bluuebunny/crossref_metadata_2025 huggingface dataset:
A Single Center Phase II Trial Of 90yttrium-Ibritumomab-Tiuxetan Produces High Response Rates As First-Line Therapy For Early Stage B Cell Indolent Lymphoma, Including Bulky Disease
Authors: Samaniego Felipe , Romaguera Jorge E , Fowler Nathan , Fanale Michelle A. , Shah Jatin J. , McLaughlin Peter , Pro Barbara , Selvaraj Vijairam , Braun Frank K , Mathur Rohit , Feng Lei , Neelapu Sattva , Kwak Larry W.
Published: October 2019

Background

90YIT is approved for the treatment of relapsed follicular lymphoma or as consolidation therapy after first line chemotherapy. Its role as therapy for the front line treatment of early stage B cell indolent lymphoma is under investigation.

Patient and Methods

Thirty-one patients with B cell lymphoma: grade 1 or 2 follicular lymphoma [FL], or marginal zone lymphoma (MZL) with stages I or II were enrolled for treatment with Zevalin (90yttrium-ibritumomab-tiuxetan [90YIT]). Patients received 90YIT according to standard procedure. Primary end points were overall response rates and progression-free survival. Initial evaluation included CT scans, PET-CT, CBC and bone marrow biopsies. Assessment of response was made at 4 months, and monitored every 3, 6 and 12 months during year 1, 2 and 3 of follow up, respectively. CBC was analyzed every 2 weeks until recovery of counts. Median and range for quantitative, absolute and relative frequencies for categorical variables were calculated. PFS was estimated by using Kaplan-Meier method.

Results

Twenty-one patients had FL and 10 had MZL. The median age was 57 years (range 28-87) with 6/31 (19%) having bulky lymphoma (greater than 5 cm diameter). The overall response rate was 100% with CR 87% and Cru 13% with a median follow up of 56 months for the censored observations. The median progression free survival time has not been reached. 10 patients relapsed; 8/20 (40%) with FL and 2/11 (18%) with MZL. The rate of relapses was 33% in bulky and 32% in nonbulky lymphoma. Adverse events were primarily hematologic; the incidence of grade 3 and 4 neutropenia, thrombocytopenia, and anemia were 45% and 16%, 19% and 16% and 0% and 3%, respectively. Non-hematologic toxicity never exceeded grade 2 according to CTCAE v3.0.

Conclusion

90YIT is well-tolerated and has achieved high response rates in patients with early stage B cell indolent lymphoma. Bulky disease did not adversely affect tumor responses.

Disclosures:

Pro: Spectrum: Honoraria.
Inhibition Of HDAC6 In Combination With Targeted Agents Results In Broad Synergistic Decreases In Viability In Non-Hodgkin’s Lymphoma (NHL) Cells
Authors: Quayle Steven N , Tamang David , Yang Min , Jones Simon S
Published: October 2019

Histone Deacetylase (HDAC) inhibitors have demonstrated significant clinical benefit as single agents in cutaneous and peripheral T cell lymphomas, and have received FDA approval for these indications. Numerous clinical studies are also ongoing to investigate the potential benefit of combining HDAC inhibitors with other standard of care and investigational agents in various subtypes of lymphoma. ACY-1215 is a first-in-class, orally available selective inhibitor of HDAC6 (approximately 11-fold selective over class I HDAC’s) that is currently in two Phase Ib clinical trials (NCT01323751 and NCT01583283) in combination with dexamethasone and either bortezomib or lenalidomide in multiple myeloma (MM) patients. Beyond MM, preclinical studies in NHL cell lines also demonstrated increased benefit from treatment with ACY-1215 in combination with the proteasome inhibitors bortezomib or carfilzomib (Amengual, et al, ASH, 2012; Dasmahapatra, et al, ASH, 2012). In addition to standard of care therapies, a number of novel targeted agents have recently demonstrated potential clinical benefit in subtypes of NHL, including agents targeting Bruton’s tyrosine kinase (eg. ibrutinib) and the phosphatidyl inositol-3’ kinase family (eg. GS-1101, IPI-145, and GDC-0941). We describe here the therapeutic potential of ACY-1215 in a collection of NHL cell lines both as a single agent and in combination with these novel targeted agents.

NHL cell lines derived from diffuse large B cell lymphomas (DLBCL; both germinal center and activated B cell type) and mantle cell lymphomas (MCL) exhibit significantly decreased viability in response to treatment with selective inhibitors of HDAC6, including both ACY-1215 and ACY-775 (approximately 300-fold selective for HDAC6 over HDAC1/2, and 1500-fold selective over HDAC3). Furthermore, combination treatment of NHL lines with ACY-1215 and either ibrutinib, GDC-0941, or GS-1101, in a dose-matrix format resulted in synergistic decreases in cell viability. The relevance of HDAC6 inhibition to this synergistic response was confirmed through the observation of similarly decreased viability when treating cells in combination with the highly selective HDAC6 inhibitor ACY-775. Detailed molecular mechanism of action studies are ongoing and will be presented. Additional studies are also underway to assess the tolerability and efficacy of combinations of these targeted agents with ACY-1215 in xenograft models of lymphoma. Overall, these data support the continued investigation of the activity of ACY-1215 in lymphoma both as a single agent and in combination with the next generation of targeted therapies for B cell lymphomas.

Disclosures:

Quayle: Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership. Tamang:Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership. Yang:Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership. Jones:Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership.
Effect Of Advanced Lytic Bone Lesions On Peripheral Stem Cell Mobilization In Patients With Multiple Myeloma
Authors: Jung Sung-Hoon , Ahn Jae-Sook , Yang Deok-Hwan , Kim Yeo-Kyeoung , Kim Hyeoung-Joon , Lee Je-Jung
Published: October 2019

The purpose of this study was to identify incidence and predictive factors of peripheral blood stem cell (PBSC) mobilization failure in patients with multiple myeloma (MM). Retrospective data of 104 patients who received cyclophosphamide plus G-CSF or G-CSF alone mobilization were analyzed. The rate of mobilization failure using two definitions of failure < 2 and < 4 ´ 106 CD34+ cells/kg following the first collection attempt was 16.3% and 33.7%, respectively. Predictors of mobilization failure were evaluated using logistic regression analysis including age, advanced osteolytic lesions, bone marrow cellularity before mobilization, platelet count, body mass index before mobilization, and mobilization method. Lytic bone lesion was assessed by conventional skeletal survey, and advanced osteolytic lesions were defined by lytic lesions more than three skeletal sites regardless of number of lytic lesions. By multivariate analysis, advanced osteolytic lesions (odds ratio mORn= 10.956, P = 0.001) and age ≥ 60 years (OR = 5.454, P = 0.017) were associated with PBSC yield lower than 2 ´ 106 CD34+ cells/kg, and advanced osteolytic lesions (OR = 5.088, P = 0.006), WBC ≤ 4,000/mL before mobilization (OR = 4.724, P = 0.005), and G-CSF only mobilization (OR 10.526, P = <0.001) were associated with PBSC yield lower than 4 ´ 106 CD34+ cells/kg. This data suggests that multiple osteolytic lesions as well as advanced age, WBC count, and mobilization method is a significant predictor of mobilization failure in MM patients.

Disclosures:

No relevant conflicts of interest to declare.
Defining Parameters That Can Guide Public Cord Blood Units (CBUs) In Korea
Authors: Lee Junglim
Published: October 2019

Backgrounds

It is well recognized that TNC and CD34 define a superior Cord blood unit (CBU). According to “the Law of Korean CB Banking and Research”, only 40% of collected CBUs meet the criteria. To improve a rate of banking, we analyzed the characteristics of CBUs and assessed predictive factors for numbers of TNC and CD34+ in collected CBUs.

Methods

From October 2003 to Dec. 2012, 3610 CBUs (2407 at the Catholic Hematopoietic Stem Cell Bank, 1203 at the Daegu Fatima Hospital Public Umbilical Cord Bank) were collected at two official Korean Public UCB banks. The variables were analysed from retrospective records at the time of donation. The associations between TNC, CD34+ and variables including gestational age (GA), gender of fetus, body weight (BW) of fetus, collected volume (CV), time from birth to preservation (TBtoP) and delivery type were analyzed by logistic regression.

Results

Our study cohort (n = 3610), the mean/median numbers of TNC, CD34+, GA, BW, TBtoP and CV were 9.50/8.76úI108 (range, 0.81-35.1), 2.78/2.10úI106 (range, 0-29.2), 39.4/39.6 weeks (range, 31-42), 3.33/3.32 Kg (range, 1.66-4.97), 0.7/1.0 days (range 0-2), 84.75/82.12g (range, 3.7-179.2) respectively.In univariate analysis, maternal/donor (M/D) characteristics that were associated with high TNC (defined as > 10úI108) included higher GA (defined as > 39weeks) [OR 1.38 (95% CI 1.19-1.60 p < 0.001)], normal delivery [OR 2.72 (95% CI 2.23-3.32 p < 0.001)], female gender [OR 1.32 (95% CI 1.14-1.53 p=0.001)], BW > 3.33Kg [OR 1.64 (95% CI 1.41-1.91 p < 0.001)] and CV > 84.75g [OR 2.37 (95% CI 2.06-2.72 p < 0.001)]. In multivariate analysis of TNC, CV > 84.75g [OR 7.53 (95% CI 2.54-22.29 p < 0.001)] and normal delivery [OR 10.41 (95% CI 1.817-59.66 p = 0.009)] were significant predictors of a high TNC and others were not significant. In case of CD34+, in univariate analysis, M/D characteristics that were associated with high numbers of CD34+ (defined as > 3.0úI106) included normal delivery [OR 1.60 (95% CI 1.32-1.94 p < 0.001)], BW > 3.33Kg [OR 1.49 (95% CI 1.27-1.73 p < 0.001)] and CV > 84.75g [OR 1.48 (95% CI 1.29-1.70 p < 0.001)]. In multivariate analysis of CD34+, normal delivery [OR 19.40 (95% CI 3.72-101.25 p < 0.001)] was also the best predictor followed by BW > 3.33Kg [OR 2.53 (95% CI 1.16-5.54 p = 0.02)].

Conclusions

M/D characteristics were associated with normal delivery which is the best predictor for TNC and CD34+ followed by CV for TNC and BW for CD34+ in collected Korean CBUs. These associations could be used to prioritize donations, collections, optimizing and financial modeling in Korean CB banks.

Disclosures:

No relevant conflicts of interest to declare.
Non Interventional Prospective Study On The Effect Of 48 Gene Variants On Different Endpoints After Allogeneic Stem Cell Transplantation For Patients With Acute Myeloid Leukemia
Authors: Koldehoff Michael , Beelen Dietrich W. , Elmaagacli Ahmet H.
Published: October 2019

Background

Single-nucleotide polymorphisms (SNPs) are molecular markers that vary significantly among different populations. Our group has earlier reported about different genetic associations of SNPs with GvHD and/or outcome after allogeneic hematopoietic stem cell transplantation (HSCT) in different retrospective studies. Here we profiled SNPs in a non interventional prospective study (Trial No DRKS00004352) about the influence of different endpoints for patients (pts) with acute myeloid leukemia who underwent allogeneic HSCT between June 2011 and February 2013.

Methods

We analyzed simultaneously 48 different genes of every patient/donor pairs in whole blood by high-throughput LightCycler® 480 real-time PCR-system using high resolution melting optimization strategies.

Results

In this cohort 20 pts received grafts from HLA-identical siblings (23%), 42 pts from matched (48%) and 26 pts from mismatched (30%) unrelated donors. Transplant consisted of unmanipulated peripheral blood stem cells (n=79, 90%) or bone marrow (n=9, 10%). Of all pts (n=88, male 48 pts and female 40 pts), 18 (21%) had relapsed and 28 (32%) died of May 2013. In the cohort the occurrence of acute GvHD (aGvHD) grade 2-4 was influenced by gene variants on recipient side of CYP 2C9 (39% vs 72%, p<0.04), IL16 (53% vs 31%, p<0.01) and MTHFR 677, 1298 (31% vs. 52%, p<0.05). Furthermore, the occurrence of severe aGvHD ≥3 was influenced by GSTP1 A/G (3% vs 20%, p<0.04), LAT (6% vs 17%, p<0.02), MBL2 codon 550 (3% vs 22%, p<0.03), and VEGF 405 G/C (15% vs 7%, p<0.02). There was no significant correlation between different gene variants of pts and the estimate for 1-year overall survival (OS). We found that the rate of 1-year none-relapse mortality (NRM) was associated favorably with the detection of variants of NOD2 genes (0% vs 26%, p<0.04) and MBL codon 220 (7% vs 32%, p<0.05) and associated adversely with the detection of variants of LAT (16% vs 34%, p<0.03). The estimate 1-year relapse rate was associated adversely with the detection of variants of IL 10 592 C/A (15% vs 45%, p<0.05) and associated favorable with the detection of variants of TLR9 genes (40% vs. 10%, p<0.02)

Conclusions

These preliminary results suggest that different gene variants have influence on the transplant settings in pts with acute myeloid leukemia.

Disclosures:

Off Label Use: HCG will be discussed as new therapy for chronic GVHD.
A Phase I/II Trial Evaluating The Use Of a Histone Deacetylase Inhibitor Panobinostat (LBH589) In Addition To Glucocorticoids In Patients With Acute Graft-Versus-Host Disease
Authors: Perez Lia , Fernandez Hugo F , Tomblyn Marcie , Locke Frederick L , Field Teresa , Pidala Joseph , Elmer Erika , Beato Francisca , Villagra Alejandro , Anasetti Claudio
Published: October 2019

Graft-versus-host disease (GVHD) remains the principal obstacle to achieve successful outcomes in allogeneic hematopoietic stem cell transplant (HCT). Glucocorticoids are the current standard initial treatment for acute GVHD with complete responses of 25% to 41%. New immunosuppressive strategies are required to improve management of acute GVHD and decrease toxicities of immunosuppressive agents. We conclude that more effective acute GVHD therapy might improve remission rates that will result in better survival after allogeneic HCT. Vorinostast, a histone deacetylase inhibitor (HDACi) have shown efficacy for acute GVHD prevention. This protocol was design to test the safety and potential efficacy of a novel HDACi, panobinostat (LBH589), administered to patients with acute GVHD within 72 hours of initiation of glucocorticoid therapy (methylprednisolone 0.8 mg/Kg/day IV or equivalent for at least 14 days) as first line therapy. Panobinostat is a potent inhibitor of deacetylases and HSP90 belonging to a structurally novel class of the cinnamic hydroxamic acid class of compounds and is one of most potent HDAC inhibitors. We have enrolled n=13 subjects, median age 52 (range, 39-63) years, male n=8/female n=5, median day of GVHD development day + 37 post HCT (26 -528 days) with overall grade GVHD II (n=8) or III (n=5). The first three patients were treated with 2.5MG/M2 intravenously (IV) weekly x 4 and subsequent subject was treated with 5MG/M2 IV weekly x 4 achieving all GVHD CR by day +15. Patients were treated with azole for fungal prophylaxis. Due to manufacturer discontinuation of IV formulation production we were obliged to amend the protocol to use PO Panobinostat. Using 10MG PO TIW 3 doses q week x 4 weeks, we treated 2 subjects which were both discontinued from study drug as evidence of GHVD progression within 7 days of Panobinostat. Due to safety concerns, the next group was treated with a reduce dose of 5 MG PO TIW q week x 4 (Level -1). So far we have accrued 7 subjects whom by day 15 achieved GVHD CR (n:4) and PR (n:3) majority being in CR (n:6/7) on day +28 and PR (n:1). Toxicities by CTCAE 4.0 criteria included reversible thrombocytopenia and neutropenia grade 1-2 possible related to study drug. We are encouraged with a GVHD CR rate of 57% on day +15 and 86% CR at study drug completion on day+36. These results suggest a role HDACi Panobinostat as a tool to improve success of glucocorticoids for GVHD treatment. Correlative studies are planned to address pharmacodynamics of Panobinostat on inflammatory cytokines, to correlate DAC enzymatic activity inhibition and clinical response and to proteins/histones acetylation in T-cell subsets.

Disclosures:

No relevant conflicts of interest to declare.
Prospective Evaluation Of Allo-HSCT From Haploidentical-Related Donors Compared With Matched Sibling Donors and Unrelated Donors For Patients With Hematological Malignancies: Results From An Open-Label Trial
Authors: Luo Yi , Xiao Haowen , Lai Xiaoyu , Shi Jimin , Tan Yamin , He Jingsong , Xie Wanzhuo , Zheng Weiyan , Zhu Yuanyuan , Ye Xiujin , Cai Zhen , Lin Maofang , Huang He
Published: October 2019

Introduction

The order of alternative donor selection for hematopoietic stem cell transplantation (HSCT) for patients with hematologic malignancies has not been addressed. We performed the first prospective trial to compare the effect of HSCT from matched sibling donors (MSDs), unrelated donors (URDs) and haploidentical- related donors (HRDs) in a contemporary protocol.

Methods

From 2008 to 2012, 234 patients with hematologic malignancies were enrolled. The treatment schedule was as follows: if a fully MSD was available, patients were assigned treatment with MSD-HSCT. If an MSD was unavailable, a suitably matched URD was used as the alternative, where a suitable match involved matching more than 8 of 10 HLA-A, -B, -C, -DRB1and DQ allele loci ( ¡Ý 8/10) and at least 5 of 6 matching HLA-A, -B, and -DRB1 antigen loci. If only URDs with > 2 mismatching allele loci were available, patients were allowed treatment with HRD-HSCT.

Results

(1) Sixty-eight patients underwent MSD-HSCT, 98 patients underwent URD-HSCT, and 68 patients underwent HRD-HSCT (Table 1). (2) Grades II¨CIV and severe aGVHD were all significantly more frequent in patients undergoing HRD-HSCT compared with those undergoing MSD-HSCT (II¨CIV: 42.6% vs 19.1%, P = 0.0015; severe aGVHD: 17.65% vs 5.88%, P = 0.03). However, the incidences of II¨CIV and severe aGVHD were comparable in patients receiving transplants from HRDs to those from URDs (II¨CIV: 42.6% vs 40.8%, P = 0.89; severe aGVHD: 17.65% vs 13.27%, P = 0.48). The incidence of cGVHD was not significantly affected by donor types. (3) The 4-year incidence of relapse was not significantly affected by donor types according to all patients (24.2% in the MSD cohort, 22.8% in the URD cohort, 11.9% in the HRD cohort, P > 0.05). However, after controlling for high-risk patients, a superior graft-versus-leukemia (GVL) effect was observed in patients undergoing HRD-HSCT compared to MSD-HSCT or URD-HSCT. In high-risk patients receiving MSD, 36.8% experienced relapse, as did 33.6% in the URD cohort, but the incidence decreased to11.1% in the HRD cohort (MSD vs HRD, P = 0.015; URD vs HRD, P = 0 .028). (4) HRD-HSCT yielded comparable rates of 4-year overall survival (OS) and disease-free survival (DFS) to MSD-HSCT ( OS: 66.4% vs 79.5%, P = 0.071; DFS: 66.4% vs 78.2 %, P = 0.109) or URD-HSCT (OS: 66.4% vs 59%, P = 0.952; DFS: 66.4% vs 58.1%, P = 0.864) (Figure 1).

Conclusion

Our data provide convincing clinical evidence to support the use of HRDs, as well as URDs, can be selected as first-line alternative donors, especially for high-risk patients.

Disclosures:

No relevant conflicts of interest to declare.
Regulatory Role Of Mitochondrial Aconitase and Isocitrate In Iron-Responsive, Erythropoietin Receptor Signaling During Anemia
Authors: Bullock Grant C , Schrott Valerie
Published: October 2019

Several clinical observations illustrate the link between iron and erythropoietin (Epo)-mediated signaling in early erythroid progenitor cells. In iron deficiency anemia (IDA) erythropoiesis is blocked at an early stage despite increased serum Epo concentrations. Intravenous iron improves the effectiveness of exogenous Epo in patients with Epo-refractory anemia of chronic disease. These clinical observations suggest that iron dominantly regulates Epo-receptor (EpoR) signaling. However, the mechanism of this iron-mediated signaling remains unclear. We recently demonstrated that 1) the aconitases, multifunctional iron-sulfur cluster proteins that convert citrate into isocitrate are essential in the iron- Epo-signaling pathway in early erythroid progenitors, and that 2) isocitrate, the downstream product of aconitase, can enhance the effectiveness of Epo during iron deficiency in vitro and in vivo in mice with IDA and in rats with the anemia of chronic inflammation. These observations suggest that isocitrate or derivatives of isocitrate that synergize with erythropoiesis stimulating agents have important therapeutic application in the treatment of anemia. New data from my lab also shows that cellular iron restriction regulates mitochondrial oxygen consumption rates differentially over time during red blood cell differentiation, suggesting a novel link between mitochondrial function and erythropoeisis. We also see an increase in mitochondrial superoxide anion production in iron deprived erythroid progenitors. Based on these data, we hypothesize that mitochondrial aconitase is an iron sensor that integrates mitochondrial redox signaling to EpoR signaling and subsequent red blood cell production. To test this hypothesis we are investigating the iron dependent mechanisms by which aconitase, isocitrate, reactive oxygen species and mitochondrial metabolic pathways alter EpoR signaling. The clinical relevance of this project lies in its potential for the development of new iron-free agonists and antagonists of red blood cell production. Agonists may benefit patients with anemia due to iron deficiency or chronic inflammation and antagonists may benefit patients with myeloproliferative neoplasms.

Disclosures:

No relevant conflicts of interest to declare.
Initial Change Of Iron Metabolic Flux Prior To Differentiation Of Erythroid Progenitors After Epoetin Beta Pegol (C.E.R.A.) Treatment
Authors: Sasaki Yusuke , Noguchi-Sasaki Mariko , Matsuo Yukari , Fukumura Junko , Sato-Inomata Fumi , Yorozu Keigo , Shimonaka Yasushi
Published: October 2019

Introduction

Erythropoiesis and iron metabolism are inextricably linked. Developing immature erythroblasts have an extremely high iron requirement, especially during hemoglobin synthesis. Hepcidin, which inhibits iron efflux by binding to iron exporter ferroportin, is suppressed to promote the supply of required iron. Suppression of hepcidin after erythropoiesis-stimulating agent (ESA) treatment is mainly in an indirect manner, especially via erythropoietic activity, but the nature of suppressive mechanism of hepcidin is still unknown. Epoetin beta pegol (C.E.R.A.) is a novel long-acting ESA, which potentially has intensive and continuous effects on reduction of hepcidin. In the present study, we investigated change of iron metabolic flux associated with enhanced erythropoiesis by C.E.R.A. to analyze the mechanism underlying suppression of hepcidin.

Methods

Initial change of iron metabolism was analyzed in C57BL/6N mice intravenously treated with 10 μg/kg of C.E.R.A. or vehicle. Hematological indices such as reticulocyte counts and iron indices including serum hepcidin and iron levels were measured. Reticulocyte hemoglobin equivalent (Ret-He) which reflects the iron status of reticulocyte was also determined. Ter119 and transferrin receptor (CD71) expression on bone marrow cells was evaluated by flow cytometry for analysis of the maturation status of bone marrow erythroblasts.

Results

C.E.R.A. suppressed serum hepcidin levels after 9 hours, while serum iron levels were significantly decreased at 9 hours followed by recovery to the control levels at 24 hours. Ter119(+)CD71(high) immature erythroblasts were decreased and CD71 expression levels on the same cells were increased at 9 hours after C.E.R.A. treatment. C.E.R.A. elevated reticulocyte counts and Ret-He levels at 48 hours.

Discussion and Conclusion

Transient decrease in serum iron levels and continuous suppression of serum hepcidin levels were observed in early phase after C.E.R.A. treatment, prior to increase in erythroblasts through proliferation and differentiation of erythroid progenitors. Furthermore, considering decrease in immature erythroblasts followed by increase in hemoglobin-rich reticulocytes, it is possible that initial change of iron metabolic flux occurred by the accelerated iron incorporation into immature erythroblasts through CD71 recycling after C.E.R.A. treatment. These results suggest that sensing initial change of iron metabolic flux leads to suppression of hepcidin after C.E.R.A. treatment, but further analysis is needed for the mechanism of increase in iron absorption into immature erythroblasts immediately after C.E.R.A. treatment independent of differentiation of erythroid progenitors.

Disclosures:

No relevant conflicts of interest to declare.
Early Detection Of Right Ventricular Diastolic Dysfunction By Pulsed Tissue Doppler Echocardiography In Iron Loaded Beta Thalassemia Patients
Authors: El-Beshlawy Amal , Agha Hala , Hamdy Mona Mohamed , Sobeih Alaa , Alzahraah Fatma , Abdelsattar Inas , Abdelmassih Antoine , Said Fadwa , Abdelaziz Osama , Eltagui Mona , Dudley Pennell
Published: October 2019

Introduction

Iron overload contributes to cardiac dysfunction in patients with beta thalassemia. Early heart iron overload can be quantified through cardiovascular magnetic resonance (CMR) T2*.

Objective

to clarify the value of tissue Doppler imaging (TDI) in early detection of global myocardial dysfunction in iron loaded thalassemia major patients diagnosed by CMR.

Patients and Methods

Two groups were included in the study; Group I: 69 asymptomatic thalassemia patients (28 females, 41 males), mean age 18.1±7.03 years (range 6 to 39 years), Group II (n=41) healthy normal controls matched for age and sex. Serum ferritin and CMR were performed to assess the degree of cardiac siderosis (T2*< 20 ms). Group I was subdivided into two subgroups; Group Ia (n=26) T2*<20ms and Group Ib (n=43) T2* >20ms. Conventional echocardiography and Doppler of LV, RV dimensions and functions and pulmonary artery pressure were evaluated. TDI measures included systolic and diastolic myocardial velocities (S', E', A' and E'/A') of the basal segments of septal, lateral LV and RV free walls.

Results

Right ventricular diastolic function assessed by tricuspid annular E'/A' was positively correlated with T2* value; the lower tricuspid E'/A' ratio the lower T2* value (r = +0.366, P=0.002). Tricuspid annular A' was significantly higher in group Ia compared to group Ib (16.7 ±5.2cm/s versus 12.1 ±4.0cm/s, P<0.001). Tricuspid E'/A'<1 was common in group Ia compared to group Ib (19/26 (73.0%) versus 3/43 (6.97%), P=0.000). Group Ia (T2*<20ms) had high serum ferritin level compared to group Ib (T2*>20ms) (6,357±2478 microgram/L versus 2,965±2289 microgram/L (P<0.0001). By multivariate logistic regression analysis, right ventricular diastolic dysfunction (tricuspid E'/A'< 1) was associated with serum ferritin and T2* level of the thalassemic patient.

Conclusion

Pulsed tissue Doppler imaging (TDI) is a promising tool for quantitative assessment of myocardial function and early detection of right ventricular diastolic dysfunction in iron loaded beta thalassemia major patients assessed by cardiovascular magnetic resonance.

Disclosures:

No relevant conflicts of interest to declare.
Features Associated With Pulmonary Hypertension In Splenectomized Patients With Hemoglobin E/β-Thalassemia Disease
Authors: Chuncharunee Suporn , Atichartakarn Vichai , Udomsubpayakul Umaporn , Archararit Napaporn
Published: October 2019

Introduction

Pulmonary hypertension (PHT) associated with thalassemia hemoglobinopathy is now an accepted clinical entity. Due to a high prevalence of thalassemia hemoglobinpathy worldwide, it is the most common entity of PHT. Despite the commonness, its pathogenesis is not yet completely understood. Although asplenia is a known risk factor, PHT does not develop in all splenectomized patients. The present study was therefore done to search for other associated features.

Patients and Methods

Sixty-one clinically stable splenectomized hemoglobin E/β-thalassemia disease (E/β-Thal) adult outpatients, on no medication aside from folic acid and who received no blood transfusion in the preceding 4 weeks, were prospectively studied. All gave written informed consent, and study protocol was approved by the institution ethics committee on studies in humans (#0774/2548). Transthoracic echocardiogram was used to evaluate cardiac function and to estimate pulmonary artery systolic pressure (PASP). PHT was defined as an estimated PASP ≥36 mmHg. Clinical features and laboratory data were dichotomized according to the presence (PHT+) or absence (PHT-) of PHT, and statistical analysis was done by STATA version 10 (Stata Corp, Texas), considering a P value<0.05 as statistically significant.

Results

Of the 61 patients, 32 (52.5%) were PHT+, 14 of whom were female. There was no gender difference between the 2 groups (p=0.246). All had normal left ventricular ejection fraction, and none had positive serology for human immunodeficiency virus. Results expressed in mean ± SD or median (range) of the various measured parameters and their statistically significant differences are shown in the following table.

Conclusions

PHT was found in 52.5% of the 61 splenectomized E/β-Thal patients with no gender preponderance. Although PHT was found mostly after the first decade of splenectomy, its prevalence did not correlate with time elapsed since the procedure. Features associated with PHT in splenectomized E/β-Thal patients were greater transfusion requirement, lower RBC counts, higher serum lactate dehydrogenase (LDH) and lower serum cholesterol levels, as a reflection of more severe disease and more severe ineffective erythropoiesis; together with a higher serum soluble vascular cell adhesion molecule-1 (VCAM-1) levels, likely from chronic hypoxemia.

Disclosures:

No relevant conflicts of interest to declare.
Binding Of Immune Serine Proteases To Nucleic Acids Enhances Their Nuclear Localization and Promotes Their Cleavage Of Nucleic Acid-Binding Protein Substrates
Authors: Whangbo Jennifer , Thomas Marshall , McCrossan Geoffrey , Deutsch Aaron , Martinod Kimberly , Walch Michael , Lieberman Judy
Published: October 2019

When released from cytotoxic T lymphocytes and natural killer cells, Granzyme (Gzm) serine proteases induce programmed cell death of pathogen-infected cells and tumor cells. The Gzms rapidly accumulate in the target cell nucleus by an unknown mechanism. Many of the known substrates of GzmA and GzmB, the most abundant killer cell proteases, bind to DNA or RNA. Gzm substrates predicted by unbiased proteomics studies are also highly enriched for nucleic acid binding proteins. Here we show by fluorescence polarization assays that Gzms bind DNA and RNA with nanomolar affinity. We hypothesized that Gzm binding to nucleic acids enhances nuclear accumulation in target cells and facilitates their cleavage of nucleic acid-binding substrates. In fact, RNase treatment of cell lysates reduced cleavage of RNA binding protein (RBP) targets by GzmA and GzmB. Moreover, adding RNA to recombinant RBP substrates greatly enhanced in vitro cleavage by GzmB, but adding RNA to non-nucleic acid binding proteins did not. For example, exogenous RNA enhanced GzmB cleavage of recombinant hnRNP C1 (an RBP) but not LMNB1 (a non-RBP). In addition, GzmB cleaved the RNA-binding HuR protein efficiently only when it was bound to an HuR-binding RNA oligonucleotide, but not in the presence of an equal amount of non-binding RNA. Thus, nucleic acids facilitate Gzm cleavage of nucleic acid binding substrates.

To evaluate whether nucleic acid binding influences Gzm trafficking in target cells, we incubated fixed target cells with RNase and then added Gzms. RNA degradation in target cells reduced Gzm cytosolic localization and increased nuclear accumulation. Similarly, pre-incubating Gzms with exogenous competitor DNA reduced Gzm nuclear localization. The Gzms form a monophyletic clade with other immune serine proteases including neutrophil elastase (NE) and cathepsin G (CATG). Upon neutrophil activation, NE translocates to the nucleus to drive the formation of neutrophil extracellular traps (NETs). NE and CATG, but not non-immune serine proteases such as trypsin and pancreatic elastase, also bind DNA with high affinity and localize to the nucleus of permeabilized cells. Consistent with this finding, competitor DNA also blocks the nuclear localization of NE. Moreover NE and CATG localization to NETs depends on DNA binding. Thus the antimicrobial activity of NETs may depend in part upon the affinity of these proteases for DNA. Our findings indicate that high affinity nucleic acid binding is a conserved and functionally important property of serine proteases involved in cell-mediated immunity.

Disclosures:

Lieberman: Alnylam Pharmaceuticals: Membership on an entity’s Board of Directors or advisory committees.
CD8+ Treg - From Mouse To Man
Authors: Holderried Tobias A.W. , Kim Hye-Jung , Lang Philipp A , Cantor Harvey
Published: October 2019

T cell-mediated regulation of the immune response to self and foreign antigens is essential to maintain immune homeostasis and prevent autoimmune tissue destruction. The majority of studies addressing this issue, both in mice and humans, have focused on the contribution of CD4+CD25+FOXP3+ regulatory T cells. The contribution of regulatory CD8+ T cells has not been appreciated until recently. Recent studies have identified a small subset of IL-15 dependent CD8+ regulatory T cells (Treg) that is essential for maintenance of self- tolerance and prevention of autoimmune disease in mice. Expression of a triad of cell surface markers – CD44+CD122+Ly49+ – has been used to distinguish and purify CD8+Treg (Kim et al., Nature 2010; Kim et al., PNAS 2011).

Here we have defined the human homologue of CD8+ Treg. The Ly49 receptor, identified as a stable surface marker on CD8+ Treg, is a member of a multigenic/multiallelic receptor family recognizing classical MHC class I molecules. The functional homologue of murine Ly49 is the killer cell immunoglobulin-like receptor (KIR). Our analysis has revealed that, while expression of KIR subtype combinations appears to be stochastic and co-expression of these KIR receptors is random, the inhibitory KIR2DL2/3 and KIR3DL1 subtypes are dominantly expressed by human CD3+CD8+ T cells. Similar to murine CD8+ Treg, CD8+ T cells that express KIR2DL2/3 or KIR3DL1 also express CD44 and CD122. Moreover, consistent with murine CD8+ Treg, incubation with IL-15 results in activation and proliferation of KIR+CD8+ T cells that maintain a stable surface phenotype. Gene array analyses in mice has indicated that Helios, a highly conserved zinc finger transcription factor and member of the Ikaros family of transcription factors, is expressed by CD44+CD122+Ly49+ CD8+ Treg. Helios is involved in T cell development and expressed by ∼70% of FoxP3+CD4+ Treg but not by mature B cells, dendritic cells or myeloid cells. Our analyses identified a Helios+ subset in the CD8+ Treg population, which (compared to the Helios- subset) embodies many of the functional characteristics of CD8+ Treg in mice. Co-expression of Helios is also apparent in some KIR+CD8+ T cells in human samples. In mice, we have shown that CD8+ Treg target CD4 TFH cells and thus maintain self-tolerance. In vitro suppression assays revealed that KIR+CD8+ cells but not KIR–CD8+ cells confer inhibitory activity on CD4+CXCR5+ TFHtarget cells in humans.

Taken together, our findings implicate KIR+CD8+ cells as the human homologue of murine CD8+ Treg, including expression of transcription factor Helios, responsiveness to IL-15 and suppression of CD4+ TFH cells. Understanding the genetic and biological features of this CD8+ T cell subset in humans opens the possibility of exploiting their regulatory activity for the development of immunotherapy in the context of autoimmune disease and cancer.

Disclosures:

No relevant conflicts of interest to declare.
Methylprednisolone Versus Intravenous Immunoglobulin As Initial Therapy In Adult Immune Thrombocytopenia
Authors: Song Ik-Chan , Jo Deog-Yeon , Choi Yoon Seok , Lee Hyo-Jin , Yun Hwan-Jung , Kim Samyong
Published: October 2019

Background

Both intravenous methylprednisolone (methyl-Pd) and intravenous immunoglobulin (IVIg) have been used as standard initial therapies in adult patients with immune thrombocytopenia (ITP). Nonetheless, few studies have addressed whether there are differences in clinical efficacy between these two treatments. In this study, we compared platelet responses, toxicities, and bleeding-related mortality associated with these two treatments in adult ITP.

Patients and Methods

Adult patients newly diagnosed with ITP, with platelet counts of less than 20,000/mL, were prospectively enrolled. Patients with comorbidities such as diabetes and uncontrolled cardiovascular disorders and who were pregnant were excluded. Patients who entered the study between 1993 and 2002 received intravenous methyl-Pd therapy (10 mg/kg/day for 3 days) followed by oral Pd (1 mg/kg/day). Patients who entered the study between 2003 and 2008 received IVIg (400 mg/kg/day for 5 days) together with oral Pd (1 mg/kg/day). After 6 weeks, the prednisolone began to be tapered in both arms. Early response and maintenance of response were assessed at 7 days and 6 months after treatment, respectively, based on the outcome criteria proposed by an international working group (Blood 2009).

Results

Of 87 patients enrolled, 77 (88.5%) were eligible for analysis. Initial platelet counts did not differ between the two groups. Early responses occurred in 30 of 39 patients (76.9%) receiving methyl-Pd versus 33 of 38 patients (86.6%) receiving IVIg (p = 0.187). Maintenance of response was observed in 28 patients (71.8%) in the methyl-Pd arm and 23 patients (60.5%) in the IVIg arm (p = 0.187). The time to a complete response in the IVIg arm (6 days; range, 1–35 days) was shorter than that in the methyl-Pd arm (13.5 days; range, 2–29 days) (p = 0.002). Side effects were mild and tolerable in both arms. Five years after treatment initiation, 5 patients (12.8%) and 7 patients (18.4%) were still under salvage treatment in the methyl-Pd and IVIg arms, respectively. During a median follow-up of 98 months (range, 33-228 months) in the methyl-Pd arm and 71 months (range, 52-180 months) in the IVIg arm, 19 patients (48.7%) and 18 patients (47.4%) patients required one or more salvage treatments, respectively (p = 0.906). No bleeding-related death was observed in either group during the follow-up period.

Conclusion

These results indicate that neither the early response rate nor the long-term outcome differs between methyl-Pd and IVIg treatments. IVIg, however, more rapidly induces a complete response than methyl-Pd.

Disclosures:

No relevant conflicts of interest to declare.
A Carboxy-Terminally Truncated G-CSF Receptor Abolishes Apoptosis Induction By Neutrophil Elastase G185R Mutant In Myeloid Cells,
Authors: Dong Fan , Qiu Yaling , Hu Nan , Friedman Alan D.
Published: October 2019

Mutations in ELANE encoding neutrophil elastase (NE) have been identified in the majority of patients with severe congenital neutropenia (SCN). SCN patients are predisposed to acute myeloid leukemia (AML), and progression from SCN to AML is accompanied by acquisition of mutations in CSF3R encoding the G-CSFR receptor (G-CSFR) in approximately 80% of the patients. The mutations, which cause receptor’s carboxy-terminal truncation, are highly specific for SCN/AML, but the reason for the prevalence of the CSF3R mutations in patients with SCN/AML is unknown. We show here that NE expression was induced by G-CSF in myeloid 32D cells expressing the wild type (WT) G-CSFR (32D/GR), but not in cells expressing an SCN-derived truncated G-CSFR, d715 (32D/d715). Unlike the WT G-CSFR, the d715 receptor was unable to activate a 1.8-kb promoter fragment of mouse Elane. The NE mutants associated with SCN have been shown to cause premature apoptosis of differentiating granulocytic precursors. To address whether the d715 receptor might abolish apoptosis induction by the NE mutants in part by suppressing their expression, we placed the cDNA encoding the SCN-derived NE G185R mutant under the control of the Elane promoter and the resultant expression construct was transfected into 32D/GR and 32D/d715 cells. G-CSF treatment resulted in increased expression of G185R mutant and premature apoptosis in 32D/GR cells, but had no effect on G185R expression and cell survival in 32D/d715 cells. We further addressed whether the d715 receptor was capable of directly suppressing apoptosis induced by the G185R mutant. As expected, forced expression of the G185R mutant in 32D/GR cells led to accelerated apoptosis in response to G-CSF, but did not negatively influence the viability of 32D/d715 cells cultured in G-CSF. To evaluate whether the d715 receptor inhibited apoptosis induction by the G185R mutant in other hematopoietic cells, we expressed the G185R mutant in murine multipotent FDCP-mix A4 cells transfected with WT G-CSFR (FDCP/GR) or d715 receptor (FDCP/d715). Although FDCP/GR cells expressing G185R mutant showed no significant increase in apoptotic cell death upon treatment with G-CSF, they exhibited increased sensitivity to tunicamycin-induced endoplasmic reticulum (ER) stress. In contrast, the G185R mutant failed to sensitize FDCP/d715 cells to apoptosis induction by tunicamycin. Thus, it appears that the d715 receptor may abolish G185R mutant-induced apoptosis by two distinct mechanisms, i.e., suppression of its expression and direct inhibition of its pro-apoptotic action. Our data may have implications for understanding the prevalence of CSF3R mutations in patients with SCN/AML. We propose that the CSF3R mutations might represent a mechanism by which myeloid cells carrying the ELANE mutations evade the pro-apoptotic effect of the NE mutants in SCN patients.

Disclosures:

No relevant conflicts of interest to declare.
Caveolin-1 Is Required For T Cell-Mediated Acute GvHD
Authors: Wohlfeil Sebastian , Anne-Kathrin Hechinger , Leonhardt Franziska , Prinz Gabriele , Schmitt-Gräff Annette , Minguet Susana , Zeiser Robert
Published: October 2019

Caveolin-1 (Cav-1) is a key organizer of membrane specializations that coordinate membrane and protein traffic in different cells including T cells. Cav-1 promotes the formation and stability of plasma membrane raft microdomains that serve as platforms for signal transduction and binds a wide array of signal transducers through interactions with its phosphorylated tyrosine 14 or the so-called scaffolding domain. Several of the proteins identified as Cav-1 binding partners have been suggested to play a role in TCR-regulated membrane dynamics and intracellular signaling. Therefore, we aimed to determine the requirement of Cav-1 for T cells during acute graft-versus-host disease (GvHD).

We observed upregulation of Cav-1 in the T cells of mice developing acute GvHD which was dependent on both tissue damage and homeostatic signals as investigated in conditioned WT recipients or RAG-/-C gamma chain-/- non-conditioned mice. By using gene targeted mice we found that Cav-1 deficiency of the donor led to improved survival (p=0.02), lower GvHD histopathology scores (p=0.004) and reduced serum levels of IL-6 (p=0.02) while Treg numbers increased when Cav-1 deficient donors were used. Comparative microarray analysis of WT compared to Cav-1 deficient T cells indicated a reduced IL-17A production in Cav-1 T cells exposed to allogeneic dendritic cells. These observations were confirmed on the protein level.

In summary we show a role for Cav-1 in T cells during GvHD as an alloantigen driven T cell response. Regarding the mechanism we describe the relevance of Cav-1 for Th-17 lineage commitment of alloreactive T cells during GvHD.

Disclosures:

No relevant conflicts of interest to declare.
TLR7 Ligand Potentiates GM-CSF-Initiated Antitumor Immunity Through Activation Of Plasmacytoid Dendritic Cells
Authors: Narusawa Megumi , Inoue Hiroyuki , Sakamoto Chika , Matsumura Yumiko , Takahashi Atsushi , Watanabe Ayumi , Inoue Tomoko , Miyamoto Shohei , Takayama Koichi , Inoue Makoto , Hasegawa Mamoru , Nakanishi Yoichi , Tani Kenzaburo
Published: October 2019

Vaccination with irradiated granulocyte macrophage-colony stimulating factor (GM-CSF)-transduced autologous tumor cells (GVAX) has been shown to induce therapeutic antitumor immunity presumably through the activated maturation of myeloid dendritic cells (DCs). However, its effectiveness is limited, and little is known about the biological properties related to GM-CSF-sensitized DCs (GM-DCs) in tumor-draining lymph nodes (TDLNs). We therefore attempted to enhance the antitumor effect of GVAX therapy by identifying the key pathways in GM-DCs in TDLNs. We initially confirmed that syngeneic mice subcutaneously (s.c.) injected with poorly immunogenic Lewis lung carcinoma (LLC) cells transduced with Sendai virus encoding GM-CSF (LLC/SeV/GM) significantly rejected the tumor growth. Using cDNA microarrays, we obtained a large number of gene expression data from CD86+ GM-DCs and control DCs in TDLNs, and found that the expressions of type I interferons (IFNs)-related genes, including IRF7 and TLR7, known to be abundantly expressed in plasmacytoid DCs (pDCs), were upregulated in GM-DCs. Indeed, to further activate pDCs, mice s.c. challenged with LLC/SeV/GM cells in combination with a TLR7 ligand, imiquimod, but not a TLR4 ligand, LPS, significantly suppressed the tumor growth compared with mice treated with LLC/SeV/GM cells alone. In contrast, pDCs-depleted mice challenged with LLC/SeV/GM cells facilitated the tumor growth, strongly suggesting that pDCs are essential immune subpopulation in exerting GM-CSF-initiated antitumor effects.

Furthermore, the additional use of imiquimod overcame the refractoriness of therapeutic vaccines with irradiated LLC/SeV/GM cells in mice with pre-established LLC tumors. Moreover, similar improvement of GVAX therapy was also observed in a mouse model of CT26 colorectal cancer. Mechanistically, mice treated with the combined vaccination displayed increased cell ratio of PDCA-1+ pDCs and expression levels of CD86, CD9, which correlate with an antitumor phonetype, and Siglec-H, which promote CD8+ T cell proliferation, in TDLNs. Indeed, allogeneic MLR test showed that bone marrow-derived pDCs matured by in vitro culture with GM-CSF plus imiquimod elicited a superior capacity of CD8+ T cell proliferation compared with those with GM-CSF only. On the other hand, the ratio of CD4+CD25+FoxP3+ regulatory T cells (Tregs) was decreased in TDLNs mice treated with the combined vaccination.

These findings collectively elucidated that pDCs play positive roles in GM-CSF-initiated antitumor immunity and that further activation of pDC by imiquimod targeting TLR7-IRF7 dependent type I IFNs pathway enhance the antitumor effects of GM-CSF-based tumor vaccination.

Disclosures:

No relevant conflicts of interest to declare.