bluuebunny/crossref_metadata_2025 huggingface dataset:
The Viral Oncoprotein vFLIP Encoded by KSHV/HHV-8 Is Responsible for the Unique Transcriptional Signature and Phenotypic Features of Primary Effusion Lymphoma
Authors: Guasparri Ilaria , Hassane Duane C , Roshal Mikhail , Cesarman Ethel
Published: October 2019

Abstract 900

Background:

Primary effusion lymphomas (PELs) are caused by the Kaposi's sarcoma herpesvirus (KSHV, also called human herpesvirus 8, HHV-8). PELs have a distinctive immunophenotype, lacking expression of most B cell antigens. In addition, PELs have a very distinct gene expression signature when compared to that of other lymphoma subtypes. While KSHV encodes almost 100 proteins, only a handful is expressed in latently infected PEL cells. One of these is vFLIP, which activates NF-kB and inhibits apoptosis and autophagy, and causes tumors in mice. In this study we evaluated the global effect of ectopic expression of vFLIP in a B cell lymphoma cell line, as well as that of suppression of vFLIP expression in a PEL cell line.

Methods:

vFLIP expression was knocked down with siRNA in the PEL cell line BC-3. Namalwa cells were stably transfected with a tetracycline-inducible vFLIP, and vFLIP expression was induced with doxycycline for 48 hours. The Namalwa cell line was selected because it contains EBV with a type I latency pattern, also a feature of most PELs. We evaluated RNA extracted from these vFLIP+ and FLIP– cells in a PEL and Burkitt lymphoma background for gene expression array analysis using the Illumina Direct Hybridization Assay. Results were validated by immunohistochemistry and flow cytometry.

Results:

The vFLIP signature in a BL cell line was analyzed by gene set enrichment analysis (GSEA), which is an analytical method to determine how gene sets from other public studies compare to our signature. 1,445 public gene signatures were compared to a rank ordered list of genes that were altered by expression of vFLIP. The top match among these previous gene signatures, for both vFLIP upregulated and downregulated genes, was the PEL signature as compared to other lymphoma subtypes and normal B cell subsets (Kelin et al, Blood 101, 2003). Thus, expression of vFLIP in BL leads to a switch from a BL expression signature to that of PEL. Furthermore, expression of vFLIP in BL cells let to an almost complete downregulation of several B cell antigens, including surface Ig light chains, CD19, as well as CD10, in conjunction with upregulation of CD138 expression.

Conclusion:

The unique phenotype of PEL has long been an intriguing feature of this disease. We have found that a single gene encoded by KSHV, vFLIP, can induce a transcriptional signature of PEL, as well as the phenotype characteristic of this disease.

Disclosures:

No relevant conflicts of interest to declare.
Pre-mRNA Splicing Mechanisms, Misregulation in Disease, and Therapeutic Strategies
Authors: Krainer Adrian
Published: October 2019

Abstract SCI-13

Most eukaryotic protein-coding genes have one or more introns, and their transcripts can undergo alternative splicing, giving rise to multiple isoforms. Accurate splicing is essential for normal gene expression, and alternative splicing is a key mechanism for expanding the proteome and regulating the expression of diverse protein isoforms. This session will review the general mechanisms of pre-mRNA splicing and the regulation of alternative splicing. In addition, the process of how abnormal splicing arises as a result of intronic or exonic mutations in particular genes, or more globally as a result of splicing-factor misregulation, as well as the contribution of splicing misregulation to cancer, will be described. Lastly the current status of targeted therapeutics development, focusing on antisense approaches to correct abnormal splicing of specific genes or to modulate alternative splicing, will be discussed.

Disclosures:

Krainer: ISIS Pharmaceuticals: Consultancy, Patents & Royalties, Research Funding.
Understanding the Role of Mutations in SF3B1 and Splicing in Chronic Lymphocytic Leukemia
Authors: Wu Catherine J.
Published: October 2019

Abstract SCI-15

A major challenge in the treatment of chronic lymphocytic leukemia (CLL) is the tremendous clinical heterogeneity of this disease, ranging from indolent disease kinetics to a rapidly fatal course despite aggressive therapy. While some prognostic markers have been established, the advent of deep-sequencing technologies has enabled identification of most or all genetic alterations underlying malignancy, which is leading to new genetic prognostic markers across cancers. We recently sequenced the exomes of 91 CLLs with matched germline controls and identified nine recurrently mutated genes, six pathways involved in CLL pathogenesis (1), as well as new prognostic factors. From this study, a striking finding was the high frequency of mutations in SF3B1, part of the catalytic core of the spliceosome. We found that 14 independent SF3B1 mutations (15% of 91 CLL samples) localized to a single region of the gene (with unknown function) with 50 percent being identical K700E mutations. This finding has been since verified by other investigators. SF3B1 mutations were significantly associated with del(11q), a cytogenetic abnormality that is associated with aggressive disease, and whose minimally deleted region contains the critical DNA damage response gene ATM. Importantly, a Cox multivariate regression model identified mutated SF3B1 as an independent predictive marker of poor prognosis. Mechanistically, CLL samples harboring SF3B1 mutation demonstrated altered pre-mRNA splicing, with increased intron retention in known spliceosome target genes. Finally, SF3B1-K700E was also identified as a recurring mutation in myelodysplasia. In short, mutations in SF3B1 are positively selected in CLL, alter splicing patterns, and represent a novel oncogenic mechanism in hematologic malignancies. Our genetic and clinical evidence suggests that altered pre-mRNA splicing is a novel cancer pathway of importance to CLL and to blood malignancies in general. An ongoing focus of investigation in our group is trying to understand the CLL-promoting activity of SF3B1 mutation by linking alterations in transcripts with functional changes in B-cell biology. Precise identification of driver transcripts and their functional characterization will enable the development of novel therapeutics that can target this pathway more efficaciously and with minimal toxicity. This is especially important in the treatment of a clinically aggressive subgroup of CLL for which effective therapies are presently unavailable. Spliceosome targeting drugs have been identified—discovered through medicinal screens of natural products with tumoricidal properties, and only later found to target the SF3b complex—and may have promise for this subset of patients. Elucidation of this novel cancer pathway has broad implications for cancer biology, from the vantage of both understanding disease mechanism and the potential for therapeutic targeting.

Disclosures:

No relevant conflicts of interest to declare.
Links Between Defective DNA Strand Break Repair and Genome Instability in Fanconi Anemia
Authors: West Stephen C.
Published: October 2019

Abstract SCI-2

All living organisms feature DNA repair pathways that safeguard the integrity of the genome, and mutations in proteins that mediate key events in DNA repair have been linked to genome instability and tumorigenesis. Homologous recombination provides an important DNA repair pathway that is needed for the restoration and restart of broken replication forks, for the rejoining of chromosome/chromatid breaks, and for the processing of DNA cross-links. Mutations in genes that encode a variety of recombination proteins are linked to breast cancers and to heritable diseases such as Bloom syndrome (BS) and Fanconi anemia (FA). In recent work, we purified the BLM protein (defective in BS), the BRCA2 (FANCND1) and PALB2 (FANCN) tumor suppressors (mutated in some cases of FA), and the newly discovered FANCP protein, also known as SLX4, and have initiated structure-function analyses to elucidate their molecular functions. How these proteins process DNA, and how they are regulated and controlled to direct the outcome of recombinational repair is now revealing unexpected insights that extend our understanding of efficient DNA repair and tumor avoidance.

Disclosures:

No relevant conflicts of interest to declare.
The Gut: Role at Steady State and Variations in Disordered Conditions
Authors: Peyssonnaux Carole
Published: October 2019

Abstract SCI-23

As the human body cannot excrete excess iron, its absorption needs to be finely regulated at the intestinal level. Ferric iron (Fe3+) is reduced to ferrous iron (Fe2+) by brush border ferric reductases, including duodenal cytochrome b (DCYTB), before being transported across the apical membrane by divalent metal transporter 1 (DMT1), which is the principal iron importer. Depending on body iron requirements, iron can be either stored bound to ferritin or exported across the basolateral enterocyte membrane into the plasma by the sole iron exporter ferroportin (FPN). Iron absorption responds to systemic signals reflecting body iron requirements and local signals in the enterocyte. At the systemic level, hepcidin is the key circulating peptide hormone maintaining iron homeostasis. Hepcidin controls plasma iron concentration by inhibiting intestinal iron absorption and iron recycling by macrophages. Hepcidin acts by inhibiting cellular iron efflux through binding to and inducing the degradation of FPN. Hepcidin transcription is upregulated by iron repletion and downregulated by iron deficiency, ineffective erythropoiesis, and hypoxia. Hypoxia-inducible factors HIF-1 and HIF-2 are heterodimeric transcriptional factors and central mediators of cellular and systemic adaptation to hypoxia. In the presence of oxygen, the HIF-α subunit is targeted to the proteasome, while in hypoxia (or iron deficiency), HIF-α is stabilized and induces the transcription of target genes. We hypothesized that HIFs, stabilized in the hypoxic intestinal epithelium, may also play critical local roles in regulating intestinal iron absorption. We generated conditional knockout mice that lacked either Hif1a or Hif2a specifically in the intestinal epithelium and found that HIF-1α was not necessary for iron absorption, whereas HIF-2α played a crucial role in maintaining iron balance in the organism by directly regulating the transcription of the genes encoding DMT1 and DCYTB. Specific deletion of Hif2a led to a decrease in serum and liver iron levels. Alterations in HIF-2 at the intestinal level can override systemic regulation via hepcidin. Interestingly, we further demonstrated that HIF-2α contributes to iron hyperabsorption in a genetic mouse model of hereditary hemochromatosis (HH). HH is a genetic disorder characterized by abnormally low hepcidin expression and excessive iron accumulation in the liver and parenchyma. These findings suggest a prominent role of HIF-2 in the physiopathological regulation of intestinal iron absorption and may provide new therapeutic perspectives for the treatment of anemias and iron overload-associated disorders.

Disclosures:

No relevant conflicts of interest to declare.
Links Between DNA Damage and Metabolism, Pathways Causing Bone Marrow Failure in Fanconi Anemia, and Therapeutic Implications
Authors: Patel Ketan J.
Published: October 2019

Abstract SCI-3

Recent work from my lab has discovered that metabolism generates reactive aldehydes. These reactive molecules are potent damagers of DNA. The consequences of this are revealed by the inactivation of enzymes that detoxify these aldehydes and the Fanconi anemia DNA repair pathway in mice and vertebrate cell lines. The scientific session presentation will discuss this work and recent unpublished research on how natural aldehydes damage blood stem cells. This work has consequences for understanding how metabolism and ethanol exposure can be genotoxic, particularly in the vast population of Southeast Asians carrying a genetic defect in aldehyde catabolism (“pink flushers”). It is also relevant to the emergence of bone marrow failure and leukemia in Fanconi anemia.

Disclosures:

No relevant conflicts of interest to declare.
New α-Granule Biology Reveals How Platelets May Regulate Angiogenesis
Authors: Italiano Joseph E. , Battinelli Elisabeth M.
Published: October 2019

Abstract SCI-35

In addition to their “classic” role in hemostasis, platelets are now known to be major contributors in wound healing, tumor growth, and angiogenesis. Stored within the α-granules is an array of angiogenic regulatory proteins, which are deposited by the secretion reaction of surface-activated platelets into the local environment of a tumor or wound. Despite the recognized importance of platelets in regulating new blood vessel growth, our understanding of how platelets modulate the angiogenic response remains unclear. Here, we consider how new α-granule biology may provide insights into how platelets regulate angiogenesis. First, we will present data on a new form of α-granule movement. We will show that actin filament assembly can power the propulsion of α-granule movement, and this may be a novel mechanism underlying the secretion reaction. Second, we show that one mechanism by which anticoagulants may impact malignancy is by disrupting the tumor cells' ability to hijack the angiogenic potential of platelets. Third, we will review the molecular basis of transport and delivery of α-granules to assembling platelets. Finally, we will discuss the concept of using “designer platelets” as a drug delivery system for targeted agents.

Disclosures:

No relevant conflicts of interest to declare.
Decoding the Chromatin Code
Authors: Jones Peter A.
Published: October 2019

Abstract SCI-4

Epigenetic processes are reinforced by interactions between covalent chromatin marks such as DNA methylation, histone modifications, and variants thereof. These marks ultimately specify the locations of nucleosomes, particularly with respect to transcriptional start sites and other regulatory regions. Understanding how the epigenome functions, therefore, requires a coordinated approach in order to reveal the mechanisms by which the chemical modifications interact with nucleosomal remodeling machines to ensure epigenetic inheritance and control of gene expression. We have developed a new methodology to simultaneously map nucleosomal positioning and DNA methylation on individual molecules of DNA. We used this nucleosomal mapping technology to ascertain alterations in nucleosomal positioning during the abnormal silencing of genes by promoter hypermethylation. These experiments show that the methylation of CpG islands at the transcriptional start sites of key tumor-suppressor genes results in the stable placement of nucleosomes at the transcription start site. Treatment with 5-azanucleoside results in an immediate inhibition of DNA methylation and a sequence of downstream events that ultimately result in the eviction of the nucleosomes from the transcription start site and the activation of gene expression.

Disclosures:

Jones: Eli Lilly: Consultancy.
Metabolic Regulation of Hematopoietic Stem Cells During Stress
Authors: Suda Toshio
Published: October 2019

Abstract SCI-42

Tissue homeostasis over the life of an organism relies on both self-renewal and multipotent differentiation of stem cells. Hematopoietic stem cells (HSCs) are sustained in a specific microenvironment known as the stem cell niche. Adult HSCs are kept quiescent during the cell cycle in the endosteal niche of the bone marrow. Normal HSCs maintain intracellular hypoxia, stabilize the hypoxia-inducible factor-1a (HIF-1a) protein, and generate ATP by anaerobic metabolism. In HIF-1a deficiency, HSCs became metabolically aerobic, lost cell cycle quiescence, and finally became exhausted. An increased dose of HIF-1a protein in VHL-mutated HSCs and their progenitors induced cell cycle quiescence and accumulation of HSCs in the bone marrow (BM), which were not transplantable. This metabolic balance promotes HSC maintenance by limiting the production of reactive oxygen species (ROS), but leaves HSCs susceptible to changes in redox status (1). We have performed the metabolomic analysis in HSCs. Upregulation of pyruvate dehydrogenase kinases enhanced the glycolytic pathway, cell cycle quiescence, and stem cell capacity. Thus, HSCs directly utilize the hypoxic microenvironment to maintain their slow cell cycle by HIF-1a-dependent metabolism. Downregulation of mitochondrial metabolism might be reasonable, since it reduces ROS generation. On the other hand, at the time of BM transplantation, HSCs activate oxidative phosphorylation to acquire more ATP for proliferation. Autophagy also energizes HSCs by providing amino acids during transplantation. ATG (autophagy-related) 7 is essential for transplantation and metabolic homeostasis. The relationship between mitochondrial heat shock protein, mortalin, and metabolism in HSCs will also be discussed.

Disclosures:

No relevant conflicts of interest to declare.
Kinomic Profiling of Pediatric Acute Myeloid Leukemia for Detailed Cellular Insights
Authors: Mahmud Hasan , Bezrouzi Pariya , ter Elst Arja , Scherpen Frank J.G. , Kampen Kim R. , De Haas Valerie , Guryev Victor , Peppelenbosch Maikel P , Johannes Frank , Kornblau Steven M. , Bont Eveline
Published: October 2019

Acute myeloid leukemia (AML) remains a life threatening malignancy in children. Considerable progress has been made in elucidating the new diagnostic and prognostic markers over the past decades. The precise etiology remains unclear. Therefore, it is essential to evaluate the activation of the components of cellular signaling pathways to understand AML signaling and to design the most successful approach for combinational therapies and new kinase inhibitors.

In this study, we used a high-throughput PepChipTMKinomics microarray system containing 976 different kinase substrates and assayed primary leukemic samples of 96 AML patients to produce an exceptionally detailed map of kinome enzymatic activities towards predefined peptide substrates. The generated profiles provide a comprehensive insight in signaling pathways active in AML patients.

As expected the activation of proteins belonging to MAPK signaling, PI3K/AKT signaling, cell cycle regulation, apoptosis and insulin signaling pathways along with the signaling receptors and immune system regulators were found. Unsupervised hierarchical cluster analysis separates the AML blast profiles based on 192 peptide activities into two clusters. Cumulative incidence of relapse (CIR) was significantly higher in the patients of cluster-2. Peptide activity patterns were independent of patient characteristics. In addition, with Gaussian network modeling, a total of 540 peptides (55%) showed at least one peptide-peptide association without a prior assumptions whereas 74 peptides (7.5%) had >39 nodes suggesting to be potential interesting signaling hubs. Among these 74 peptides, 10 peptides were identified in cluster-1 and 50 peptides were in cluster-2. Thus, this total analysis defined peptides correlated to low incidence for relapse, for examples AKT1, HGFR, RGS7 and to high incidence for relapse for instance, proteins involve in MAPK pathways (RAF1, RAC1,14-3-3 eta) and cell cycle regulation and cellular growth (c-Myc, FOXO3A, RBL1).

In conclusion, our study demonstrates the feasibility of peptide activity profiling to identify two active signaling network clusters in pediatric AML correlated to CIR. Highly correlated peptides belonging to cluster-2 provide stronger leads for selection of novel targets in future therapeutics.

Disclosures

No relevant conflicts of interest to declare.
Identification of a Dynamic Core Transcriptional Network in t(8;21) AML Regulating Differentiation Block and Self-Renewal
Authors: Ptasinska Anetta , Assi Salam , Martinez-Soria Natalia , Imperato Maria Rosaria , Piper Jason , Cauchy Pierre , Pickin Anna , James Sally , Hoogenkamp Maarten , Tenen Daniel G , Ott Sascha , Westhead David R , Cockerill Peter N , Heidenreich Olaf , Bonifer Constanze
Published: October 2019

One of the best-characterized chromosomal rearrangements found in AML is the t(8;21) translocation which accounts for approximately 10% of all AMLs and fuses the DNA-binding domain of the hematopoietic master regulator RUNX1 to the almost the entire ETO protein. The resulting RUNX1/ETO fusion protein lacks the transactivation domain of RUNX1 resulting in major differences in the biological activities of RUNX1 and RUNX1/ETO. RUNX1 normally recruits transcriptional activators and binds to DNA as a heterodimer with CBFb which is important for high-affinity DNA binding. The RUNX1/ETO fusion protein also interacts with CBFb but functions as a RUNX1/ETO tetramer, and like ETO itself, it also interacts with NCOR and SIN3A co-repressors. The leukemogenic fusion protein RUNX1/ETO forms a complex with other transcription factors and the binding of this complex to RUNX1 target genes causes a block in myeloid differentiation.

Here we investigate the dynamic changes in global transcription factor binding patterns that occur immediately following depletion of RUNX1/ETO. To that end, we combined digital footprinting, ChIP-sequencing for multiple factors and transcriptome analysis to identify the core transcriptional network of t(8;21) AML cells, and then characterized changes in these networks upon RUNX1/ETO knockdown. These analyses revealed a dynamic equilibrium between RUNX1/ETO and RUNX1 complexes competing for the same genomic sites. We show by sequential ChIP that both complexes have similar transcription factor compositions, but differ in their preference for the recruitment of co-activators and co-repressors. Using a novel digital DNaseI footprinting approach we show that both t(8;21)-positive cell lines and patient-derived primary AML cells share the same pattern of binding site occupancy. Within this core transcriptional network, RUNX1/ETO-bound loci are predominantly associated with gene repression. Furthermore, loss of RUNX1/ETO establishes a new differentiation-associated transcriptional network dominated by de novo binding of C/EBPa resulting from the up-regulation of CEBPA gene expression. Our results demonstrate that the block in myeloid differentiation in t(8;21) is caused by the dynamic interference of RUNX1/ETO with cis-regulatory elements destined to bind alternate factor assemblies during myeloid differentiation including both RUNX1 and C/EBPa.

Disclosures

No relevant conflicts of interest to declare.
Haploidentical Hematopoietic Stem Cell Transplantation in Pediatric Patients: Comparison of Early Post-Transplant Outcome According to in Vitro Depletion Method
Authors: Im Ho Joon , Koh Kyung Nam , Suh Jin Kyung , Choi Eun Seok , Jang Seongsoo , Park Chan-Jeoung , Seo Jong Jin
Published: October 2019

Currently, haploidentical hematopoietic cell transplantation (HHCT) is considered an established option for patients who have diseases curable with HCT but who lack a suitable donor. We compared the early post-transplant outcomes of the two transplant groups using different in vitro depletion method.

Between July 2008 and June 2014, 49 pediatric patients underwent in vitro T cell-depleted HHCT. Of 49 patients, 28 received CD3-depleted stem cells (CD3-HHCT) and 21 received TCRαβ-depleted grafts (TCRαβ-HHCT). Among 28 patients of CD3-HHCT, nine had hematologic malignancy [HM, one with ALL in non-CR, six with AML (3 CR1, 2 CR2, 1 non-CR), and two with MDS-RCMD], 18 had non-malignant disease (one with Fanconi anemia, 16 with acquired SAA, and one with CDA), and one had refractory neuroblastoma. Among 21 patients of TCRαβ-HHCT, 16 had HM [five with ALL (2 CR1, 2 CR2, 1 CR3), five with AML (2 CR1, 2 CR2, 1 non-CR), one with mixed lineage leukemia in non-CR, one with MDS-RCC, two with JMML, and two with NHL (1 CR2, 1 CR3)], two had SAA, and three had solid tumors [two with RMS (1 CR2, 1 refractory), and one with Ewing sarcoma in CR3].

Of 28 patients who received CD3-HHCT, two patients failed to achieve primary engraftment, and five patients experienced graft rejection (GR) within 28 days post-transplant. No patients of TCRαβ-HHCT experienced graft failure (GF). Early GF (primary GF and GR) was more common in CD3-HHCT compared to TCRαβ-HHCT (P=0.011). The cumulative incidence of acute GVHD ≥ grade II was comparable between two groups (33.3% for CD3-HHCT and 27.0% for TCRαβ-HHCT). Extensive chronic GVHD occurred in three patients from CD3-HHCT and one from TCRαβ-HHCT. T and T4 cell counts at 2 months post-transplant were higher in TCRαβ-HHCT than that of CD3-HHCT (P=0.007 for T and P=0.074 for T4). In CD3-HHCT, four patients died of non-relapse causes (two of CMV disease, one of encephalopathy, and one of autoimmune hemolytic anemia) and one died of leukemia. In contrast, five patients from TCRαβ-HHCT died of disease but, none died of non-relapse cause.

Of 20 patients (18 from CD3-HHCT and two from TCRαβ-HHCT) with non-malignant disease, only one patient of CD3-HHCT died of TRM. As for 29 patients (10 from CD3-HHCT and 19 from TCRαβ-HHCT) with malignant disease, the EFS at 1 year for CD3-HHCT and TCRαβ-HHCT were 60.0% and 56.6%, respectively (P>0.05).

In this study, TCRαβ-depleted HHCT showed a better early post-transplant outcome in terms of engraftment, immune recovery, and NRM, compared to CD3-HHCT. However, further study is warranted to evaluate the efficacy of TCRαβ-HHCT in preventing relapse in advanced malignancy.

Disclosures

No relevant conflicts of interest to declare.
Impact of Persistent Left Ventricular Regional Wall Motion Abnormalities in Childhood Cancer Survivors after Anthracycline Therapy: Assessment of Global Left Ventricular Myocardial Performance By 3D Speckle-Tracking Echocardiography
Authors: Okuma Hirotsugu , Yagasaki Hiroshi , Shimozawa Katsuyoshi , Mugishima Hideo , Noto Nobutaka
Published: October 2019

Background: Previous studies of childhood cancer survivors demonstrated the existence of transient or persistent left ventricular (LV) regional wall motion abnormalities (WMA) after treatment with anthracyclines, but the differential impact on deformation of persistent regional WMA against global LV myocardial performance is unknown.

Methods: To investigate whether childhood cancer survivors after treatment with anthracyclines with persistent LV regional WMA have a poorer LV myocardial performance compared to those without WMA, 34 long-term childhood cancer survivors (mean age 14.6±4.0 years) with a median cumulative anthracycline dose 234.7mg/m2 (range 80-625mg/m2) who had been off treatment for ≥ 5 years and a preserved LV ejection fraction (EF) (>55%), and 12 healthy control subjects, were studied by using 3D speckle-tracking echocardiography (3D STE). The 34 patients were divided into two groups according to the existence of regional WMA: group 1 (with WMA, n=14), group 2 (without WMA, n=20). 3D STE was performed to assess LV global longitudinal strain (GLS), global circumferential strain (GCS), global radial strain (GRS), global area strain (GAS), LV torsion, LV end-diastolic volume (LVEDV), LV end-systolic volume (LVESV), LVEF, and LV systolic dyssynchrony index (SDI: % of SD of time to peak of the 16-segment/R-R interval).

Results: Of the 14 patients with WMA, regional WMA was seen at mid-anterior septum in 13 patients (92.8%) and apical septum in 1 patient (7.2%). There was no statistical difference in LVEDV, LVESV, LVEF, GLS, LV torsion or SDI derived from GLS, GCS, GAS among the 3 groups. In contrast, there were significant differences in GRS (p<0.001), GAS (p<0.018), GCS (p<0.025), and SDI derived from GRS (p<0.01) among the 3 groups. Compared with group 2, group 1 had significantly reduced GRS (14.3±6.1% vs. 33.1±10.1%, p=0.003), GCS (-23.5±3.7% vs. -33.9±6.5%, p=0.026), GAS (-34.3±5.1% vs. -45.41±6.6%, p=0.034, respectively), and greater SDI derived from GRS (16.5±5.1% vs. 6.9±2.9%, p<0.01, respectively). Moreover, existence of WMA was correlated with GRS (p<0.0001), SDI derived from GRS (p<0.0001), LVEF (p=0.036), and cumulative dose (p=0.049). Multiple linear regression analysis identified GRS as a significant determinant of the existence of WMA (β=0.751, p=0.001).

Conclusion: Childhood cancer survivors after anthracycline therapy with persistent regional WMA show a poorer LV myocardial performance with mechanical dyssynchrony compared to those without regional WMA, despite a preserved LVEF.

Disclosures

No relevant conflicts of interest to declare.
The Reduction of Erythropoietin Stimulating Agent (ESA) Use Due to the FDA Black Box Warning
Authors: Noxon Virginia , Bennett Charles L , Wu Jun
Published: October 2019

Background: Erythropoietin stimulating agents (ESAs), have been readily used to treat chemotherapy related anemia in cancer patients since 2003. ESAs are a useful substitute for blood transfusions for these patients. However, there were unacknowledged toxicities for ESAs when they were first administered. In March 2007, these toxicities were acknowledged by the FDA when a black box warning was issued for ESAs in these patients. This study aims to observe ESA utilization trends along with VTEs to determine the effect of the black box warning.

Methods: The 2002-2010 merged South Carolina Medicaid- Cancer Registry dataset with Medicaid claims up to 2012 was used to determine the effect of the FDA black box warning. Breast, non-small cell lung and colorectal patients were determined from the cancer registry. Of these, those who had chemotherapy were identified. ESA users were identified from medical and pharmacy claims. The final sample contained those who had their first ESA claim after chemotherapy and who were not dual enrolled in Medicare.

Results: Of the 1,645 cancer patients with chemotherapy, 1,310 (80%) had at least one ESA claim. Total ESA claims by year showed a peak in 2006 at 2,243 claims followed by a decrease to 1,347 in 2007 and 714 in 2006. A month by month breakdown of ESA claims showed the peak in May 2006 at 250 and eventually decreasing to 7 in December 2012. Total VTE events for these patients showed a peak in 2007 at 811 followed by a steep decline to 351 in 2009 and 154 in 2012.

Conclusion: The FDA black box warning may have been effective in reducing ESA utilization. There is also the potential delayed response to the clinical trial data for VTEs and ESA utilization. The meta-analysis in 2008 by Dr. Bennett also seems to have had an impact on reducing ESA utilization and VTE events.

Figure 1 Figure 1.

Disclosures

No relevant conflicts of interest to declare.
An Intergenic SNP in the β–globin Gene Cluster Is Associated with Hyperuricemia and Influences Gene Transcription in Vitro
Authors: Kumkhaek Chutima , Shriner Daniel , Doumatey Ayo P. , Rotimi Charles N. , Rodgers Griffin P.
Published: October 2019

Hemolysis as a suggested cause of hyperuricemia is based upon the fact that red blood cells contain uric acid. The development of hyperuricemia also generates from an increased synthesis of nucleic acids occurring as part of the erythropoietic response to hemolysis in hemoglobin disorders such as sickle cell anemia, α-thalassemia, and β-thalassemia. In addition, multiple genome-wide association studies (GWAS) have reported significant association between uric acid levels and specific genomic loci. However, the mechanism of hyperuricemia still remains controversial and it is also unknown whether African Americans have higher prevalence of hyperuricemia due to genetic vs environmental risk factors. Here, we used joint admixture mapping and association testing to identify genetic variants associated with serum uric acid levels in African American. Interestingly, we detected 6 SNPs (rs2855125, rs2855126, rs11036415, rs11036496, rs4320977, and rs4348933) in an intergenic region of the β–globin cluster on chromosome 11 are associated with high levels of serum uric acid in populations of African ancestry. Next, we explored the potential regulatory role of intergenic SNPs associated with hyperuricemia using luciferase reporter gene activity assays and electrophoretic mobility shift assays (EMSA). Each SNP-containing DNA fragment was amplified by PCR using human genomic DNA and inserted into a firefly luciferase reporter vector, pGL3-basic vector. 293T or K562 cells were co-transfected with these constructs and a Renilla luciferase vector to control for transfection efficiency. Expression of firefly luciferase driven by each SNP-containing DNA fragment was measured by a dual luciferase reporter assay and normalized by Renilla luciferase expression. SNPs rs2855126, rs11036496 and rs4348933 on chromosome 11 had significantly greater expression levels of firefly luciferase than pGL3-basic-transfected cells in both 293T and K562 cells. Of these, the SNP rs2855126 ancestral C allele (associated with higher serum uric acid levels) showed significantly higher luciferase activity than the derived G allele. Functionally, the luciferase activities from these constructs were determined to be very similar in both cell lines used. Alleles altering expression were further assessed for binding of nuclear extracted proteins by EMSA. We found specific gel shift bands for SNPs rs2855126, rs11036496, and rs4348933, suggesting these SNPs are situated in the binding site of potential transcription factors. These data provide new insights into the potential contribution of imbalanced β-globin gene expression to hyperuricemia.

Disclosures

No relevant conflicts of interest to declare.
The CD21low B Cell Population Is Composed of an Anergic and an Immunostimulatory Germinal Center-like B Cell Subset
Authors: Shimabukuro-Vornhagen Alexander , García Márquez María , Fischer Rieke , Wennhold Kerstin , Iltgen Juliane , Theurich Sebastian , von Bergwelt-Baildon Michael S.
Published: October 2019

In recent years we have gained an increased understanding of the complexity of B cell biology and function. It has become increasingly recognized that apart from antibody production B cells exert many more function. B cells serve as antigen-presenting cells (APC), they contribute to immunoregulation and represent an important source of cytokines and chemokines. A deeper understanding of the role of B cells in the pathophysiology of human diseases has been hampered by the lack of well-defined functional B cell subsets. We therefore aimed to identify novel human B cell subsets which could serve as biomarkers or targets of therapeutic intervention. Using a transcriptomic approach combined with flowcytometric immune assessment of healthy human subjects and patients we were able to identify several functional B cell subpopulations with relevance to human disease. We were able to define a CD21low CD86pos human B cell subset with strong antigen-presenting capacity which gradually develops from conventional resting B cells under the continuous stimulation via CD40. These cells were phenotypically and functionally distinct from CD21low CD86neg B lymphocytes, which represent anergic B cells. Using calcium flux assays and phospho-specific flow cytometry we were able to show that the CD21low B cell subsets displayed distinct signaling states. Both CD21low B cell subpopulations had an impaired response to B cell receptor stimulation. However, CD21low CD86pos B cells had higher basal calcium levels and basal phosphorylation of BCR-associated signaling molecules such as Syk and Erk. Contrary to CD21low CD86neg B cells, which demonstrated poor antigen-presenting capacity, CD21low CD86pos B cells were potent immunostimulatory antigen-presenting cells. CD21low CD86pos B cells were increased in acute inflammation and autoimmune diseases such as rheumatoid arthritis. CD21low CD86neg B cells, on the other hand, were increased in chronic inflammatory conditions such as chronic HIV infection. The balance between the CD21low B cell subsets varied with the functional state of the B cell compartment in inflammatory conditions and could be used to classify the functional state of the B cell compartment. In summary, we have identified several novel human B cell subsets with distinct functions. Given the large number of B cell-directed drugs which are in clinical development or already approved it seems likely that an increased knowledge of the human B cell subsets will not only provide important insights into the pathology of immune-mediated diseases but will also result in novel therapeutic strategies.

Disclosures

No relevant conflicts of interest to declare.
Regulation of Stimulated and Basal Release of Weibel-Palade Bodies By Syntaxin-3 Containing SNARE-Complexes
Authors: Van Breevoort Dorothee , Wahedi Maryam , Schillemans Maaike , Carter Tom , Huveneers Stephan , van den Biggelaar Maartje , Voorberg J.J. , Bierings Ruben
Published: October 2019

Vascular endothelial cells contain unique rod-shaped secretory granules, called Weibel-Palade bodies (WPBs), which contain a number of haemostatic, angiogenic and inflammatory mediators. Several components that are critical for regulated WPB exocytosis have been identified, including the small GTPase Rab27A and its effector synaptotagmin-like protein 4-a (Slp4-a), but the mechanism remains unclear. We have previously identified syntaxin binding protein 1 (STXBP1) as an endogenous Slp4-a binding partner involved in WPB release, along with the SNARE proteins syntaxin-2 and -3. In this study we investigated the possible role of syntaxin-2 and -3 in WPB exocytosis.

We characterized the subcellular location of these syntaxins in endothelial cells using immunocytochemistry. Syntaxin-2 was primarily associated with the plasma membrane where it localized at VE-caderin-based adherens junctions and at integrin-based adhesions to the extracellular matrix. Interestingly, the t-SNARE syntaxin-3 was primarily associated with WPBs. To further explore its role in WPB biology we mapped the endothelial interaction partners of syntaxin-3 through an unbiased mass spectrometry approach using pull downs of lentivirally-expressed mEGFP-syntaxin-3 with anti-GFP nanobeads. Among its interaction partners are various SNAREs and associated proteins such as syntaxin binding proteins 2 and 5 (STXBP2/5), N-ethylmaleimide-sensitive factor (NSF), SNAP23 and α-SNAP, suggesting we successfully pulled down a SNARE complex and its regulatory machinery that are involved in exocytosis. We further addressed the role of syntaxin-3 in stimulated WPB exocytosis using siRNA-mediated knockdowns and found that depletion of syntaxin-3 led to a significant potentiation of Ca2+- as well as cAMP-mediated VWF secretion. In contrast, we observed a decrease in basal (unstimulated) VWF secretion after silencing of syntaxin-3, which amounts to a significantly increased intracellular VWF content. The potentiation of VWF secretion after depletion of syntaxin-3 was almost completely attributable to an increased WPB pool size; when corrected for increased WPB content the probability of stimulated release of a WPB was unaltered in the absence of syntaxin-3.

Our data position syntaxin-3 as a WPB-linked SNARE-protein that regulates basal secretion of VWF.

Disclosures

No relevant conflicts of interest to declare.
Studies on the Formation of Heinz Bodies. I. Methemoglobin Production and Oxyhemoglobin Destruction
Authors: HARLEY JOHN D. , MAUER ALVIN M.
Published: October 2019

A study of the changes in hemoglobin of erythrocytes incubated with various test substances has been reported. The concentrations of oxyhemoglobin and methemoglobin in these erythrocytes were measured and the term "intact" hemoglobin was introduced, in order to distinguish these pigments from later degradation products of hemoglobin. Certain agents which are known to cause Heinz body formation and erythrocyte destruction in vivo have been shown to cause methemoglobin formation and "intact" hemoglobin destruction in vitro, and some of these agents were also shown to produce these changes in vivo. The possible significance of these findings in relation to the role of methemoglobin formation in Heinz body production has been discussed.
CONGENITAL HYPOPLASTIC ANEMIA ASSOCIATED WITH MULTIPLE DEVELOPMENTAL DEFECTS (FANCONI SYNDROME)
Authors: ESTREN S. , SUESS JOHN F. , DAMESHEK WILLIAM
Published: October 2019

A sporadic example of Fanconi syndrome (congenital hypoplastic anemia in association with other congenital defects) is reported.

The significance of the simultaneous occurrence of multiple congenital defects in the pathogenesis of congenital hypoplastic anemia is discussed.
THE USE OF PHOTO-ELECTRIC TURBIDOMETRY IN THE DETERMINATION OF RED CELL COUNTS, HEMATOCRITS, AND HEMOGLOBIN
Authors: WHITLOCK J. H.
Published: October 2019

1. Blood turbidometry is recommended as a screening technic for distinguishing between anemic and nonanemic individuals.

2. Blood turbidometry must be supplemented by other technics for an exact diagnosis of the type of anemia. However, with such help it makes its own contribution to the accuracy of the diagnosis.

3. Blood turbidometry alone would seem to be capable of following an anemic individual’s response to therapy once the proper diagnosis is established.

4. Determination of hemoglobin concentration by turbidometry appears as a distinct possibility. However, further investigation is necessary to validate its utility.

5. Evidence is accumulated that the shadow-volume relationship is a constant in several species.
INCONSTANCY AND VARIABILITY OF THE VASCULAR FRAGILITY TEST EVEN IN PURPURIC CONDITIONS
Authors: ROSKAM J. , RENARD CH. , SWALÜE L.
Published: October 2019

Two unusual cases of purpura simplex raise the problem, as yet unsolved, of the pathogenesis of the purpuric eruption. Together with a previous observation of hemorrhagic purpura made by Roskam, they show the inconstancy and variability of the vascular fragility test.
ULCERS OF THE LEG IN MEDITERRANEAN DISEASE
Authors: ESTES J. EARLE , FARBER EUGENE M. , STICKNEY J. M.
Published: October 2019

Ulceration of the skin of the legs may occur in Mediterranean disease. Such ulceration cannot be distinguished grossly from that occurring in sickle cell anemia and congenital hemolytic icterus. The outstanding histologic feature (noted at biopsy of one of these ulcers) is the prominent deposition of iron in the cutis.
THE EFFECT OF QUANTITATIVE AND QUALITATIVE PROTEIN DEFICIENCY ON BLOOD REGENERATION. I. WHITE BLOOD CELLS
Authors: GUGGENHEIM K. , BUECHLER EDITH
Published: October 2019

1. The effect of diets, varying in quantity or quality of protein, on white blood cell regeneration was studied in leukopenic rats, the leukopenia having been induced by a protein-free diet.

2. Diets containing different amounts of casein (3, 6, 9 and 18 per cent, respectively), were fed ad libitum. At the 3 per cent level, a further decrease occurred of white blood cells, whereas the other three diets initiated a regeneration of leukocytes, its degree being more or less in proportion to the casein content.

3. In experiments with diets containing 18 and 30 per cent of casein, the amount of protein eaten and not its level in diet was the decisive factor in the regeneration of leukocytes. The white blood cell regenerating effect of a diet containing an optimal level of protein, may be neutralized when given in restricted amounts.

4. Diets containing nutritionally inferior proteins, fed at 9 per cent level, also impaired normal regeneration of leukocytes. The white blood cell regeneration afforded by the proteins investigated was found to increase in the following order: maize, gelatin, wheat, casein, processed soya, peanut, meat, egg.

5. In white blood cell regeneration promoted by dietary protein, granulocytes were found to react to a greater degree than lymphocytes and monocytes.
Correction of poor platelet transfusion responses with leukocyte-poor HL-A-matched platelet concentrates
Authors: Herzig RH , Herzig GP , Bull MI , Decter JA , Lohrmann HP , Stout FG , Yankee RA , Graw RG Jr
Published: October 2019

Matching donor-recipient pairs for HL-A antigens provides a logical starting point for selecting donors for recipients with extensive prior transfusion histories. However, during the course of continued exposure to even HL-A-matched platelet concentrates, further sensitization occurs, as indicated by progressively poorer post-transfusion increments and transfusion reactions. There is evidence that such sensitization may be due to non-HL-A antigens. Finally, it is postulated that the poor post-transfusion platelet increments obtained when standard platelet concentrates are used result from the leukoagglutinin antigen-antibody reaction involving the platelet as an “innocent bystander.” The standard platelet concentrate can be purified by a simple method of centrifugation (178 g times 3 min), removing about 96% of the contaminating white blood cells with concomitant loss of about 21% of the platelets. The use of these leukocyte-poor platelet concentrates can restore compatible transfusion increments in highly alloimmunized thrombocytopenic recipients. The luekocyte-poor concentrates can diminish undesirable transfusion reactions following imcompatible platelet transfusions.
Reasons for first line ART modification over the years during the ART scale up in Uganda
Authors: Castelnuovo B. , Mubiru F. , Kalule I. , Kiragga A.
Published: October 2019

Background

During the initial scale up of ART in sub-Saharan Africa, prescribed regimens included drugs with high potential for toxicity (particularly stavudine). More recently a growing number of patients requires second line treatment due to treatment failure, especially following the expansion of viral load testing. We aim to determine the reasons and risk factors for modification of first line ART across the years.

Methods

We included patients started on standard first line ART (2NRTI + 1 NNRTI) between 2005 and 2016 at the Infectious Diseases Institute, Kampala, Uganda. We described the reasons for treatment modification categorized in (1) toxicity (2) treatment failure (3) other reason (new TB treatment, new pregnancy). We used Cox proportional hazard to identify factors associated with treatment modification due to toxicity.

Results

We included 14,261 patients; 9114 (63.9%), were female, the median age was 34 years (IQR: 29–40), 60.8% were in WHO stage 3 and 4. The median BMI and CD4 count were 21.9 (IQR: 19.6–24.8) and 188 cell/µL (IQR: 65–353) respectively; 27.5% were started on stavudine, 46% on zidovudine, and 26.5% on a tenofovir containing regimens. We observed 6248 ART modifications in 4868/14,261 patients (34.1%); 1615 were due to toxicity, 1077 to treatment failure, 1330 to contraindications, and 1860 patients following WHO recommendation of phasing out stavudine and substituting with another NRTI. Modification for drug toxicity declined rapidly after the phase out of stavudine (2008), while switches to second line regimes increased after the implementation of viral load monitoring (2015). Patients with normal BMI compared to underweight, (HR: 0.79, CI 0.69–0.91), with CD4 counts 200–350 cells/µL compared to < 200 cells/µL (HR: 0.81− CI 0.71–0.93), and started on zidovudine (HR: 0.51 CI 0.44–0.59) and tenofovir (HR: 0.16, CI 0.14–0.22) compared to stavudine were less likely to have ART modification due to toxicity. Older patients (HR: 1.14 per 5-year increase CI 1.11–1.18), those in WHO stage 3 and 4 (HR: 1.19, CI 1.06–1.34) were more likely to have ART modification due to toxicity.

Conclusions

Toxicity as reason for drugs substitution decreased over time mirroring the phase out of stavudine, while viral load expansion identified more patients in need of second line treatment.