significance in multivariate analysis.

CONCLUSION:

Older patients receiving newer therapies appear to have a better outcome, which is still lagging behind the younger patients. A poor performance score at diagnosis predicts for eventual poor outcome.

Disclosures:

No relevant conflicts of interest to declare.
Family History of Venous Thromboembolism As a Risk Factor for Thrombosis in Multiple Myeloma Patients: A Population-Based Study,
Authors: Kristinsson Sigurdur Y , Goldin Lynn , Turesson Ingemar , Bjorkholm Magnus , Landgren Ola
Published: October 2019

Abstract 3987

Background:

Patients with multiple myeloma are at an increased risk of venous thromboembolism (VTE), especially when treated with thalidomide and lenalidomide. The etiology of this is largely unknown, but probably involves both genetic and environmental factors. Family history of VTE is a known risk factor for VTE in the general population, including known inherited thrombophilic abnormalities. The influence of a family history of VTE as a potential risk factor for VTE in multiple myeloma patients is unknown. To expand our knowledge on this topic, we conducted a large population-based study based on all multiple myeloma patients diagnosed in Sweden 1958–2004.

Patients and Methods:

We assessed the impact of family history of VTE as a risk factor for VTE among 21,067 multiple myeloma patients and 83,094 matched controls. Data on multiple myeloma patients was gathered from the Swedish Cancer Registry, information on first-degree relatives from the national Multigenerational Registry, and occurrence of VTE from the nationwide Patient Registry. We calculated odds ratios (OR) and 95% confidence intervals (CI) using chi-square.

Results:

Of the 21,067 multiple myeloma patients included in the study (54% males, median age at diagnosis 71 years), 66% had an identifiable first-degree relative. VTE was diagnosed in 1,429 multiple myeloma patients, and 921 had a family history of VTE. Compared to multiple myeloma patients without a family history of VTE, multiple myeloma patients with a family history of VTE had a 2.2-fold (95% CI 1.8–2.7; p<0.001) higher risk of VTE. Among 4,986 controls that were diagnosed with VTE, 316 had a family history of VTE. Controls with a family history of VTE had a 1.5-fold (95% CI 1.3–1.7; p<0.001) increased risk of VTE compared to controls without a family history of VTE. The difference of the impact of family history of VTE on the risk of VTE in multiple myeloma patients versus controls was significant.

Summary and Conclusions:

In this large population-based study including more than 20,000 multiple myeloma patients, we found family history of VTE to have a larger impact on VTE risk in multiple myeloma than in matched controls. Our findings confirm that genetic factors contribute to thrombophilia in multiple myeloma and may have therapeutic implications regarding thromboprophylaxis and treatment.

Disclosures:

No relevant conflicts of interest to declare.
In Vitro Generation of Influenza-Virus Specific T Cells for Adoptive Immunotherapy,
Authors: Gaundar Shiva , Blyth Emily , Clancy Leighton , Simms Renee , Gottlieb David
Published: October 2019

Abstract 4040

Influenza viruses cause fatal respiratory infections in stem cell transplant patients. Specific T cells provide long-lived adaptive immunity to influenza and the potential for generating such cells for clinical use was investigated. The inactivated influenza vaccine (Fluvax, CSL Australia) was used as the antigen source. Only reagents and culture media approved for clinical manufacture were used. Monocyte-derived dendritic cells (MoDC) pulsed with Fluvax were used to stimulate autologous PBMC at a responder to stimulator ratio of 10:1. On Day 7, a second stimulation was performed. 20U/ml IL-2 was added from Day 7 and 50U/ml IL-2 from Day 14 onwards. Media exchanges were performed as required using fresh medium containing IL-2.

Over 21 days of culture, a mean fold increase of 26.3 in cell number was observed (n=7). Cultures were mainly T cells (mean 92.9%) with effector and central memory phenotypes. CD4 cells dominated (mean 78.1%) with a lower percentage of CD8 cells (mean 14.9%). Following expansion, CD154 was expressed on 8.1% of CD4 cells (<1% in unstimulated cells). Both CD4 (mean 26.3%) and CD8 (mean 3.2%) cells produced one or more of the Th1 cytokines IFNg, TNFa and/or IL-2 when restimulated with Fluvax antigen pulsed MoDC. Most responding CD4 cells produced all three cytokines simultaneously, while CD8 cells primarily produced IFNg alone. A mean of 11.1% CD4 and 9.1% of CD8 cells mobilized CD107 on the cell surface when restimulated with Fluvax. Th1 cytokines were produced in response to stimulation with antigens derived from individual H1N1, H3N2 and Brisbane virus strains by the CD4 subset in all of 6 cases and CD8 subset in 2 of 6 cases. In addition, both CD4 and CD8 cells proliferated on restimulation with Fluvax or antigen from H1N1, H3N2 or Brisbane strains.

In conclusion, we demonstrate a clinically applicable method that yields high numbers of highly reactive T cells specific for influenza viruses including the H1N1 strain. Cells express a phenotype of activated antigen specific cells capable of B cell helper function. We propose that reconstructing host immunity through adoptive transfer of donor derived influenza virus specific T cells possibly combined with early post-transplant influenza vaccination will reduce the frequency of influenza-related deaths in the period of severe immune suppression that follows allogeneic stem cell transplant.

Disclosures:

Off Label Use: Use of influenza vaccine for in vitro cell generation.
Simultaneous Isolation and Enrichment of Multi Specific and Multi Functional T-Cells Populations for Treatment of Patients Undergoing Allogeneic Hematopoetic Stem Cell Transplantation,
Authors: Kruse Meike , Bonig Halvard , Kapp Markus , Germeroth Lothar , Einsele Hermann , Grigoleit Götz Ulrich
Published: October 2019

Abstract 4056

Allogeneic hematopoetic stem cell transplantation (aHSCT) is a treatment option for a variety of diseases in particular heamatological malignancies. Due to an ongoing immunosuppression to prevent graft versus host reaction (GvHD), disease relapses as well as viral infections are major causes of morbidity and mortality after aHSCT.

T cell responses against different tumor-associated and tumor-specific antigens could be detected not only in patients with malignant diseases but also in healthy donors. We investigated the selection of MART-1 (Melanoma Antigen Recognized by T-cells), Proteinase 3 and WT-1 (Wilms Tumor- Antigen) specific T cells from the blood of healthy donors as basis for a tumor-specific T cell transfer in the context of aHSCT.

With a newly established protocol, based on streptamer selection, we isolated simultaneously multi-functional and multi-specific T-cell populations. We selected tumor-antigen-specific CTL′s (Cytotoxic T- Lymphocytes) mentioned above and also antiviral T-cells namely against CMV, EBV and AdV from a single blood donation. In this simultaneous selection with up to 7 different epitopes in one step, even the tumor specific T cells, which are known to be rarely detected among healthy donors, could be enriched to an amount sufficient for a direct T cell transfer.

Purity achieved after selection was at least 82% and up to 98,87%, which minimizes the risk for GvHD after clinical application. The possibility to transfer these selected CTL`s to patients after stem cell transplantation improves the graft versus tumor effect as well as the anti infective T cell immunity without a relevant risk for GvHD.

Furthermore, the selected multi specific T cell populations could be expanded in vitro without loss of specificity and include different phenotypes such as central memory and memory effector cells, which may provide long lasting immunity.

Moreover, starting with a leukapheresis, we successfully transferred our selection protocol in a closed system according to current good manufacturing practice (cGMP) requirements, which allows clinical application in the future. With that, it opens new perspectives in cellular immunotherapy against malignancies and viral infection for patients after aHSCT.

Disclosures:

No relevant conflicts of interest to declare.
What Predicts High Risk Acute Gvhd At Diagnosis?: Identification of Those At Highest Risk by a Novel Acute Gvhd Risk Score,
Authors: MacMillan Margaret L. , Defor Todd E. , Weisdorf Daniel J.
Published: October 2019

Abstract 4062

To define high risk acute graft-versus-host disease (GVHD) at its onset, we examined the initial GVHD stage and grade of 864 patients at the University of Minnesota who received uniform therapy with prednisone 60 mg/m2/d. We compared the prognostic utility of the Minnesota (MN) (modified from Consensus) versus Center for International Blood and Marrow Transplant Research (CIBMTR) GVHD organ stage-derived grading systems. As neither GVHD grading system optimally predicted outcomes, a novel acute GVHD risk score was devised by combining the MN and CIBMTR systems. Using multiple regression analysis, we could dichotomize patients into (high risk, HR, n=86) acute GVHD with initial grade IIIC, IID or IVD who were significantly less likely to respond to steroid therapy by day 28 than patients with standard risk (SR, initial grade IA-IIIB, n=778) GVHD (Univariate complete response [CR]+ partial response [PR] at day 28: HR 43% [95% CI 33–53%] vs. SR 68% [95% CI 65–71%], p <.001; Multivariate HR Relative Risk [RR] of overall response [CR+PR], 0.3, 95% CI, 0.2–0.5, p<.001). The probability of treatment-related mortality (TRM) at 2 years was significantly higher in patients with HR than SR acute GVHD (51% [95% CI 40–62%] vs. 38% [95% CI 35–41%], p <.01) (figure).

In multivariate analysis, patients with HR acute GVHD had a 2 fold increase risk of TRM (RR, 2.0, 95% CI, 1.4–2.8, p<.001) and similarly higher 2 year mortality (RR, 1.5, 95% CI, 1.1–2.1, p=.004). Using this novel acute GVHD Risk Score, we can identify initially HR GVHD as either skin stage 4, lower gastrointestinal (GI) stage 3+, liver stage 3+, or skin stage 3 and lower GI or liver stage 2+ GVHD. Patients with HR acute GVHD at onset have a poor prognosis, require alternative initial therapy and should be the focus of novel therapeutic trials.

Disclosures:

Weisdorf: Genzyme: Consultancy, Research Funding.
Umbilical Cord-Derived Mesenchymal Stem Cells for Treatment of Steroid-Resistant Severe Acute Graft-Versus-Host Disease
Authors: Chen Guanghua , Yang Ting , Fu Chengcheng , Miao Miao , Jin Zhengming , Tang XiaoWen , Han Yue , He Guangsheng , Zhang Xuhui , Ma Xiao , Chen Feng , Hu Xiaohui , Xue Shengli , Wang Ying , Qiu Huiying , Sun Aining , Depei Wu
Published: October 2019

Abstract 4536

Objective

To evaluate the safety profile and efficacy of umbilical cord-derived mesenchymal stem cell infusion in patients with steroid-resistant, severe, acute graft-versus-host disease (aGVHD).

Methods

A total of 19 patients with steroid-resistant severe aGVHD received mesenchymal stem cell infusion treatment. We analyzed the treatment response, transplantation-related mortality, events associated with infusion and relapse rate.

Results

Two patients with grade II, 5 patients with grade III and 12 patients with grade ‡W aGVHD received a total of 58 infusions of mesenchymal stem cell. The mean total dose of mesenchymal stem cell was 2.13×106 (range 0.6–7.2×106) cells per kg bodyweight. 7 patients received one infusion, 2 patients received two infusions, and 10 patients received three or more infusions. 11 patients had a complete response and 4 had a partial response and 4 had no response. No patients had side-effects during or immediately after infusions of mesenchymal stem cell and no ectopic tissue was detected to date. 11 patients survived and 8 died, 4 for aGVHD, 1 for infection and 2 for aGVHD with concomitant infection and 1 for underlying leukemia relapse. The cell viability of freshly prepared mesenchymal stem cell is 93% (92%-95%) by trypan blue staining. The cell viability of controlled-rate freezed and thawed cells mesenchymal stem cell is 72% (70%-74%).

Conclusion

Infusion of umbilical cord-derived mesenchymal stem cell expanded in vitro is an effective therapy for patients with steroid-resistant, severe aGVHD without negative impact on relapse. Freshly prepared mesenchymal stem cells are superior to freezed and thawed cells in terms of cell viability.

Disclosures:

No relevant conflicts of interest to declare.
VITAMIN D Insufficiency Affects Time to FIRST TREATMENT (TFT) In EARLY CHRONIC LYMPHOCYTIC LEUKEMIA (CLL)
Authors: Molica Stefano , Digiesi Giovanna , Antenucci Anna , Levato Luciano , Mirabelli Rosanna , Gentile Massimo , Giannarelli Diana , Sperduti Isabella , Morabito Fortunato , Conte Laura
Published: October 2019

Abstract 4601

Recent data suggest that vitamin D insufficiency is related to inferior prognosis in patients with chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL). We evaluated whether 25(OH)D serum levels affect time to first treatment (TFT), a disease-specific end point, in 130 previously untreated CLL patients with early disease (i.e., Binet stage A). Measurement of 25(OH)D was conducted by means of a direct, competitive chemiluminescence immunoassay using the DiaSorin LIAISON 25(OH)D TOTAL assay (DiaSorin, Inc., Stillwater, Minnesota). Levels of 25(OH)D did not reflect Rai substages (P=0.27), Beta2-microglobulin (P=0.52), absolute lymphocyte count (P=0.21), FISH chromosome abnormalities (P=0.11), mutational status of IgVH (P=0.43), ZAP-70- (P=0.53) or CD38-expression (P=0.82).Overall, 42 patients (31.8%) had severe vitamin D insufficiency (< 10 ng/mL), 66 (50%) had mild to moderate insufficiency (10–24 ng/mL), and 24 (18.1%) had 25(OH)D levels within the optimal range(25–80 ng/mL), with no relationship between the month of sample collection and 25(OH)D level (P=0.188). A patient stratification according to these 3 groups led to significant difference in terms of TFT, with vitamin D insufficient patients having the shortest TFT (P=0.02). With respect to continuous 25(OH)D levels and clinical outcome, TFT was shorter as 25(OH)D decreased until a value of 13.5 ng/mL at which point the association of 25(OH)D and TFT remained constant. As a matter of fact, the 25(OH)D value of 13.5 ng/mL identified two patients subsets with different TFT risk (HR=1.91; 95%CI=1.06–3.44;P=0.03). In multivariate analysis the variable entering the model at a significant level were mutational status of IgVH (P<0.0001), serum thymidine kinase ((P=0.02) and absolute lymphocyte count (P=0.03). Thus confirming the Mayo clinic experience, our data provide further evidence that 25(OH)D levels may be an important host factor influencing TFT of Binet stage A patients. Since the effect of 25(OH)D insufficiency may be may be relevant in this subset of patients who are generally observed for years before starting therapy future trials should address the role of vitamin D therapy in delaying disease-progression.

Disclosures:

No relevant conflicts of interest to declare.
Analysis of Mantle Cell Lymphoma Patients Reveals Novel Regulatory Circuits Involving MicroRNAs and Their Targets
Authors: Kulvait Vojtech , Pospisil Vit , Vargova Karin , Trneny Marek , Klener Pavel , Stopka Tomas
Published: October 2019

Abstract 4624

Mantle cell lymphoma (MCL) represents B-cell lymphoma derived from the mantle zone that surrounds normal germinal center follicles. Pathophysiology of this hardly curable disease involves t(11,14)(q13,q32) translocation which leads to upregulation of Cyclin D1(CCND1). Recently, microRNAs were demonstrated to significantly modify MCL pathogenesis (Jian-Jun Zhao et al. 2010) and therapy responsiveness (Jiang et al. 2010). In order to broaden our knowledge of regulatory pathways in MCL we searched for differentially expressed microRNAs and their differentially expressed mRNA targets between MCL samples and control samples. Samples consist of magnetically separated B cells derived from peripheral blood. We used 1) Microarray mRNA hybridization [Affymetrix Human Genome U133 Plus 2.0 Array, N(MCL)=5, N(control)=5] and 2) microRNA profiling [TaqMan® Array Human MicroRNA Card A v2.0 technology, N(MCL)=5, N(control)=5] followed by statistical analysis [limma (Smyth 2005)]. Differentially regulated targets of the deregulated microRNAs were selected from the databases involving both predicted (Betel et al. 2007) or confirmed (Hsu et al. 2010) target mRNAs. Among the most significant upregulated microRNAs (exceeding 10 fold) are miR-9, miR-124 and miR-183. We have found that upregulated miR-9 has confirmed downregulated target gene PRDM1 (PR domain zinc finger protein 1) which plays a role in B cell maturation (Turner et al. 1994) and may act as a tumor suppressor (Pasqualucci et al. 2006). Upregulation of miR-9 and downregulation of its targets was recently demonstrated in Burkitt lymphoma (Onnis A et al. 2010) and Hodgkin lymphoma (Nie K et al. 2008). Two additional upregulated microRNAs, miR-124 and miR-183, yet not associated with lymphomas, have downregulated target gene Integrin beta-1 (CD29) which regulates survival (Fukumori et al. 2003). Among most significant downregulated microRNAs is miR-101 (FC=-7). Downregulated microRNA miR-101 has known oncogene N-Myc (MYCN) as upregulated confirmed target and DNA (cytosine-5)-methyltransferase 3A (DNMT3A) as upregulated target. Overexpression of a well known hematopoietic oncogene MYCN and epigenetic repressor DNMT3A may represent additional pathogenesis-related factors in MCL. Our data support importance of the candidate mechanisms involving microRNAs and their target programs in pathogenesis of MCL. They are currently extended on a larger patient cohort by analyzing expression and functional significance of the candidate microRNAs. (Grants: NT10310-3/2009, MPO FR-TI2/509, NPVII 2B06077, MSM 0021620806, LC 06044, SVV-2011-262507).

Disclosures:

No relevant conflicts of interest to declare.
Analysis of Methylation Status of ZAR1, GATA4, CDH22, SOX3, SLC16A5, PFN2, EHD3 and TBPL1 in Hematological Malignancies
Authors: Kobayashi Yujin , Hatta Yoshihiro , Takeuchi Jin , Fujiwara Kyoko , Shinojima Yui , Kawashima Hiroyuki , Igarashi Jun , Nagase Hiroki
Published: October 2019

Abstract 4631

Background:

DNA cytosine methylation in CpG dinucleotides is an important epigenetic event and critical for the control of gene expression, and appears to play a crucial role in tumorgenesis in hematologic malignancies. To identify novel tumor-specific differentially methylated regions in human leukemia/lymphoma, we performed methylation analysis of new target lesions in hematological malignancies.

Methods:

The aberrant methylation at 8 new candidate human homologous genomic regions (ZAR1, GATA4, CDH22, SOX3, SLC16A5, PFN2, EHD3 and TBPL1), where hypermethyloation status was identified through a mouse-skin cancer model study by the RLSG method, were analysed with quantitative DNA methylation analysis using the Sequenom MassARRAY system. 8 human leukemia/lymphoma cell lines (HL-60, KG-1, Jurkat, MOLT-4, NALM-6, K562, Ramos, and Raji), and bone marrow or peripheral blood samples from 20 patients with leukemia before treatment were obtained for samples (10 in AML, 4 in Ph- ALL, 2 in Ph+ ALL, 2 in CML-CP, 1 in CML-BC, and 1 in CLL). Normal lymphocyte cells from four healthy individuals were used as normal controls. Each data were assigned as hypermethylaion when the average methylation levels of the entire target regions were more than 50%.

Results:

In ZAR1, SLC16A5 and EHD3, hypermethylation level was seen in all leukemia/lymphoma cell lines. GATA4, CDH22 and SOX3 showed hypermethylaion in all tumor cell lines except for AML cell line KG-1. PFN2 showed hypermethylation in all cell lines except for KG-1, T-ALL cell line Jurkat and MOLT-4. No aberrant methylation among tumor and normal cells was observed in TBPL1. Interestingly, DNA methylation patterns of ZAR1, GATA4, CDH22, and SOX3 were different between the clinical specimens of AML and ALL. Hypermethylation of those genes were frequently observed in ALL samples but less in AML. The Mann-Whitney U-test showed that the differences were significant in all ZAR1, GATA4, CDH22, and SOX3 (p < 0.01, respectively). DNA samples from CML-CP patients showed no abnormal methylation patterns in all genes. DNA from T-CLL patient showed hypermethylation only in EHD3. No abnormal methylation pattern was observed in SLC16A5 and PFN2 in clinical specimens. None of the normal control samples showed aberrant methylation in any genes.

Conclusion:

We identified regions aberrantly methylated with high frequency among the new candidate genes in leukemia and lymphoma, and demonstrated a distinct methylation pattern between tumors and normal lymphocytes. Aberrant DNA methylation of ZAR1, GATA4, CDH22 and SOX3 may be associated to differentiation to lymphoid populations of leukemia.

Disclosures:

No relevant conflicts of interest to declare.
Prospective Evaluation of Hemostatic Abnormalities in Overt DIC Due to Various Underlying Diseases
Authors: Wada Hideo , Hatada Tsuyoshi , Kawasugi Kazuo , Nobori Tsutomu
Published: October 2019

Abstract 4652

[Introduction]

Disseminated intravascular coagulation (DIC) is often found in patients with infections, leukemia, solid tumors, trauma, aneurysms or obstetricdiseases. The causes of DIC associated with leukemia are considered to be an elevated expression of tissue factor, tissue type plasminogen activator or anexin II in leukemic cells. The factors thought to be responsible for DIC associated with sepsis are an elevated inflammatory response, including an increased production of inflammatory cytokines.

[Materials and Methods]

In this study, 692 patients with suspected DIC were prospectively evaluated for various types of underlying diseases using the DIC diagnostic criteria established by International Society of Thrombosis and Haemostasis (ISTH) and Japanese Ministry Health Labor and Welfare (JMHLW), and the usefulness of hemostatic markers were examined for each patient with DIC due to various underlying diseases.

[Results]

The main underlying disease of DIC was infectious diseases, hematologic malignancies, and solid tumors, and a high resolution rate from DIC was observed in obstetric diseases and hematologic malignancies. The diagnosis of DIC was related to a poor outcome in trauma/burn victims and those with infectious disease. In the main underlying disease, it is suggested that DIC would be excluded in patients with hematologic malignancies or solid tumors with a platelet count of more than 1×108/ml and in the patients with fibrinogen and fibrin degradation products (FDP) of less than 10 mg/L, and fibrinogen of less than 100 mg/dl, suggesting the presence of DIC. The prothrombin time was a sensitive marker, but fibrinogen levels were not sensitive for DIC due to infectious diseases. The plasmin plasmin inhibitor complex in hematologic malignancy, and soluble fibrin monomer complex, antithrombin and thrombomodulin in patients with infectious disease, were sensitive markers for the diagnosis of DIC.

[Discussion and conclusion]

Although hemostatic markers were useful for the diagnosis of DIC, the usefulness varied depending on the different underlying diseases. The global coagulation tests are therefore considered to be useful for the diagnosis of DIC, and hemostatic molecular markers are useful for examining the pathophysiology of patients with DIC.

Disclosures:

No relevant conflicts of interest to declare.
Importance of Individualized Replacement of Factor VIII Concentrate According to Recovery Test in Hemophilia A Patient During Surgery
Authors: Shim Ye Jee , Choi Yong-Mook , KIm Ji Yoon , Lee Kun Soo
Published: October 2019

Abstract 4654

Purpose:

There is a common guideline for surgery in hemophilia A patients - to maintain their factor VIII (FVIII) levels over 100% during and 24 hours after operation by infusion of FVIII concentrates. However their responses to coagulation factors are very different from each other even with the lack of inhibitor. Thus we demonstrated the correlation between individual FVIII recovery and prognosis after surgery.

Methods:

We reviewed the operation cases in hemophilia A patients who had recovery tests before surgery in Kyungpook National University Hospital from January 2001 to July 2011. The blood sample for recovery test was acquired at 15 minutes after initial infusion of FVIII concentrate from the opposite arm. The patients with inhibitors were excluded. The average hospitalization period and consumption of FVIII concentrates were analyzed.

Results:

A total 24 surgery cases in 16 hemophilia A patients were reviewed. They were divided into 3 groups; A (recovery/expected FVIII > 0.8), B (recovery/expected FVIII < 0.8, but they were managed by immediate additional replacement of FVIII concentrates), and C (recovery/expected FVIII < 0.8 and the extra supplementations of coagulation factors were delayed over 3 days). Type of operation included neurosurgeries (craniotomy/removal of hematoma 3, ventriculo-peritoneal shunt 2, ventriculostomy 1), orthopedic procedures (arthroplasty 4, open reduction/internal fixation 3, bone graft 1, drilling of bone 1), and gastrointestinal operations (subtotal gastrectomy 2, appendectomy 2, hemorrhoidectomy 2, rectosigmoidectomy 1, cholecystectomy 1, herniorrhaphy 1). The total consumption of coagulation factors and the dose per body weight were lower in group A compared with B and C (respectively p=0.028 and p=0.003, CI 95%). However the hospitalization periods were not different between group A and B (p=0.956, CI 95%). And in group B, the hospitalization period, total consumption of coagulation factors, and the dose per body weight were all lower than C (respectively p=020, p=023, and p=048, CI 95%). No complication was noted in group A and B while 2 cases of hematoma infection were developed in group C.

Conclusion:

The low FVIII recovery under 80% of expected may contribute higher consumption of coagulation factors after surgery in hemophilia A patients. However if the immediate supplemental FVIII replacement was performed, the medical expense would be conserved without complication. Thus the individualized replacement of FVIII concentrates according to the recovery test is very important for hemophilia A patients who need operation.

Disclosures:

No relevant conflicts of interest to declare.