bluuebunny/crossref_metadata_2025_split huggingface dataset:
Myeloid Derived Suppressor Cells (MDSCs) Are Increased and Exert Immunosuppressive Activity In CML Patients At Diagnosis
Authors: Giallongo Cesarina , Parrinello Nunziatina , Tibullo Daniele , La Cava Piera , Romano Alessandra , Chiarenza Annalisa , Stagno Fabio , Vigneri Paolo G. , Palumbo Giuseppe A. , Raimondo Francesco Di 
Published: October 2019

Abstract

Introduction
In some solid tumors it has been demonstrated that a subpopulation of myeloid cells, defined as “myeloid-derived suppressor cells” (MDSCs), plays an important role in inducing T cell tolerance by production of arginase 1 (arg1) that depletes microenvironment of arginine, an essential aminoacid for T cell function. Since chronic myeloid leukemia (CML) patients have high levels of immature myeloid cells it is of interest to investigate if these cells have MDSCs phenotype and activity. The aim of this study was to analyze MDSCs and investigate their activity in CML patients.

Methods
MDSCs were analyzed in peripheral blood (PB) of 20 healthy donors (HD) and 30 CML patients at diagnosis. In 21 patients MDSCs were also measured during TKI treatment. Granulocytic MDSCs (G-MDSCs) were identified as CD11b+CD33+CD14-HLADR- cells, while the monocytic MDSCs (Mo-MDSCs) as CD14+HLADR by cytofluorimetric analysis. Arg1 expression was assessed using real time PCR and Western Blot. Arg activity was measured in granulocyte lysates using a colorimetric test after enzymatic activation and arginine hydrolysis. Microvesicles (MV) were isolated from CML serum at diagnosis (n=5) by sequential ultracentrifugation.

Results
CML patients showed high levels of Mo- and G-MDSCs at diagnosis in comparison to HD (41±8 and 82,5±12,2% respectively for CML vs 9±2,1 and 55±5,3% for HD; p<0.001), while after TKIs therapy both subpopulations decreased, returning to normal values. T-reg (CD4+ CD25high Foxp3+ cells) were also significantly increased in CML patients at diagnosis in respect to HD (9±2% vs 6,1±0,8%, p<0.001) with a significant correlation with the percentage of Gr-MDSCs (r=0,6254; p<0.001). Both in PB and purified granulocytic cells, Arg1 expression showed a 30 fold increase in CML at diagnosis compared to HD (p<0.001) and decreased after therapy. The same data were confirmed by Western Blot analysis. Arg enzymatic activity in granulocytes resulted also increased in CML (n=10) compared to HD (n=10) (p<0.001). The suppressive function of CML G-MDSCs was demonstrated by their ability to inhibit the proliferation of CFSE+ HD T cells (p<0.001). In addition, an increase of Mo-MDSCs in vitro was observed after incubation of HD monocytes with CML sera (29±13%; p<0.0001) or MV (8±2,8%; p<0.05).

Conclusions
MDSCs are increased in CML patients at diagnosis and decrease during TKIs treatment. CML granulocytes have high arg1 activity and immunosuppressive activity. Moreover, CML serum as well as CML microvesicles increase the percentage of HD Mo-MDSCs.

Disclosures:
No relevant conflicts of interest to declare.
Dysregulation Of Bcl2 Family Proteins Induced By JAK2V617F Mutation Contributes To The Abnormal Expansion Of Neoplastic Initiating Cells
Authors: Figueiredo-Pontes Lorena Lobo , Welner Robert S , Mishima Yuta , Yamamoto Mihoko , Mullally Ann , Tenen Daniel G. , Kobayashi Susumu 
Published: October 2019

Abstract
The treatment of myeloproliferative neoplasms (MPN) harboring JAK2V617F mutation remains non-curative with the current strategies despite the development of JAK2 inhibitors, which improve the symptoms of the disease but do not effectively reduce the allele burden of the mutation. In addition, it has been shown that, although JAK2 inhibitors can control the expansion of the myeloerythroid precursors that contribute to the clinical phenotype, they fail to eliminate the most primitive stem cells, which are responsible for the initiation of the disease. The differential expression of genes/proteins between the hematopoietic stem cells (HSC) and the more committed bone marrow progenitors may reveal, at least in part, the contribution of each cell type to the pathogenesis of the disease. We have shown that JAK2 inhibition induces down-regulation of the anti-apoptotic protein Bcl-xL and up-regulation of the pro-apoptotic BH3-only protein, Bim, in human cells harboring the JAK2V617F mutation, suggesting a key role of these Bcl-2 family proteins as potential targets for JAK2-mediated apoptosis. Therefore, we aimed to study the contribution of the Bcl-2 family proteins to MPNs by the use of a conditional JAK2V617F knock-in model, particularly focusing on MPN initiating cells. The heterozygous Jak2V617F expressing animals (Jak2+/VF) develop a lethal MPN characterized by an elevated hematocrit and splenomegaly due to extramedullary hematopoiesis, with a prominent expansion of early (Lin- CD71+ Ter119+) and late (Lin- CD71- Ter119+) erythroid cells in the spleen, and increased pre-megakaryocyte-erythrocyte (Lin- Sca1- cKIThi CD41- FcgRII/III- CD105- CD150+), megakaryocyte (Lin- Sca1- cKIThi CD41+ CD150+) and erythroid (Lin- Sca1- cKIThi CD41- FcgRII/III- CD105+ CD150+) precursors in the bone marrow. HSC numbers (Lin- Sca1- cKIThi CD105+ CD150+) are also increased in Jak2+/VF as compared to the Jak2+/+ controls. Gene expression analysis revealed that Bcl-xL was upregulated in sorted LSKs (Lin- Sca-1+ cKIThi) containing HSCs but not in the more committed progenitors (CMP, GMP, MEP). By contrast, the pro-apoptotic Bim was downregulated in CMPs and MEPs but not at the LSK cells containing HSCs. Expression of Bcl-2 showed no difference between Jak2+/VF and Jak2+/+ mice. These data suggest that signals from JAK2V617F mutation may be cell-stage specific, which may explain why JAK2 inhibitors fail to eliminate MPN initiating cells. Modulating the overexpression of Bcl2 antiapoptotic proteins at the MPN initiating cells level could potentially render these cells sensitive to the JAK2 inhibition.

Disclosures:
No relevant conflicts of interest to declare.
Generation Of FANCA-/- Human CD34+ Hematopoietic Stem Cells By shRNA Knockdown
Authors: Sun Zejin , Enzor Rikki , Rio Paula , Clapp D. Wade , Hanenberg Helmut 
Published: October 2019

Abstract
Fanconi anemia (FA) is a recessive DNA repair disorder characterized by bone marrow (BM) failure, genomic instability, and a predisposition to malignancies. Natural gene therapy due to molecular self-correction of hematopoietic stem cells (HSCs) has been reported in a minority of FA patients, suggesting that due to the in vivo selection advantage of the corrected cells, FA is an excellent model disease for stem cell gene therapy. However, the scarcity of autologous HSCs from FA patients for research purposes is one of the major road blocks to preclinical studies with human cells. Here, we developed a lentiviral vector with EGFP as marker gene that co-expresses two distinct shRNA sequences against FANCA under two different human promoters (H1 and U6). In vitro analysis in primary human fibroblasts showed that stable integration of this construct was highly efficient to induce the typical FA cellular phenotypes as assessed by (1) FANCD2 ubiquitination deficiency and (2) a characteristic G2/M arrest upon DNA damage induced by DNA crosslinking reagent Mitomycin C (MMC). We then transduced human cord blood (CB) CD34+ cells with this lentiviral vector and demonstrated a reduced survival of clonogenic cells in progenitor assays at 20nM MMC: 70% (scrambled control shRNA) vs. 23% (FANCA shRNA). This vector pseudotyped with a foamyviral envelope was then used to transduce CD34+ CB cells on fibronectin CH296. The next day, genetically modified cells were transplanted into NOD.Cg---Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. When analyzing the percentage of EGFP+ cells in the human graft (hCD45+ cells), we noticed a progressive decline of EGFP+ cells from 29% on day 5 to 5% at 4 months after transplantation in the peripheral blood of the recipient mice, mimicking the progressive BM failure in FA patients. In contrast, engraftment over time was stable in CD34+ cells transduced with scrambled control shRNA vector (33% on day 5 vs. 34% at 4 months). The human progenitors isolated from the BM of NSG recipient mice at sacrifice 4 months after initial transduction and transplantation are still hypersensitive to MMC, with a much lower survival rate of 34% at 20nM MMC in the FANCA shRNA group as compared to 78% in the scrambled control shRNA group, thus confirming the knockdown by the lentiviral shRNA construct is stable. In summary, the novel double shRNA lentiviral vector is capable of inducing all major hallmarks of FA cells in normal human CB CD34+ cells, thus providing unlimited FA-like cellular materials including NSG mice-repopulating HSCs for preclinical gene therapy and basic stem cell biology research in FA.

Disclosures:
No relevant conflicts of interest to declare.
Smac Mimetic-Mediated Sensitization Of Childhood ALL Cells For Drug-Induced Apoptosis Independent Of TNFα and NFκB Signaling Pathways
Authors: Schmelz Karin , Weichert Nina , Proba Jutta , Erdmann Marie-Sophie , Hundsdoerfer Patrick 
Published: October 2019

Abstract
Targeting inhibitor of apoptosis proteins (IAPs) using small molecular Smac mimetics (SM) has been shown to offer a novel promising treatment strategy for resistant malignant diseases including childhood acute lymphoblastic leukemia (ALL). The effect of SM alone has been shown to be associated with endogenous TNFα expression, therefore tumor cells can be classified into sensitive or resistant against apoptosis induction by SM alone. In SM sensitive tumor cells the effect of SM has been shown to be mediated mainly by degradation of cellular IAP (cIAP) and activation of TNFα and NFκB signaling pathways but not inhibition of XIAP.
We show here, that sensitivity of ALL cells to SM alone (as well as TNFα expression) is highly variable. Nevertheless even in ALL cells resistant against SM alone, treatment with SM resulted in significant sensitization for drugs used within standard induction therapy for childhood ALL. Sensitization for drug-induced apoptosis by SM was not only mediated by activation of the intrinsic (cleavage of caspase 9) but also extrinsic apoptosis pathway (cleavage of caspase 8). Surprisingly, SM-induced cIAP degradation alone was not sufficient for caspase 8 activation and apoptosis induction. Consistently, SM-mediated sensitization for drug-induced apoptosis was independent of TNFα and NFκB signaling pathways. We demonstrate that caspase 8 activation by combined treatment with SM and cytostatic drugs is blocked by inhibition of caspase 3 and caspase 9 and therefore occurs downstream of intrinsic apoptosis pathway activation.
In conclusion, our data argue for a model comprising inhibition of XIAP-mediated blockade of caspase 3/9 as the central effect of SM in chemo-sensitization of childhood ALL cells resistant against SM-alone.

Disclosures:
No relevant conflicts of interest to declare.
Impact Of Allogeneic Stem Cell Transplantation On The Health and Economic Burden Of Sickle Cell Disease In Children
Authors: Dalal Jignesh , Lee Brian R , Hall Matt , Woods Gerald M , Kalpatthi Ram 
Published: October 2019

Abstract

Background
Hospitalization is the major driver of health care utilization and costs in sickle cell disease (SCD) [Kauf et al. Am J Hematol 2009]. Allogeneic stem cell transplantation (alloSCT) is the only curative therapy available for sickle cell disease. However, it is a highly specialized, resource intense and expensive medical procedure. The objective of our study is to analyze the impact of alloSCT on the hospitalization patterns and resource utilization in children with SCD.

Methods
We used the Pediatric Health Information System (PHIS), a database of clinical and financial data from free-standing children's hospitals in the US. Using ICD-9 procedure and diagnosis codes, patients under the age of 21 who underwent alloSCT at one of the 26 PHIS hospitals from 2000-2011 were identified. We abstracted data on hospitalizations, alloSCT, resource utilization and costs. Pre and post alloSCT hospitalizations were calculated from day 0 (actual alloSCT procedure date) and does not include initial transplant hospitalization. The PHIS database provides an encrypted patient medical record number, thus we were able to follow patients over time. This allowed for a better visualization of the patient's hospitalizations trend over 11 years.

Results
From 2000 to 2011, 186 unique pediatric patients with SCD who underwent alloSCT were identified. The median hospital length of stay (LOS) and the median total hospital charges for the initial BMT hospitalization were 38 days (31, 51) and $143,000 ($109,000, $123,000) respectively. The mean hospitalization rate was significantly higher one year after alloSCT compared to one year before the transplantation (5.8 vs. 3.1 respectively, p=0.01). However, when we expanded the analysis over 2 years, there was no significant difference in the hospitalizations between the pre and post alloSCT groups (4.8 vs. 6.8 respectively, p=0.2). Similarly, the median total hospital charges per patient were significantly higher 1 year post-alloSCT compared to one year pre-alloSCT (Table 1) whereas it was not significant when analyzed 2 years pre/post-alloSCT (Table 2). In addition, both the hospitalization rate and costs decrease consistently for several years post alloSCT suggesting a long term benefit (Figure 1A & 1B).

Conclusions
AlloSCT for SCD increases the hospitalizations and costs in the first post transplantation year. This is consistent with the previous report of increased hospitalizations in the immediate post-transplant period of alloSCT for all pediatric disorders (Shulman et al. Pediatr Blood Cancer 2013). However, over the long term, alloSCT likely improves the quality of life (by decreasing the hospitalization rate) and economic burden of pediatric sickle cell population. Further studies associating various clinical morbidities (pre & post-transplant) and healthcare utilization are needed.

Disclosures:
No relevant conflicts of interest to declare.
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

Abstract

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.
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

Abstract
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

Abstract

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.