bluuebunny/crossref_metadata_2025 huggingface dataset:
Hypoxia-Inducible Factor (HIF)-1a Is A Therapeutic Target in Myeloma-Induced Angiogenesis and Bone Destruction in Vivo.
Authors: Bolzoni Marina , Storti Paola , Donofrio Gaetano , Airoldi Irma , Guasco Daniela , Toscani Denise , Peronaci Marco , Martella Eugenia , Lazzaretti Mirca , Agnelli Luca , Patrene Ken , Mancini Cristina , Amiot Martine , Neri Antonino , Colla Simona , Anderson Judith , Aversa Franco , Roodman G. David , Giuliani Nicola
Published: October 2019

Abstract 2947

It has been previously reported that bone marrow (BM) microenvironment is hypoxic in multiple myeloma (MM) patients and that hypoxia inducible factor (HIF)-1α is overexpressed by MM cells. However, the potential role of HIF-1α as a therapeutic target in MM is not known and is currently under investigation.

In this study we explored the effect of persistent HIF-1α inhibition by expression of a lentivirus shRNA pool in MM cells on proliferation, survival and transcriptional and pro-angiogenic profiles of MM cells either in vitro or in vivo in mouse models. A HIF-1α Lentivirus shRNA pool was used for HIF-1α stable knock-down in human myeloma cell lines (HMCL)s and the pKLO.1 lentiviral vector was used as the empty control vector. HMCLs were infected and then selected with puromycin. Selected clones were screened for HIF-1α, HIF-1β, HIF-2α and HIF-3α. The transcriptional profiles were evaluated in the HMCL JJN3 cells transduced with shRNA forHIF-1α (JJN3-anti-HIF-1α) and on those infected with the control vector pKLO.1 (JJN3-pKLO.1) by U133 Plus2.0 Arrays (Affymetrix®) either in hypoxic or normoxic conditions. Microarray data were further validated by quantitative real time PCR and by ELISA assays for protein levels. Finally the effect of HIF-1α inhibition in MM cells was assessed in vivo in NOD/SCID mice both in subcutaneous and intratibial models. Together with tumor volume and weight, microvascular density was evaluated by CD34 immunostaining. Cortical bone thickness was determined by microQcT in the intratibial mouse model.

Among the genes significantly modulated by HIF-1α inhibition (327 and 361 genes in hypoxic and normoxic condition, respectively), we found that the pro-angiogenic molecules VEGF, IL8, IL10, CCL2, CCL5, MMP9 were down-regulated by HIF-1α inhibition. Interestingly some pro-osteoclastogenic cytokines were also inhibited including IL-7 and CCL3/MIP-1α. In the in vivo mouse models, we found that mice, injected either subcutaneously or intratibially with JJN3-anti-HIF-1α, showed a dramatic reduction in the weight and volume of the tumor burden compared to mice inoculated with the JJN3-pKLO.1. A significant reduction in the number of vessels X field and VEGF immunostaining were observed in both mouse models. Moreover in the intratibial experiments HIF-1α inhibition significantly blocked MM-induced bone destruction.

Overall our data indicate that HIF-1α suppression in myeloma cells significantly blocks MM-induced angiogenesis and reduces the MM tumor burden and bone destruction in vivo suggesting that HIF-1α is a potential therapeutic target in MM.

Disclosures:

No relevant conflicts of interest to declare.
Long-Term Tolerance to Kidney Allografts After Induced Rejection of Donor Hematopoietic Chimerism in a Preclinical Canine Model.
Authors: Mathes David , Graves Scott Stoll , Georges George E. , Kuhr Christian , Chang Jeff , Butts Tiffany , Storb Rainer
Published: October 2019

Abstract 2991

Allogeneic hematopoietic cell transplantation provides a reliable method for inducing tolerance towards solid organ grafts. However, this procedure can result in graft-versus-host disease (GVHD) thereby limiting its application. Here we test the hypothesis that mixed chimerism can be intentionally reverted to host hematopoiesis without rejection of a kidney graft. Recipient dogs were given 2 Gy total body irradiation (TBI) before and a short course of immunosuppression after marrow infusion from dog leukocyte antigen-identical littermates. All dogs achieved stable mixed chimerism. After a mean of 20 weeks, one cohort of dogs received kidney transplants from their respective marrow donors. Subsequently, recipients were reconditioned with 2 Gy TBI and given autologous granulocyte-colony stimulating factor-mobilized leukocytes (recipient leukocyte infusion) that had been collected before marrow transplant. Dogs receiving a second TBI and recipient leukocyte infusion without a kidney transplant rejected their donor hematopoietic graft within 3 weeks. Dogs that received kidney grafts, followed by a second TBI and recipient leukocyte infusion, rejected their marrow graft without rejecting their transplanted kidneys for periods greater than one year. Mixed chimerism may be clinically reverted to 100% recipient without rejection of a kidney allograft. This model has potential applications in understanding the mechanism of split tolerance. This finding may have application towards minimizing the risk of GVHD in solid organ transplant patients given hematopoietic cell transplantation from HLA-identical donors.

Disclosures:

No relevant conflicts of interest to declare.
Dynamic Changes and Impact of Myeloid Derived Suppressor Cells in Allogeneic Bone Marrow Transplantation in Mice.
Authors: Wang Dapeng , Yu Yu , Semple Kenrick M , Haarberg Kelley M.K. , Fu Jianing , Kaosaard Kane , Anasetti Claudio , Srinivas Nagaraj , Gabrilovich Dmitry , Yu Xue-Zhong
Published: October 2019

Abstract 2999

Myeloid-derived suppressor cells (MDSCs) are a group of myeloid cells comprised of hematopoietic progenitor cells and immature macrophages, dendritic cells and granulocytes. MDSCs accumulate in inflammatory diseases and various cancers. In this study, we investigated the dynamic changes and effects of MDSCs in graft-versus-host disease (GVHD) development and/or tumor relapse after allogeneic bone marrow transplantation (BMT). Using murine models of syngeneic and allogeneic BMT, we found that MDSCs transiently accumulated in the blood and spleen of recipients, but returned to the physiological levels shortly after BMT without GVHD. On the contrary, the levels of blood MDSCs always elevated after BMT with GVHD in the recipients of allogeneic BM+T cells. The MDSC accumulation was positively related with the severity of GVHD. In addition, MDSC accumulation was further increased upon tumor relapse. Although MDSCs isolated from both syngeneic and allogeneic BMT recipients inhibited T-cell proliferation under allogeneic stimulation ex vivo, MSDCs from GVHD recipients were significantly much suppressive compared to recipients without GVHD. Moreover, adding functional MDSCs in donor graft alleviated GVHD, whereas partial depletion of MDSCs in vivo using all-trans retinoic acid exacerbated GVHD(Figure 1A and B). These results indicate MDSCs may serve as a biomarker for acute GVHD and tumor relapse after allogeneic BMT. The accumulated MDSCs are not sufficient to completely prevent GVHD although they do ameliorate GVHD. Hence, manipulating MDSCs could be implicated in allogeneic BMT for controlling GVHD or tumor relapse.

Figure 1 Figure 1.

Disclosures:

No relevant conflicts of interest to declare.
PI3K-Independent Signaling Pathways Contribute to ICOS-Mediated T-Cell Costimulation in Acute Graft-Versus-Host Disease in Mice.
Authors: Li Jun , Leconte Julie , Semple Kenrick , Heinrichs Jessica , Anasetti Claudio , Suh Woong-Kyung , Yu Xue-Zhong
Published: October 2019

Abstract 3003

ICOS provides an important costimulation to promote T-cell activation and function. Using a knock-in mouse strain, termed ICOS-YF, in which the cytoplasmic tail of ICOS cannot activate phosphoinositide 3-kinase (PI3K), we have shown that ICOS-PI3K signaling axis is critical for the generation of follicular helper T cells. We also observed that, in both CD4+ and CD8+ T cells, ICOS could potentiate TCR-mediated calcium flux in a PI3K-independent manner in vitro. Although ICOS can potentiate TCR-mediated calcium flux independent of PI3K, its biological significance is unclear. To address this question, we studied the function of ICOS-YF T cells in comparison with ICOS wild-type (WT) and knock-out (KO) T cells in MHC-mismatched bone marrow transplantation (BMT) models. Severity of acute graft-versus-host disease (GVHD) was evaluated based on recipient survival, body weight change, and pathologic scores. Consistent with the data previously published by us and others, ICOS KO T cells had significantly reduced ability to cause acute GVHD as compared to WT T cells. We further observed that YF T cells were significantly more capable in causing GVHD than KO T cells, but less capable than WT T cells. Mechanically, the levels of serum TNFa and IFNg were similar in the recipients of YF or KO T cells, but significantly lower than those of WT T cells. However, on the per-cell basis, YF CD8+ T cells expressed similar levels of intracellular IFNg as WT T cells, but significantly higher than KO T cells. We further compared the ability of CD4+ or CD8+ T cells alone in the induction of acute GVHD, and found that CD4+ T cells from YF and KO mice were similarly impaired in their capacity to induce acute GVHD. In contrast, the pathogenic capacity of CD8+ T cells from YF mice was comparable to that of WT cells, whereas KO CD8+ T cells were significantly less pathogenic. These results suggest that although both CD4+ and CD8+ T cells depend on ICOS costimulation, the downstream signaling pathways they utilize are distinct: CD4+ T cells depend on ICOS-PI3K signaling whereas CD8+ T cells are more dependent on PI3K-independent pathways, probably calcium signaling. Taken together, our study reveals a complexity in ICOS signaling mechanisms in T cell activation and GVHD induction.

Disclosures:

No relevant conflicts of interest to declare.
Mirnas and Gene Expression Profiles in CD34+ Cells Are Dependent On the Source of Progenitor Cells Employed in Transplantation.
Authors: Báez Alicia , Martin-Antonio Beatriz , Prats-Martín Concepción , Álvarez-Laderas Isabel , Barbado María Victoria , Caballero-Velázquez Teresa , Piruat Jose Ignacio , Carmona Magdalena , Pérez-Simón Jose Antonio , Urbano-Ispizua Alvaro
Published: October 2019

Abstract 3020

Introduction:

Hematopoietic progenitors cells (HPCs) used in allogenic transplantation (allo-HSCT) may have different biological properties depending on their source of origin: mobilized peripheral blood (PB), bone marrow (BM) or umbilical cord (UC), which may be reflected in miRNAs or gene expression. The identification of different patterns of expression could have clinical implications. The aim of this study was to determine differences in miRNAs and gene expression patterns in the different sources of HPCs used in allo-HSCT.

Materials and Method:

CD34 + cells were isolated by immunomagnetic separation and sorting from 5 healthy donors per type of source: UC, BM and PB mobilized with G-CSF. A pool of samples from PB not mobilized was used as reference group. We analyzed the expression of 375 miRNAs using TaqMan MicroRNA Arrays Human v2.0 (Applied Biosystems), and gene expression using Whole Human Genome Oligo microarray kit 4×44K (Agilent). The expression levels of genes and miRNAs were obtained by the 2-ΔΔCTmethod. From expression data hierarchical clustering was performed using the Euclidean distance. To identify genes and miRNAs differentially expressed between the different sources of HPCs statistical Kruskal Wallis test was applied. All analysis were performed using the Multiexperiment Viewer 4.7.1. The function of the miRNAs and genes of interest was determined from the various databases available online (TAM database, Gene Ontology and TargetScan Human).

Results:

Forty-two miRNAs differentially expressed between the different sources were identified. As compared to BM or UC, in mobilized PB most miRNAs were overexpressed, including the miRNA family of miR515, which is characteristic of embryonic stem cells. On the other hand, 47 genes differentially expressed between the different sources were identified. Interestingly, a similar pattern of expression was observed between movilized PB and UC as compared to BM. Interestingly, 13 of these genes are targets of the miRNAs also identified in this study, which suggests that their expression might be regulated by these miRNAs.

Conclusion:

There are significant differences in miRNAs and gene expression levels between the different sources of HPCs

Disclosures:

No relevant conflicts of interest to declare.
Unmanipulated Haploidentical Bone Marrow Transplantation and Post-Transplant Cyclophosphamide for Hematologic Malignanices Following A Myeloablative Conditioning.
Authors: Bacigalupo Andrea P , Raiola Anna Maria , Dominietto Alida , Van Lint Maria Teresa , Gualandi Francesca , Lamparelli Teresa , Bregante Stefania , Ghiso Anna , Grazia Carmen Di , Varaldo Riccardo
Published: October 2019

Abstract 3034

Despite a large number of unrelated donors (UD), not more than 30% of patients who have activated a donor search, undergo an allogeneic UD stem cell transplant. HLA haploidentical family members are being increasingly considered as an alternative donors, both using T cell depleted or T cell replete grafts. Post-transplant high dose cyclophosphamide (PT-CY), introduced by the Baltimore group, has shown very promising results following non myeloablative conditioning regimens. We are now reporting 50 patients with high risk hematologic malignancies, who received a myeloablative regimen, followed by unmanipulated haploidentical bone marrow transplant (hBMT) and PT-CY. The myeloablative conditioning consisted of thiotepa (10 mg/kg), busulfan (9,6 mg/m2̂), fludarabine (150 mg/m2̂)(n=35), or total body irradiation (9,9–12 Gy), fludarabine (120 mg/m2̂) (n=15). The median age was 42 years (18–66); 23 patients were in remission and 27 had active disease; 10 patients were receiving a second allograft. Graft versus host disease (GvHD) prophylaxis consisted in PT-CY on day+3 and +5, cyclosporine (from day 0), and mycophenolate (from day +1).

The median nucleated cell dose was 3.6 ×108̂/kg (range: 1,4 – 7,7). The median time to neutrophil counts of >0.5×109/L was 18 days (range, 13–30 days) and to platelet counts of >20×109/L 23 days (range, 14 – 58 days), respectively. There was no correlation between infused number of nucleated cells and days of neutrophil engraftment. The cumulative incidence of engraftment was 90%for neutrophils and 86% for platelets. Three patients died before engraftment, and 2 patients had autologous recovery: 45 patients (90%) had full donor chimerism on day +30. The cumulative incidence of grade II-III acute GvHD was 12%, and of moderate chronic GvHD 10%. With a median follow up for surviving patients of 333 days (149–623), the cumulative incidence of transplant related mortality is 18%, and the rate of relapse 26%. The actuarial 22 months disease free survival is 68% for patients in remission and 37% for patients with active disease (p<0.001). Causes of death were pneumonia (n=3), haemorrhage (n=3), sepsis (n=3) and relapse (n=7).

In conclusion, a myeloablative conditioning regimen followed by h-BMT with PT-CY, results in a low risk of acute and chronic GvHD and encouraging rates of transplant related mortality and disease free survival.

Disclosures:

No relevant conflicts of interest to declare.
Autologous Re-Transplantation for Patients with Relapsed Multiple Myeloma: A Single Center Experience with 200 Patients.
Authors: Neben Kai , Sellner Leopold , Heiss Christiane , Benner Axel , Raab Marc S , Hillengass Jens , Hose Dirk , Lehners Nicola , Egerer Gerlinde , Ho Anthony D , Goldschmidt Hartmut
Published: October 2019

Abstract 3086

Background:

Therapeutic options for patients with relapsed multiple myeloma (MM) after autologous stem cell transplantation (ASCT) include novel agents, conventional chemotherapy or salvage ASCT, with no standard of care.

Design and Methods:

We retrospectively reviewed all myeloma patients who relapsed after upfront ASCT and received an autologous re-transplantation as salvage therapy at our center over a period of 15 years (n=200). The aim of our study was to evaluate the role of salvage ASCT in terms of efficacy and to define patients who may benefit most from this treatment approach.

Results:

The median follow up after salvage ASCT was 57.1 months. Median progression-free survival (PFS) and overall survival (OS) after salvage ASCT were 15.2 and 42.3 months, respectively. Overall response rate (≥partial response) was 80.4% at day 100, excluding 6 patients who died before assessment. Factors associated with improved PFS and OS after salvage ASCT included remission duration of >18 months after upfront ASCT, bortezomib- or lenalidomide containing therapies for reinduction, response to reinduction, ISS stage I prior to salvage ASCT and year of salvage ASCT 2005 or thereafter. The remission duration after upfront ASCT and the ISS prior to salvage ASCT were combined to create a risk-stratification model with three distinct prognostic groups.

Conclusions:

Salvage ASCT causes low morbidity and is capable of achieving sustained disease control in patients with myeloma. The use of lenalidomide and bortezomib for reinduction has improved the results after salvage ASCT, suggesting that novel agents and salvage ASCT are complementary rather than alternative treatment approaches. Stratification by duration of remission after upfront ASCT and ISS allows prediction of survival after salvage ASCT. To circumvent the problem of stem cell harvest in intensively pretreated patients, we recommend collecting and cryopreserving sufficient stem cells for both upfront and salvage ASCT early in the course of myeloma treatment.

Disclosures:

No relevant conflicts of interest to declare.
Haploidentical Mismatched Allogeneic Versus Autologous Hematopoietic STEM CELL Transplantation in Adult Patients with ACUTE Myeloid Leukemia (AML) in First Complete Remission (CR1): A Pair-Matched Analysis From the Acute Leukemia Working Party of EBMT.
Authors: Gorin Norbert C. , Labopin Myriam , Ciceri Fabio , Velardi Andrea , Santarone Stella , Arcese William , Mohty Mohamad
Published: October 2019

Abstract 3093

In the absence of a matched family donor, adult patients with AML in CR1 are nowadays usually offered, as a first choice, an allogeneic transplant from an alternative donor (haplomismatched donor or cord blood cells), but there has been thus far no demonstration that outcome when using an alternative donor would be superior to outcome following an autologous stem cell transplantation (ASCT).

Between January 2000 and December 2010, 113 AML patients transplanted in CR1 with an haplo-mismatched family donor and 4444 patients autografted were reported to the EBMT registry. For the purpose of this analysis, we performed a matched pair analysis (113 haplo/226 autos) using age and interval from diagnosis to transplant as matching factors. The median follow up was 18 months (range, 1–128). Patients allografted were transplanted more recently (median year of transplant: 2008 vs 2004, p<10e-4), and they received more frequently total body irradiation in the pretransplant regimen (58% vs 17%). Engraftment following an haplo- mismatched transplant occurred in 93% of cases vs 99% after ASCT and recovery of ANC>500/mm3 was slightly delayed (15 days vs 13 days, p<10e-4). The outcome at two years, following an haplo mismatched transplant versus an ASCT was: LFS 44±5% vs 54±4% (p=0.008); OS 44±6 % vs 68±4% (p<10e-4); Non Relapse Mortality (NRM) 37±5% vs 2±2 ( p<10e-4) and Relapse Incidence (RI) 19±4% vs 43±4% (p<10e-4). Following an haplo mismatched transplantation the incidence of chronic extensive GVHD was 24+/− 6%. By multivariate analysis adjusted on year of transplantation, source of stem cells and TBI, the RI was no longer statistically different between the 2 groups, NRM was higher following haplo (HR=11.8; p<0.0001) and LFS was better following ASCT (HR=0.52; p=0.004).

Though this study did not account for cytogenetics and molecular markers (data collection is ongoing), these results suggest that outcome following ASCT in CR1 may be better compared to outcome following a haplo-mismatched transplantation. Recent developments in both transplant approaches may change the results in the near future and may justify a randomized prospective study.

Disclosures:

No relevant conflicts of interest to declare.
The Benefit of Allogeneic Hematopoietic Cell Transplantation in Older Patients with Acute Myeloid Leukemia Is Restricted to Those in First Complete Remission At the Time of Transplantation.
Authors: Michelis Fotios V. , Messner Hans A. , Atenafu Eshetu G , Kim Dennis Dong Hwan , Kuruvilla John , Lipton Jeffrey H. , Uhm Jieun , Loach David , Gupta Vikas
Published: October 2019

Abstract 3097

Allogeneic hematopoietic cell transplantation (HCT) is a potentially curative treatment for some patients with acute myeloid leukemia (AML). Whether older patients with AML benefit from curative potential of HCT similar to younger patients is not well understood. To understand this issue, we evaluated the impact of age and remission status on 242 consecutive patients that underwent HCT between 1999 and 2011 in our program. Based on age and remission status, patients were divided into 4 groups: Gp 1, CR1 age <60 (n=116); Gp 2, CR1 age ≥60 (n=32); Gp 3, CR2 age <60 (n=78); and Gp 4, CR2 age ≥60 (n=16).

Median age of all patients was 48 years (range 18–71), 123 patients (51%) were male. Peripheral blood stem cells were used in 178 patients (74%), bone marrow in 64 patients (26%). Donors were matched related (n=155, 64%) or matched unrelated (n=87, 36%). Median follow up of survivors was 65 months (range 12–145). No significant difference was found in terms of cytogenetic risk distribution between the 4 groups (p=0.14). Of the 48 patients ≥60 years of age, 46 (96%) received reduced-intensity conditioning regimens.

Survival at 2-years in Gp 1, Gp 2, Gp 3, Gp 4 was 59%, 43%, 43% and 23%, respectively (Fig 1). Corresponding relapse free survival (RFS) was 59%, 34%, 40%, and 19%, respectively. Cumulative incidence (CI) of relapse in the four groups was 14%, 34%, 21% and 25% respectively. The corresponding CI of non-relapse mortality (NRM) was 27%, 31%, 38% and 56% respectively. In a univariate analysis, the hazard ratios for survival for Gp 2, Gp 3 and Gp 4 were 1.488, 1.533 and 2.718 in reference to Gp 1, respectively.

Our data demonstrate that patients ≥60 years with AML in CR1 benefit from curative potential of HCT. Due to high NRM, patients ≥60 years in CR2 do not appear to benefit from curative potential of HCT. Therefore, if in an older patient HCT is indicated, attempts should be made to deliver the HCT in CR1.

Disclosures:

No relevant conflicts of interest to declare.
Association of Functional Single Nucleotide Variation in the NLRP3 Gene with Survival Outcomes After Unrelated Bone Marrow Transplantation.
Authors: Takami Akiyoshi , Espinoza J. Luis , Matsuo Keitaro , Morishima Yasuo , Onizuka Makoto , Kashiwase Koichi , Fukuda Takahiro , Kodera Yoshihisa , Nakao Shinji
Published: October 2019

Abstract 3140

NLRP3 is an intracellular trigger of IL-1β production that plays important roles in the regulation of inflammation and apoptosis. A single nucleotide variation in the 3'-untranslated region of the NLRP3 gene, rs10754558 (+29940G>C), is linked to several immunological diseases. When we examined the impact of the NLRP3 genotype in a cohort consisting of 392 pairs of patients with hematologic malignancies and their unrelated HLA 12/12 matched bone marrow donors transplanted through the Japan Donor Marrow Program, the recipient NLRP3 GG genotype was found to be associated with a significantly worse 5-year overall survival (OS) rate (34% vs. 50%, P=0.006) (Fig. 1) and a trend toward a higher transplant-related mortality (TRM) rate (39% vs. 27%, P=0.09) than the recipient CC or CG genotype. The recipient GG genotype remained statistically significant in the multivariate analysis for OS (hazard ratio [HR], 1.86; 95% confidence interval [CI], 1.22 to 2.22; P=0.004) and TRM (HR, 2.28; 95% CI, 1.20 to 4.35; P=0.01). The donor NLRP3 genotype did not significantly influence the transplant outcomes. Next, we investigated the functional relevance of the NLRP3 +29940G>C variant. When leukocytes from healthy individuals were stimulated in vitro with NLRP3 ligand, the leukocytes with the NLRP3 GG genotype produced significantly more IL-1β than those with the NLRP3 CC or CG genotype (Fig. 2). These findings substantiate the functional relevance of the NLRP3 variant, and suggest that the higher IL-1β secretion in the peri-transplant period by recipients with the NLRP3 GG genotype likely accounts for their poor transplant outcomes. NLRP3 genotyping could therefore be useful in predicting prognoses and creating therapeutic strategies for improving the final outcomes of patients who undergo allogeneic hematopoietic stem cell transplantation.

Disclosures:

No relevant conflicts of interest to declare.
Regions on Chromosomes 1 and 14 Are Associated with VWF:Ag Levels in African Americans Using Mapping By Admixture Linkage Disequilibrium
Authors: Paterson Andrew D , Roslin Nicole M , Christopherson Pamela A. , Bellissimo Daniel B , Flood Veronica H , Haberichter Sandra L. , Montgomery Robert R , Investigators Zimmerman Program
Published: October 2019

Background: VWF:Ag is highly heritable, however, despite genome-wide linkage and association analysis, mostly in European derived populations, the genetic basis for VWF levels is only partially understood. African Americans (AfAm) have significantly higher VWF:Ag than European Americans: although variants in VWF and ABO can account for some of this, it is not clear whether they explain all of this ethnic difference. A powerful method to identify chromosomal regions that account for ethnic differences is mapping by admixture linkage disequilibrium (MALD). MALD uses markers that differ markedly in allele frequency between ancestral populations, estimates the global ancestry for each individual, and compares local ancestry between subjects with different trait values to global estimates to identify regions of the genome that account for the ethnic differences of a trait.

Hypothesis: Loci that are associated with VWF:Ag levels in AfAm can be mapped using MALD.

Methods: From 188 AfAm healthy volunteers from the Zimmerman Program for the Molecular and Clinical Biology of VWD, 96 were selected to have 98≤VWF:Ag≥ 164 IU which represent the <25% and >75% of VWF:Ag distribution. They were genotyped using Illumina HumanCoreExome genome-wide SNP chip. SNPs were selected for MALD if they had allele frequency difference between ancestral population (YRI and CEU from HapMap) of >0.4. Related and non-admixed individuals were excluded. Extreme trait analysis was performed using ADMIXMAP.

Results: 16 males and 68 females (mean age 41 years, SD=13) of whom 41 had with Ag≤98and 43 with Ag ≥164 IU were subjected to analysis with 2254 ancestry informative markers across the genome with a mean spacing of 1.8 cM (1.3 Mb). Estimated African ancestry was 79% (SD=13%, range 24-96%), while European ancestry was 20% (SD=12%, range 4-72%). Comparing the subjects at extremes of VWF, regions on chrs 1 (110-147 Mb) and 14 (22-40 Mb) had |Z| scores >3 (p<0.0025) which is the conventional cut-off in MALD studies. At chrs 1 and 14, greater African admixture was associated with higher VWF:Ag, consistent with the expected direction. No significant admixture signal was observed at the VWF (p=0.39) or ABO (p=0.48) loci, which may represent low power due to small sample size.

Discussion: These pilot results require replication in a larger number of independent subjects, as well as fine-mapping to identify the underlying variants responsible for differences in VWF between individuals.

Disclosures

No relevant conflicts of interest to declare.
Risk of Stroke in Patients with Multiple Myeloma: A Nationwide Population-Based Study
Authors: Liu Chia-Jen , Liu Yao-Chung , Hong Ying-Chung , Ku Fan-Chen , Gau Jyh-Pyng , Chiou Tzeon-Jye , Tzeng Cheng-Hwai
Published: October 2019

Background. Patients with multiple myeloma are at risk of thromboembolism. However, studies investigating the relationship between multiple myeloma and ischemic stroke are limited. Therefore, we conducted a nationwide population-based study to investigate the risk of stroke among patients with multiple myeloma and the association between myeloma-related therapy and development of stroke.

Material and methods. We recruited patients with newly diagnosed multiple myeloma aged 20 years or older without antecedent cerebrovascular events from Taiwan's National Health Insurance database between January 1, 2004 and December 31, 2011. Hazard ratios (HRs) of stroke risk for patients with multiple myeloma compared with an age-, sex-, and comorbidity-matched cohort were calculated by Cox proportional regression analysis. Additionally, therapeutic agents were put into Cox models as time-dependent covariates to avoid immortal time bias.

Results. This study consisted of 2,296 patients with multiple myeloma and 22,960 matched individuals, with a median age of 65 years (interquartile range 55-73). One hundred thirteen cerebrovascular events developed among 2,296 patients with multiple myeloma, with a follow-up of 4,119.2 person-years. The stroke incidence was 1.23 times higher in the multiple myeloma cohort than in the matched cohort (27.4 vs. 22.2 per 1,000 person-years), with an age-, sex-, and comorbidity-adjusted HR of 1.38 (95% confidence interval [CI] 1.14-1.67; P < 0.001). The multivariable Cox proportional hazard models showed bortezomib treatment as an independent risk factor determining the subsequent stroke in patients with multiple myeloma (adjusted HR 2.01; 95% CI 1.22-3.28; P = 0.006). This effect was not seen with steroids, thalidomide, or other chemotherapy agents.

Conclusion. Our study reveals an increased risk of stroke among patients with multiple myeloma compared with the matched cohort. Bortezomib treatment is associated with a higher risk of stroke in patients with multiple myeloma.

Figure. Incidence of stroke occurrence in patients with multiple myeloma and the matched cohort Figure. Incidence of stroke occurrence in patients with multiple myeloma and the matched cohort

Disclosures

No relevant conflicts of interest to declare.
A Single Nucleotide Polymorphism Affects the Binding Affinity of H2A.X and Regulates Latexin Transcription and Hematopoietic Stem Cell Function
Authors: Liang Ying
Published: October 2019

Hematopoietic stem cells (HSCs) provide life-long production of blood cells and undergo self-renewal division in order to sustain the stem cell pool. Precisely regulated HSC pool maintenance and blood cell production is vital for organismal survival. We have identified a protein, latexin (Lxn), as a novel regulator of HSCs in mice, whose natural variation in the expression is inversely correlated with HSC population size. However, the molecular mechanisms involved in transcriptional regulation of Lxn in HSCs have not been clearly defined, and the genetic factors contributing to its natural variation are unknown. Here we report that an upstream regulatory sequence of the Lxn gene controls the promoter activity, and histone protein H2A.X interacts with the promoter and acts as a transcriptional activator. We identify a functional single nucleotide polymorphism (SNP), rs31528793, in the Lxn promoter that is associated with the differential binding of H2A.X, as identified by mass spectrometry. SNP rs31528793 attenuates the ability of H2A.X to bind and activate the Lxn promoter, as shown by chromatin immunoprecipitation and promoter luciferase assay. Moreover, overexpression of H2A.X increases the endogenous Lxn level and decreases HSC number and self-renewal. Concomitant blockage of Lxn activation significantly reverses these phenotypic changes, suggesting Lxn is one of the downstream targets and functional mediators of H2A.X. This study, for the first time, reveal the regulatory mechanism involved in Lxn transcription and uncover the functional role of naturally occurring genetic variants in determining differential gene expression and phenotypic diversity in HSC population.

Disclosures

No relevant conflicts of interest to declare.
Prognostic Score Based on Clinical and Biochemical Surrogates of Disease Status in DLBCL Trated with Chemo-Immunotherapy
Authors: Marcheselli Luigi , Bari Alessia , Pozzi Samantha , Marcheselli Raffaella , Cox M. Christina , Baldini Luca , Luminari Stefano , Sacchi Stefano
Published: October 2019

Introduction.

In the past years there were many attempts to replace or improve the IPI score for overall survival evaluation (OS) in diffuse large B cell lymphoma (DLBCL). We focused our attention on prognostic factors, studied in the past or in recent years, that could be used as surrogates of the disease status. We considered the serum albumin (SA) level as surrogate of age, comorbidity and disease severity; LDH as surrogate of cell activity and disease stage; absolute monocyte count (AMC) as surrogate of inflammatory status and absolute lymphocyte count (ALC) as surrogate of immune status of patients.

Patients and method

We collected data of 322 patients comprehensive of IPI factor, SA, AMC and ALC recorded in the "Gruppo Itliano Studio Linfomi" (GISL) database from 2003 to 2012. The score was obtained summing the factors SA <3.7 g/dL, LDH >UNL, AMC>630 /uL and ALC <840 /uL, any with weight one. The factors were not the results of statistical procedure but were chosen on the basis of clinical and pathophysiological considerations, about the role played by these factors in the DLBCL. The OS was estimated using the Kaplan-Meier method; the discriminate ability of the score was checked by means of log-rank test and using the ROC curve, sensibility and specificity at 3-years of follow-up.

Results

All the 322 patients included in the study were treated with R-CHOP and R-CHOP like regimens. The median age at diagnosis was 67 years (range 22-86) and 54% were males; SA <3.7 was identified in 44% of the patients, LDH>UNL in 51%, AMC >630 in 33% and ALC <840 in 23%. The sum of those four factors defined a score with three levels of risk: low (0-1, n=69, 21%), intermediate (2, n=94, 29%) and high (3-4, n=159, 49%). The OS at 5-yrs was 96%, 76% and 46% for score 0-1, 2 and 3-4, respectively The log-rank between score 0-1 and 2 was 11.2 (p=0.0008) and 16.9 between score 2 and 3-4 (p<0.0001). The ROC curve at 5-yrs was 72.1%, with 75.8% of sensitivity and 68.5% of specificity. The score showed a moderate accordance with IPI coded 0-1, 2 and 3/5 (k-statistic 0.37).

Conclusion

This approach showed that it is possible to define a simple prognostic score taking into account simple factors, easily available in every hospital, and related to the disease status. This selection was based on the clinical and pathophysiological knowledge accumulated over the years and, also, avoiding complicated statistical procedures for the selections of the prognostic factors. As it appears in the graph (Fig.1) , patients can be clearly stratified in three groups with deeply different survival outcome, based only on biochemical factors.

Figure 1 Figure 1.

Disclosures

No relevant conflicts of interest to declare.
Clonal Heterogeneityand Evolutional Paths in Multiple Myeloma Revealed By QM-FISH in Single Cell of Sequential Analysis
Authors: Qin Xiaoqi , An Gang , Qin Yu , Xu Yan , Feng Xiaoyan , Zang Meirong , Sui Weiwei , Deng Shuhui , Yi Shuhua , Zhou Wen , Qiu Lugui
Published: October 2019

Background: Clonal heterogeneity is a hallmark of many solid and hematologic malignancies. Clonal heterogeneity may foster clonal evolution and affect personalized care strategies in multiple myeloma(MM). In order to grasp this ubiquitous phenomenon and its effect on MM patients’ prognosis, we report on sequential analysis by quantitative multicolor-fluorescence in situ hybridization (QM-FISH) in single cell of myeloma patients who were longitudinally followed.

Methods: Bacterial artificial chromosomes (BAC) clones that contain target genes including TP53, Rb1, CKS1B, CYLD and cIAP, which belonged to secondary genetic variation of MM and were searched in the data base UCSC Genome Bioinformatics. Multicolor FISH probes were prepared by linking fluorescein labeled dUTP and dCTP to target genes by nick translation. We performed QM-FISH in single cell of 42 specimen from 17 myeloma patients who were longitudinally followed and compared the difference between myeloma patients at initial diagnosis and progression of disease.

Results: Sequential samples revealed that MM may follow three clonal evolution patterns in the course of disease progression. In these 17 patients, myeloma clone in 5 patients had no changes over the time, 6 patients acquired one or more molecular abnormalities and only one predominant clone over the course, 6 patients had more than 1 clone in the course and all clones competing and tiding, or acquired one or more molecular abnormalities resulted in new clones emerging and all involved clones waxing and waning. Furthermore, we analyzed clone architecture in samples at initial diagnosis and 15/17(88.2%) cases had multiple subclones architecture at initial diagnosis. 3 patients of the first group (3/5, 60%) had more than 1 subclone, and all patients of the latter two groups had multiple subclones at initial time. Clonal heterogeneity may contribute to clonal evolution

Conclusions: The outcome of tracking clonal heterogeneity and evolutional paths by QM-FISH is effective and feasible. Myeloma patients may follow 1 of 3 pathways to progress. Clonal heterogeneitymay foster lonal evolution in the course of disease progression.

Disclosures

No relevant conflicts of interest to declare.
Ectopic HOXB4 Expression Expands the Pool of Hemogenic Endothelium-Initiating Progenitor Cells during Pluripotent Stem Cell Differentiation
Authors: Teichweyde Nadine , Kasperidus Lara , Horn Peter A , Klump Hannes
Published: October 2019

Generation of hematopoietic stem cells (HSCs) from pluripotent stem cells, in vitro, holds great promise for future somatic gene and cell therapy. So far, HSCs capable of long-term multilineage reconstitution in mice have only been obtained when the homeodomain transcription factor HOXB4 was ectopically expressed during pluripotent stem cell differentiation (Kyba et al. Cell 109(1): 29-37, 2002; Pilat et al. Proc Natl Acad Sci USA 102(34): 12101-12106, 2005; Lesinski et al. Stem Cells Transl Med 1(8): 581-591, 2012). However, the primary "target" cell of HOXB4 during hematopoietic development, in vitro, is not yet known. Its identification is a prerequisite for unambiguously identifying the molecular circuits driving HSC development, at least in vitro.

To pin down this cell, we retrovirally expressed HOXB4 or a Tamoxifen-inducible HOXB4-ERT2 fusion protein in different reporter and knock-out mouse embryonic stem cell (ESC) lines. For these experiments, ESCs were differentiated for 6 days as embryoid bodies (EBs), dissociated and subsequently cocultured on OP9 stroma cells in medium supplemented with 100 ng/ml mSCF, 40 ng/ml mTPO, 100 ng/ml hFlt3L and 40 ng/ml hVEGF (STFV) for further 3 days. Use of a Runx1(-/-) ESC-line containing a doxycycline-inducible Runx1 coding sequence (“iRunx1”; kindly provided by G. Lacaud, Manchester) uncovered that HOXB4 acts during formation of the hemogenic endothelium (HE) from which HSCs arise. Without Runx1 induction, which arrests hematopoietic development at the HE-stage, ectopic HOXB4 expression mediated an approximately 30-fold increase in the number of circular endothelial, bona fide HE-sheets being Flk1+VE-Cadherin+Tie2+ (mean values: control: 11+/-4.8 n=7; HOXB4: 301+/-47 n=7; P<0.0001, unpaired, 2-sided Student´s t-test) and expressing Sox17 and Lmo2. This observation suggested an expansion of HE progenitors, detectable from day 5 of EB differentiation on. Determination of their frequencies within the VE-Cadherin+ population revealed a HOXB4-mediated increase from 1:360 cells (control) up to 1:15 cells (HOXB4; 95% C.I. = 1:12-1:21). After additional Runx1 induction, the endothelial cells morphologically underwent an Endothelial-to-Hematopoietic Transition (EHT) as verified at the single cell level by time-lapse microscopy. Concomitantly, they upregulated transcription of Gfi1, Gfi1b and Pu.1, initiated surface expression of the pan-hematopoietic marker CD45 and generated hematopoietic colony forming cells (CFC), thus proving their identity as real hemogenic endothelial cells.

Taken together, our results strongly suggest that HOXB4 first and foremost promotes hematopoiesis by substantially increasing the number of hemogenic endothelium progenitors during mouse pluripotent stem cell differentiation.

Disclosures

No relevant conflicts of interest to declare.
JMJD1c, a Histone Demethylase, Is Required for the Maintenance of MLL-AF9 AML Leukemic Stem Cells
Authors: Leung Halina , Wang Jianlong , Wang Jenny Yingzi
Published: October 2019

While most leukemic cells are initially sensitive to chemo- and radiotherapy, leukemic stem cells (LSC) persist and are therefore considered to be the basis for disease relapse in acute myeloid leukemia (AML) (Nat Biotechnol, 25: 1315-1321, 2007). The discovery of epigenetic mutations being the cause of a number of cancers has increased interest in their therapeutic potential (Nat Rev Genet, 7:21-33, 2006; Cell, 128: 683-692, 2007). Although the clinical importance of epigenetic dysregulation has been recognised in various cancers, including MLL-rearranged AML, this topic is currently a novel area of study.

Our microarray analysis comparing genes differentially expressed between LSC and normal hematopoietic stem cells (HSC) identified JMJD1c, a histone demethylase, as a potential LSC-specific target. An analysis of a comprehensive patient outcome database showed that high levels of JMJD1c were associated with significantly poorer survival in patients with AML (P = 0.0029), making it clinically relevant and emphasising the therapeutic potential of JMJD1c. Furthermore, the JMJD1c mRNA level was significantly higher in MLL-rearranged than in non-MLL AML patient xenograft samples.

Western blot and quantitative PCR confirmed the microarray results, where JMJD1c was significantly upregulated in MLL-AF9 LSC compared to Hoxa9/Meis1a derived LSC and HSC. Overexpression of JMJD1c in Hoxa9/Meis1a pre-LSC significantly increased the clonogenic activity and proliferation compared to control cells. Subsequent microarray experiment identified Hox genes as downstream targets of this epigenetic regulator (fold change > 2, P < 0.05), suggesting the regulatory role of JMJD1c in the self-renewal pathway. Consistent with in vitro observations, there was a significant increase in disease progression as shown by leukemia onset in mice with Hoxa9/Meisa1 pre-LSC transduced with JMJD1c cDNA compared to control (P < 0.0001; at least 12 mice per cohort). Stable knockdown of JMJD1c in MLL-AF9 cells using shRNA significantly impaired the proliferation, clonogenic activity and induced cell differentiation in vitro. In vivo survival studies in mice showed that JMJD1c knockdown severely impaired the maintenance of MLL-AF9 induced leukemia (P < 0.0001; 12 mice per cohort).

In conclusion, our studies have identified JMJD1c as an important driver in leukemia maintenance and strongly suggest that JMJD1c possesses an oncogenic role in MLL-AF9 leukemogenesis. Targeting this oncogene may enable us to directly target LSC as a strategy for overcoming drug resistance in AML patients.

Disclosures

No relevant conflicts of interest to declare.
Discovery and Characterization of Super-Enhancer-Associated Dependencies in Acute Myeloid Leukemia
Authors: Corces-Zimmerman Michael Ryan , Eaton Matthew , Lopez Jeremy , Ke Nan , Fritz Christian , Olson Eric , Majeti Ravi , Loven Jakob
Published: October 2019

Misregulation of genes that play an important role in transcription and chromatin biology is a salient feature of many hematological cancers, including acute myeloid leukemia (AML). Numerous genome-wide analyses in AML have provided evidence linking recurring genetic mutations to epigenomic alterations and disease-specific gene expression programs but the prognostic value and clinical utility of these findings remain unclear. Recently, a novel class of densely clustered cis-regulatory elements termed super-enhancers have emerged as key effectors initiating and maintaining cell type-specific gene expression in a variety of physiological and disease settings, including cancer. Tumor-specific super-enhancers regulate key oncogenes and other cancer-essential genes, providing a novel target discovery strategy for detecting both known and unrecognized cancer dependencies of high diagnostic and therapeutic value. Here we describe the discovery and characterization of super-enhancer domains across a cohort of nearly 50 AML patients and relevant normal hematopoietic stem and progenitor cell controls. We identified unique, clone-specific cancer targets encoding protein kinases, chromatin regulators, and lineage-specific transcription factors, including key drivers of AML such as FLT3, CDK6, and MYB. In addition, we have identified clusters of AML cases with shared super-enhancer domains suggesting convergence on common key drivers of AML. We provide the biological and disease relevance of super-enhancer-associated genes in the context of tumor cell state and drug-target discovery and establish a molecular rationale for developing therapies based on these new insights.

Disclosures

Eaton: Syros Pharmaceuticals: Employment, Equity Ownership. Lopez:Syros Pharmaceuticals: Employment, Equity Ownership. Ke:Syros Pharmaceuticals: Employment, Equity Ownership. Fritz:Syros Pharmaceuticals: Employment, Equity Ownership. Olson:Syros Pharmaceuticals: Employment, Equity Ownership. Loven:Syros Pharmaceuticals: Employment, Equity Ownership.