bluuebunny/crossref_metadata_2025 huggingface dataset:
Variable Support Towards ASCO Recommendations for Venous Thromboembolism (VTE) Prophylaxis and Treatment in Patients with Cancer
Authors: Matzdorff Axel , Schwindel Uwe , Mueller Michael , Hutt Hans Joachim
Published: October 2019

Objectives: To determine the support of physicians for the recently published ASCO evidence-based clinical practice guidelines for venous thromboembolism (VTE) prophylaxis and treatment in patients with cancer (J Clin Oncol. 2015;33:654-6).

Methods: ASCO key recommendations were presented as part of three CME seminars to physicians not specialized in hem/onc or hemostasis. In addition two specialists in the field discussed a list of arguments for and against each of the recommendations considering (1) quality of scientific evidence underlying each recommendation, (2) feasibility in daily practice including sensitivity to patients preferences, and (3) medicolegal implications and reimbursement aspects. After each presentation attendees were asked to fill out a questionnaire on how much they supported the ASCO recommendations and each of the pro&con arguments.

Results: A total of 89 physicians attended the three meetings. 56 questionnaires were returned. The ASCO recommendations with the highest degree of support were:

‒ Patients undergoing major cancer surgery should receive prophylaxis for 7-10 days and even 4 weeks after major abdominal or pelvic surgery with high-risk features (84% pro)

‒ LMWH should be given for the initial treatment as well as for long-term secondary prophylaxis (80% pro).

The recommendations with the lowest degree of support were:

‒ Patients with cancer should be educated about signs and symptoms of VTE. They should be periodically assessed for VTE (59% pro).

‒ Anticoagulation should not be used to extend survival of patients with cancer in the absence of other indications (56% pro).

Conclusion: After presenting ASCO recommendations as part of CME training the degree of physicians' support was still at best moderate and sometimes even close to neutrality. In daily medical practice scientific evidence has no priority to clinical practicability, patients preferences, medicolegal aspects and cost-restrictions. Guideline authors should complement their recommendations with advice from their own practice on how to implement guidelines in daily care and how they manage financial and medicolegal restrictions. In addition there is an unmet need for more education on VTE prophylaxis and treatment among physicians not specialized in hem/onc or hemostasis who are caring for cancer patients.

Disclosures

Matzdorff: AMGEN - e. Honoraria: Honoraria; Aspen Germany: Honoraria; Bayer: Equity Ownership; Boehringer Ingelheim: Honoraria; Behring: Honoraria; Bristol Myers Squibb: Honoraria; GlaxoSmithKline: Honoraria; LEO Pharma: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Equity Ownership, Honoraria. Schwindel:LEO Pharma GmbH: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria; Amgen: Honoraria; Nordic Pharma: Honoraria. Hutt:LEO Pharma GmbH: Employment.
Increased Outpatient Care Can Reduce Hospital Stays for Sickle Cell Patients
Authors: Landau Danny A. , Steininger Kaitlyn , Landis Erica
Published: October 2019

Introduction: Sickle cell disease is associated with frequent hospitalizations and emergency room visits when patients enter vasooclusive crisis. This is associated with lengthy hospitalization that can be time life interrupting for patients, increase hospital cost, and limit bed availability for other patients. Some patients with sickle cell have higher inpatient requirements than others due to the frequency of crisis. Recognizing the issues associated with this, we began scheduling patients for frequent outpatient IV fluids and pain medication to try and prevent ER visits and lengthy hospital stays. We are reporting the experience of three of our patients who have had the most frequent hospitalizations and emergency room visits.

Methods: Patients who had the highest rate of hospitalizations and emergency room visits were offered regular outpatient intravenous and pain medication during the week (up to four times per week) on an as needed basis. Chairs were reserved for these purposes so on demand therapy could be offered without significant delays. The number of ER visits and hospital stays were counted from 12 months before and 12 months after we started offering this. For one of the three patients, data on length of hospital stay could not be accurately determined due to a lengthy hospitalization for infection rather than sickle cell pain crisis, but his emergency room visits were still counted. Basic hospitalization cost for daily hospital charge was also considered, but additional costs such as drug administration, diet, imaging, etc. were not.

Results: In the 12 months prior to starting regular outpatient IV fluids, there were 48 aggregate emergency room visits compared to 26 in the 12 months after, a reduction of 38%. Inpatient hospital days before were 91 before and 46 post (49%). Cost for hospital days were reduced from $224497 to $113480 (49%).

Conclusion: While offering more outpatient support to our sickle cell patients with the highest hospital demand, we can cut hospital days in half, and limit ER visits and also reduce the cost for inpatient care. Increasing outpatient support, when feasible, should be considered for sickle cell patients who require regular hospitalizations.

Figure 1. Figure 1.

Disclosures

No relevant conflicts of interest to declare.
Impact of a Cancer Associated Thrombosis Clinic on Reducing Hospitalization for Venous Thromboembolism: The Cleveland Clinic Experience
Authors: Afzal Amber , Khorana Alok A. , Ornstein Moshe , McCrae Keith
Published: October 2019

INTRODUCTION:

Cancer patients with venous thromboembolism (VTE) have threefold increase in all cause hospitalization and days of hospitalization than cancer patients without VTE. Hospitalization with VTE is also associated with higher overall all cause inpatient cost (PMID 23430767). We hypothesized that a centralized cancer-associated thrombosis clinic instituted at the Cleveland Clinic in August 2014 would reduce hospital admissions for this indication and impact the cost of healthcare.

METHODS:

The study population comprised two cohorts: cancer patients admitted with a new diagnosis of VTE to the Cleveland Clinic Main Campus between August 1 and December 31 in 2013 before initiation of the cancer-associated thrombosis (CAT) clinic and between August 1 and December 31 in 2014 after initiation of CAT clinic. Patients who were accepted as transfers from outside hospitals were excluded from the study.

RESULTS:

In the pre CAT clinic study period, there were 101 admissions. Of these, 51 (50%) were admitted primarily for a thromboembolic event whereas the rest had VTE as a secondary diagnosis. In the post CAT clinic study period, there were 94 admissions. Of these, 42 (44.7%) were admitted primarily for a thromboembolic event. The baseline characteristics of the two study groups are detailed in table 1.

There was a reduction in hospital admissions for VTE by 9 admissions in the post CAT clinic period (RR 0.89, RRR 11%, p 0.252). The median length of hospital stay per patient was 3 days, reducing the total days of hospitalization by 27 days in post CAT clinic period. Figure 1

CONCLUSION:

We demonstrated that a centralized CAT clinic can reduce but not eliminate admissions for VTE. Larger health care systems should consider the development of such a service for the management of VTE in cancer patients as it can lead to significant cost savings and reduce unnecessary exposure of this vulnerable patient population to hospital environment

Table 1. Characteristic Pre CAT clinic period (N) Post CAT clinic period (N) Patients admitted with VTE as primary diagnosis 51 42 Median age (years) 62.41±12.85 61.6±13.4 Female patients 22 (44%) 26 (39%) Solid tumor diagnosis 42 (82.4%) 36 (70.6%) Hematological malignancy 9 (17.6%) 6 (11.8%) DVT 16 (31.4%) 12 (28.6%) PE 35 (68.6%) 30 (71.4%) PE patients with high PESI score (Class III/IV) 33 (94%) 28 (93%) Median length of hospital stay (days) 3 (1-27) 2.5(1-16)

Figure 1. Figure 1.

Disclosures

Khorana: Pfizer: Consultancy, Honoraria; sanofi: Consultancy, Honoraria; Leo Pharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Boehringer-Ingelheim: Consultancy, Honoraria; Daiichi Sankyo: Consultancy, Honoraria. McCrae:Janssen Scientific Affairs: Membership on an entity's Board of Directors or advisory committees.
Mutations in the Poly(A)-Specific Ribonuclease (PARN) Impair Telomerase RNA 3' End Maturation in Dyskeratosis Congenita Patients
Authors: Moon Diane H. , Segal Matthew , Boyraz Baris , Guinan Eva , Hofmann Inga , Cahan Patrick , Tai Albert K , Agarwal Suneet
Published: October 2019

Mutations in 11 genes have been described in patients with dyskeratosis congenita (DC) and related telomere diseases, and account for ~60% of cases. Amongst these, loss-of-function mutations in the poly(A)-specific ribonuclease (PARN) have most recently been found in patients with DC and idiopathic pulmonary fibrosis. PARN has no known role in telomere biology. Why PARN deficiency should mimic telomere diseases remains unclear. Based on its 7-methylguanylate (m7 G) cap recognition and poly(A) deadenylase functions, PARN's primary role is considered to be in regulating mRNA metabolism. Recent studies implicate PARN in non-coding RNA biogenesis, including maturation of small nucleolar RNAs (snoRNAs) via the deadenylation of oligo-adenylated (oligo(A)) intermediates. In two DC patients with biallelic defects in the PARN gene, we found decreased levels of the telomerase RNA component (TERC). TERC is the essential non-coding RNA template and scaffold of the telomerase holoenzyme. TERC possesses an m7 G cap but unlike mRNAs does not have a long poly(A) tail; rather, TERC contains a box H/ACA motif as is found in some snoRNAs. We hypothesized that TERC is regulated by PARN in a manner similar to snoRNAs, given their shared 3′ box H/ACA architecture. Here, usingsomatic cells and induced pluripotent stem (iPS) cells from DC patients with PARN mutations, we show that PARN is required for the 3′ end maturation and accumulation of TERC RNA. In PARN-mutant iPS cells and PARN-deficient cell lines, we find impaired telomerase activity and telomere maintenance. Deep sequencing of TERC 3' ends reveals that PARN is required for the removal of genomically-encoded extensions and post-transcriptionally acquired oligo(A) tails that target nuclear RNAs for destruction. In keeping with this, TERC transcripts from PARN-mutant patient cells decay at an accelerated rate. The diminished TERC steady-state levels, increased oligo(A) forms of TERC, and defects in telomere maintenance in patient cells are rescued by ectopic expression of PARN. Global transcript analysis by RNA-Seq in patient cells and PARN-deficient cell lines compared to controls reveals no protein coding mRNAs that consistently manifest a fold-change exceeding the change in TERC levels. Our data indicate that PARN functions in TERC biogenesis via deadenylation of oligo-adenylated nascent transcripts, which promotes 3' end maturation and stability. Given these findings and the phenotype of patients with PARN mutations, we speculate that a major, non-redundant function of PARN in human cells is regulating the maturation of TERC and other non-coding RNAs, more so than mRNA metabolism. Our results reveal a novel role for PARN in the biogenesis of TERC, and provide a mechanism linking PARN mutations to defective telomere maintenance in DC and related diseases.

Disclosures

No relevant conflicts of interest to declare.
The Fusion Gene Landscape in Multiple Myeloma, with Clinical Impact
Authors: Cleynen Alice , Szalat Raphaël , Samur Mehmet Kemal , Parmigiani Giovanni , Munshi Nikhil C. , Avet-Loiseau Hervé
Published: October 2019

Background: Gene fusions play an important role in aberrant cellular biology as well as development and progression of cancer. Expression of fusion genes such as PML-RAR drives the transformation in APL and provides important targets for therapy. However, in multiple Myeloma (MM), a heterogeneous disease characterized by genomic instability, frequent gains and losses of DNA, and a diverse mutational landscape, only the well characterized MMSET-IGH fusion product has been reported. Here we investigate the fusion gene landscape in multiple myeloma, and its possible impact on survival.

Method: Deep RNA-Seq was performed on purified MM cells from 430 newly-diagnosed MM patients, 20 normal individuals and 71 cell-lines; data were analyzed for gene expression profiles, long-non coding RNA signatures, and both novel and known fusion genes using two common algorithms: TopHat and MapSplice. MM characteristics, cytogenetic and FISH as well as clinical survival outcomes were also analyzed and correlated with genomic data.

Results: After filtering candidate fusions linking genes belonging to the same family, we identified 416 different candidates in myeloma patients, 40 % of which identified either IGH or Kappa as a partner. IGH fusion partners included the previously described and validated WHSC1 and B2M genes, as well as over 50 new candidates, while more than 70 different partners were found to be fused with Kappa. These genes exhibit functional enrichment of positive regulators of the cytokine-mediated signaling pathway, negative regulation of myeloid cell differentiation, negative regulation of interleukin-6 production, as well as others. 31% of patients presented no fusions, and another 32% presented a single fusion event. The other 37% presented at least 2 fusion candidates, with up to 27 different candidates. Similar patterns were observed in cell-lines, with 196 unique candidates identified, only 16% of which involving IGH or kappa. However, all partners were found in at least one patient as well. Only 12% of cell-lines exhibited no fusion, and another 14% presented only one fusion. On average, 3 fusions were identified per cell-line, with a maximum of 10. Validation of some these fusion genes is required to understand their functional role. Importantly, although having IgH-or kappa-related fusions did not affect patient outcome by themselves, patients with high numbers of fusion candidates had worse event-free survival.

Conclusion: Our data describes a diverse and rich fusion gene landscape in Multiple Myeloma. Similar to mutational profiles, there is no predominant fusion gene driving the disease process. Association of poor prognosis with a higher number of fusions may indicate that genomic instability plays an important role in the biology of Multiple Myeloma.

Disclosures

No relevant conflicts of interest to declare.
Mesenchymal Stem Cell As a Salvage Treatment for Patients with Refractory Bronchiolitis Obliterans Syndrome after Allogenetic Hematopoietic Stem Cell Transplantation
Authors: Zhao Ke , Fan Zhiping , Huang Fen , Xiang Peng , Liu Qifa
Published: October 2019

Background Bronchiolitis obliterans syndrome (BOS) is the most detrimental late pulmonary complications after allogeneic hematopoietic stem cell transplantation, as it is characterized by non-responsiveness to treatment, leading to high morbidity and mortality. Mesenchymal stem cell (MSC) has been considered as an effective treatment for refractory aGVHD, but the response to treat cGVHD, especially refractory BOS, is rarely reported. This study evaluated the efficacy of MSC from bone marrow (BM) of a third-party donor to BOS patients.

Methods Fifty-three patients with refractory BOS were enrolled in our prospective study, including 29 patients in MSC group and 24 patients in non-MSC group. All patients had previously failed to at least 2 lines of immunosuppressive therapy. MSCs were given at a median dose of 1×106 cells/kg once weekly for 4 weeks as one cycle treatment. The responsiveness of MSC was evaluated after one cycle, non-responsive patients discontinued MSC treatments, others continued to accept another cycle of MSC treatments.

Results Fifty-three patients developed BOS at a median time of 172 days (range, 94 to 398). After a total of 169 doses of MSC were administered, with a median of 6 (range:3-11) doses per patient, the overall response (OR) rate in MSC group was 75.9%, including CR in 13.8% and partial response (PR) in 62.1%. Compared with MSC group, OR rate in non-MSC group was 16.7%, including CR in 4.2% and PR in 12.5% (P=0.000; P=0.233; P=0.000, respectively). Furthermore, the efficacy of MSC to refractory BOS was significantly related with the severity of pulmonary function (mild VS severity, P=0.039). During the median follow-up time of 814 (range:228-2499) days post-transplantation, 7 patients were dead in MSC group, while 18 patients died in non-MSC group. The 5-year OS post-transplantation in MSC group were 68.6%±5.7%, compared with 21.2%±7.1% in non-MSC group (P=0.033). No patients experienced any toxic side effects and other secondary tumors after MSC treatment.

Conclusion MSC derived from BM of third-party donors is a promising treatment for patients with refractory BOS.

Disclosures

No relevant conflicts of interest to declare.
Hematopoietic Peripheral Circulating Blood Stem Cells As an Independent Marker of Good Transfusion Management in Patients with Beta-Thalassemia
Authors: Napolitano Mariasanta , Gerardi Calogera , Di Lucia Anna , Accardo Pietro Andrea , Rizzuto Luigi , Maria Ferraro , Tarantino Giuseppe , Siragusa Sergio , Buscemi Filippo
Published: October 2019

Aim Aim of the current study was to prospectively evaluate the potential role of peripheral circulating CD34+ stem cells as new independent marker of appropriate hemopoietic balance in patients with thalassemia major and intermedia.

Materials and methods Peripheral blood samples from patients with thalassemia major (TM) and intermedia (TI) were drawn. Peripheral circulating CD34+ stem cells, CF-GEMM, CFU-GM and BFU-GM were assayed with monoclonal antibodies for CD34 and clonogenic tests, according to standard procedures and ISHAGE method (BD stem cell enumeration kit, Becton Dickinson; H4434, Stem Cell Technology). Demographic and clinical data were recorded from each enrolled subject. Results were compared with healthy controls.

Results Overall, 56 patients with thalassemia major (median age:35 years, range:13-52 years) and 13 with thalassemia intermedia (median age:44 years, range:27-67) were evaluated. Annual red blood cells transfusion requirements ranged from 10 to 65 in all the patients except three with thalassemia intermedia, that were transfusion independent. One patient with TM did not accept transfusion for religion (Table 1). A statistically significant increase in peripheral circulating stem cells was observed in all the patients, in comparison with healthy controls. Mean peripheral circulating CD34+cells were 6.9±4.5/mmc in patients with TM and 11.8±14.8/mmc in patients with TI, significantly higher than mean CD34+ cells of healthy volunteer blood donors values (3.5±2.9/mmc, p=0.014 and p= 0.051 respectively for TM and TI). Only in patients with TI an increase in CFU-GEMM (3.0±4.8 vs 0.75±2.05, p=0.0001) was observed. Patients not treated with transfusions showed the mean highest levels of circulating stem cells (CD34: 32.5±14.8/mmc, BFU-E: 41.3±22.8, CFU-GM: 19.6±5.6, CFU-GEMM 9.0±6.1). At multivariate analysis, peripheral circulating CD34+ stem cells did not correlate with age, sex, number of red blood cells units transfused, hemoglobin levels, history of splenectomy and hypothyroidism.

Conclusion Circulating peripheral CD34+stem cells are increased in patients with the highest hemopoietic stress (TI, transfusion independent), thus their determination could represent a useful independent marker of clinical response to transfusion in patients with beta thalassemia.

Table 1. Baseline data of patients with thalassemia Thalassemia major (n=56) Thalassemia intermedia (n=13) Males 28 7 Females 28 6 Mean Age (range) 35 (13-52) 42(27-67) BMI (range) 21.5 (15.7-27.3) 21.4 (14.7-33.5) Splenectomy 31 13 Hypothyroidism 9 1 Smokers 32 31

Disclosures

No relevant conflicts of interest to declare.
First Case of Leukemia in a Child Suffering from Cyclic Neutropenia with ELANE Mutation
Authors: Zeidler Cornelia , Mellor-Heineke Sabine , Klimiankou Maksim , Skokowa Julia , Welte Karl
Published: October 2019

Congenital neutropenia (CN) and Cyclic neutropenia (CyN) are rare hematological conditions in which ELANE mutations have been found. The discrimination of Cyn from CN is based on the cycling neutrophil counts which decrease to <0.2 x 109/L, usually at 21 day intervals. Infectious episodes are typically less severe in CyN compared to infections in CN patients. While in patients suffering from ELANE-CN an increased risk of leukemic transformation of more than 10 percent is well documented, no AML has been reported in the European SCNIR cohort of 38 patients with ELANE -CYN so far.

Here we report the first case of AML secondary to ELANE -CyN in a 17 year old girl. Severe neutropenia was first diagnosed at the age of four weeks during a septicemia with pseudomonas, but no serial blood counts were collected. G-CSF treatment was initiated at an age of 2 years after recurrent otitis media, pneumonia and skin abscesses. Infectious events occurred infrequently under G-CSF treatment. At an age of 14 years she was referred to us after a severe infectious event (liver abscess) and an unexplained high range of ANCs (0 to more than 10x109/µl) under G-CSF treatment with varying doses. Sequential blood counts confirmed the diagnosis of CyN. Molecular genetic testing revealed a heterozygous ELANE mutation, both parents are ELANE negative. Previous bone marrows varied between a maturation arrest on the promyelocyte stage with absence of neutrophils and up to 16 percent segmented neutrophils. No cytogenetic aberrations were detected prior to AML. AML was diagnosed recently when she presented with decreasing platelets and hemoglobin. Bone marrow at this time showed 14% blasts and presence of monosomy 7 plus trisomy 21 in the cytogenetic evaluation.

Molecular genetic evaluation of the bone marrow revealed presence of a CSFR3 and RUNX1 mutations in the leukemic cells.

Conclusion

Our patient presented with a secondary AML after ELANE positive cyclic neutropenia. Cytogenetics, CSFR3 and RUNX1 mutations in the bone marrow at the time of leukemia diagnosis have been described in the pathway of leukemogenesis in Congenital neutropenia with ELANE, HAX1 or other underlying mutation.

Our patient is the first case of leukemia in ELANE -CyN and proofs that ELANE -CyN is another pre-leukemic condition. However, the risk of myeloid transformation is very low.

Disclosures

No relevant conflicts of interest to declare.
DNA Editing: A Primer
Authors: Mali Prashant
Published: October 2019

RNA-guided Cas9 nucleases based on the prokaryotic CRISPR-Cas systems provide an unprecedented ease in being able to edit the genomes of diverse organisms. As a single unifying factor capable of co-localizing RNA, DNA, and protein, I believe tools and techniques based on Cas9 will grant exquisite control over cellular organization, regulation, and behavior. Here I will describe work on development of the CRISPR-Cas9 targeting methodology, and detail current and prospective genome-engineering methodologies.

Disclosures

No relevant conflicts of interest to declare.
Modified IPS Cells for Hemoglobinopathies
Authors: Townes Tim M. , Ding Lei , Chang Chia-Wei , Lai Yi-Shin , Sun Joe , Pawlik Kevin
Published: October 2019

We have recently used CRISPR-Cas to correct the HBB gene in induced Pluripotent Stem Cells (iPSC) derived from patients with Sickle Cell Disease (SCD) and to correct the JAK3 gene in iPSC derived from patients with Severe Combine Immune Deficiency (SCID). Off-target mutations were minimized, if not eliminated, by use of paired guide RNAs and the Cas9 nickase. When erythroid progenitors (EP) produced from corrected SCD iPSC are transplanted into NSG mice, a complete switch from gamma- to beta-globin gene expression occurs within 24 hours and high levels of beta mRNA are synthesized; the betaA:betaS ratio is 60:40. Hematopoietic progenitors produced from corrected SCID iPSC can be differentiated into T cell populations that express a full repertoire of T Cell Receptors (TCRs). These results suggest that CRISPR-Cas enhanced gene replacement may provide safe and effective therapies for many erythroid and lymphoid disorders. In an alternative approach, we electroporated a preformed, biochemical complex composed of a guide RNA (gRNA), a modified recombinant Cas9 (mrCas9) and a single stranded oligodeoxnucleotide (ssODN) into sickle iPSC or primary sickle CD34+ hematopoietic progenitors. Sixty-five percent of sickle iPSC colonies contained at least one corrected allele. When homozygous corrected colonies were differentiated into erythroid progenitors and transplanted into NSG mice, human erythroid cells expressed 100% HbA. When primary sickle CD34+ hematopoietic progenitors were electroporated with the gRNA/mrCas9/ssODN complex and differentiated into erythroid cells in vitro, HbA was expressed at 30-40%. These results suggest that primary bone marrow CD34+ cells that are isolated from patients with sickle cell disease, beta-thalassemia, SCID, DBA and other hematopoietic disorders may be electroporated with specific gRNA/mrCas9/ssODN complexes and transplanted into patients to rapidly, safely and effectively treat these debilitating disorders.

Disclosures

No relevant conflicts of interest to declare.
Engineering the Cancer Genome
Authors: Jacks Tyler
Published: October 2019

Over the past several years, my laboratory has employed gene targeting technology to create a series of mouse models of cancer that share genetic and pathological features of the cognate diseases in humans. We have focused particularly on models of lung cancer, both adenocarcinoma and small cell lung cancer. These models have been subjected to various types of molecular characterization, including mRNA profiling and exome/whole-genome sequencing. These efforts have defined genes and pathways that promote tumor progression and metastasis, including in the case of the adenocarcinoma model down-regulation of the lineage-restricted transcription factor Nkx2.1 and other transcriptional regulators that are involved in inducing and enforcing various differentiation states that might limit tumor progression. We have also studied the involvement of subpopulations of tumor cells in this model in which the Wnt signaling pathway is active. Data from lineage tracing experiments are consistent with this population having increased tumor propagating potential. In order to functionally characterize these and other cancer genes, we have recently developed methods to mutate genes in developing tumors using the CRISPR/Cas9 system. Using tri-functional lentiviral, we have developed tumors that have undergone Cas9-mediated mutation of a series of genes of interest. Several examples of the application of this method will be reviewed. As another example of cancer genome manipulation, we have engineered tumors of the lung and other sites to express specific T cell antigens and/or NK cell ligands. These models have allowed a detailed investigation of adaptive and innate immune responses to a developing tumor. We have used the models to explore mechanisms of immune suppression in cancer and develop methods to elicit improved anti-tumor immune responses.

Disclosures

No relevant conflicts of interest to declare.
New Approaches for Mapping Epigenome Dynamics
Authors: Henikoff Steven
Published: October 2019

The protein complexes that package our genomes must be mobilized for active processes to occur, including replication and transcription, but until recently we have only had a static, low resolution view of the "epigenome". Genomes are packaged into nucleosomes, octamers of four core histones wrapped by 147 base pairs of DNA. Nucleosomes present obstacles to transcription, which over genes is the RNA Polymerase II (RNAPII) complex, and one current challenge is to understand what happens to a nucleosome when RNAPII transcribes through the DNA that it occupies. We study this process by developing methods for following nucleosomes as they are evicted and replaced. Among the factors that we have implicated in the process is torsional stress, which we can now measure genome-wide. RNAPII movement can unwrap nucleosomes and thus destabilize them, causing them to be occasionally evicted and replaced. Interestingly, we find that destabilization of nucleosomes during transcription is enhanced by anthracycline compounds, widely used chemotherapeutic drugs that intercalate between DNA base pairs, thus suggesting a new mechanism for cell killing during chemotherapy. We are also interested in what happens to RNAPII during its encounter with a nucleosomes. In vitro, RNAPII cannot transcribe completely through a nucleosome, but rather stalls as it tries to unwrap the DNA from around the core. We have been studying this process in vivo, and have developed a simple method for precisely mapping RNAPII genome-wide. We have used this method to show exactly where RNAPII stalls as it unwraps a nucleosome in vivo, surprisingly in a different place in vivo from where it stalls in vitro. We also have discovered that a variant histone, H2A.Z, which is found in essentially all eukaryotes, helps to reduce the nucleosome barrier to transcription, and in this way may modulate transcription. Other protein components of the epigenome involved in dynamic processes are nucleosome remodelers, which use the energy of ATP to slide or even evict nucleosomes from DNA. Some remodelers help RNAPII get started and others help it overcome the nucleosome barrier to transcription, and by mapping them at base-pair resolution, we can gain insight into how they act. We have also applied our high-resolution mapping tools to transcription factors, which bind DNA at specific sites to regulate transcription and other processes. Our ability to achieve high spatial and temporal resolution mapping of the binding and action of nucleosomes, transcription factors, remodelers and RNAPII provides us with a detailed picture of epigenome dynamics. By using these tools we are beginning to understand how DNA sequence and conformation are recognized for regulation of transcription and other epigenomic processes.

Disclosures

No relevant conflicts of interest to declare.
Noncellular Niche Influences: Nervous System Mediators, Metabolic Mediators, and Hypoxia/Oxidative Stress
Authors: Méndez-Ferrer Simón
Published: October 2019

Hematopoietic stem cells (HSCs) traffic between bone marrow and circulation, what allows for life-saving clinical transplantation. Our previous work has shown that HSC numbers in blood follow circadian oscillations that are regulated by the central pacemaker in the brain, which reaches bone marrow nestin+ mesenchymal stem cells through peripheral sympathetic nerves. In the perinatal bone marrow, HSC-niche forming mesenchymal stem cells might be different from those that form the skeleton and some of them might be neural crest-derived, like peripheral neurons and supporting glial cells. Thus, tight regulation of the bone marrow stem-cell niche in vertebrates might build upon developmental relationships of its cellular components. We have found recently that cholinergic nerves regulate HSC maintenance, proliferation and migration in divergent niches. We will present unpublished evidence of how both branches of the autonomic nervous system cooperate to regulate HSC maintenance and function in spatially and temporally distinct niches. Moreover, we have shown recently that damage to this regulatory network is essential for the manifestation of myeloproliferative neoplasms. In these diseases, previously thought to be driven solely by mutated HSCs, protecting the HSC niche might represent a novel therapeutic strategy. Patients with myeloproliferative neoplasms have a higher risk of developing acute leukemia. However, at this stage, leukemic cells might be less sensitive to the normal control by the microenvironment and, instead, acute myelogenous leukemic cells might transform the bone marrow niches to support their own survival. We will discuss potential contributions of HSC niches to myeloproliferative neoplasms and MLL-AF9-driven acute myeloid leukemia.

Disclosures

Off Label Use: Potential use of selective estrogen receptor modulators and beta3-adrenergic agonists in myeloproliferative neoplasms.
Educating the Innate Immune System
Authors: Worthen Scott , Mei Junjie , Liu Yuhong , Deshmukh Hitesh
Published: October 2019

The microbiome regulates the development of innate immunity G. Scott Worthen, Junjie Mei, Yuhong Liu, Ning Dai, Hitesh Deshmukh Circulating granulocytes are maintained within a fairly narrow window for each individual during homeostasis. This regulatory system is nonetheless capable of dramatic shifts during stress. The mechanisms that govern control over granulocyte number remain incompletely understood. Recent information, however, provides clues to the feedback control systems that regulate homeostatic and emergency granulopoiesis. The first clues have come from adult mice with abnormalities in granulocyte function. Defects in Leukocyte Integrins or chemokine receptors result in marked increases in circulating (?2 integrin) or bone marrow (Cxcr2) neutrophils, associated with increased circulating IL-17 and G-CSF. This result appears to be due to a functional inability to arrive at sentinel site(s) whence they are attracted by (among others) Cxcl5. Strikingly, a single infusion of normal WT neutrophils resets this feedback loop. One signal for expression of IL-17 and G-CSF appears to be the gut microbiome. Antibiotic treatment also resets the system, reducing neutrophil numbers and cytokine expression. Thus, at sentinel site(s), the supply of functional neutrophils is balanced against perceived threat from the microbiome. In neonates, exposure to commensal organisms is an immediate result of birth from a sterile environment into a dirty one. We have described postnatal granulopoiesis in murine neonates, a dramatic increase in circulating and bone marrow neutrophils, that lasts for 5-7 days. Human infants similarly have been shown to demonstrate a postnatal increase in circulating neutrophils for 72 hrs after birth. Antibiotic-exposed mouse pups fail to develop postnatal granulopoiesis, as do mice deficient in IL17ra, MyD88, TLR4, or G-CSF even if not exposed to antibiotics, indicating a pathway that requires exposure to LPS that induces IL17 and G-CSF, as in adults. In contrast to adults, however, commensal bacteria are required for host defense. Antibiotic-exposed pups are exquisitely sensitive to E. coli sepsis, which can be partially reversed by transfer of cecal contents or exogenous G-CSF. Furthermore, small quantities of LPS, fed by gavage, can also partially protect. Thus, neonates use exposure to commensal bacteria and their products to trigger the rapid expansion and functional maturation of granulocyte development, and in so doing, prepare the neonate for potential exposure to pathogens. Adults maintain this system, where it is at least partially responsible for maintenance of granulocyte homeostasis.

Disclosures

No relevant conflicts of interest to declare.
Reacting to Inflammatory Signals
Authors: Carlesso Nadia
Published: October 2019

Specialized cellular niches in the bone marrow (BM) modulate critical functions of the hematopoietic stem and progenitor cells, such as self-renewal, cell-fate decisions and the balance between proliferation and differentiation. Constituents of the BM niche, such as osteoblasts, endothelial cells and macrophages, respond to inflammation with secretion of pro-inflammatory cytokines, and serve as effectors of the inflammatory circuitry; thus their excessive activation can potentially impact on the regulation of hematopoietic cells, and on the initiation and progression of myeloid malignancies.

Our current understanding of the mechanisms for Bcr/Ab-negative myeloproliferative neoplasia (MPN) involves recognition of driver mutations (targeting i.e. Tet2, Jak2, Mpl, or Asxl) and of an inflammatory microenvironment. However, despite accumulating evidences on the role of inflammation and of the microenvironment in the maintenance and progression of myeloid malignancies, it is still unclear how the inflamed BM niche contributes to disease establishment and progression. Similarly, the causes leading to an inflammatory microenvironment in MPN are not fully understood.

We use genetically-controlled animal model of BM inflammation to study the contribution of the inflammatory microenvironment to MPN. We found that loss of Notch signaling in the BM microenvironment results in an inflammatory state of the BM niche promoting myeloproliferation and marrow fibrosis. Using this model, we discovered that loss of Notch/RBPJ signaling leads to transcriptional upregulation of the pro-inflammatory microRNA miR-155, especially in BM endothelial and mesenchymal cells, resulting in miR-155-dependent inhibition of the NF-κB inhibitor κB-Ras1. Decreased levels of kB-Ras1 resulted in the heightened and persistent activation of NF-κB and in the increased production of NF-kB dependent pro-inflammatory cytokines, leading to uncontrolled myeloproliferation. Deletion of miR-155 in the stroma of RBPJ-/- mice prevented the development of the myeloproliferative disease, and patients affected by MPN exhibit elevated expression of miR155 in their BM. Therefore, we hypothesize that the Notch/miR155/NF-kB axis regulates the level of the inflammatory tonus in the BM niche, and that persistent deregulation of this pathway may contribute to the establishment and progression of MPN. In this presentation, we will discuss our findings and ongoing studies in the context of other recent insights on the crosstalk between tumor cells and the BM niche, and their implications for disease initiation, progression and therapeutic approaches.

Disclosures

No relevant conflicts of interest to declare.
Disease Response Guiding Therapy in Acute Lymphocytic Leukemia: Pivotal Role of Minimal Residual Disease
Authors: Pulsipher Michael A.
Published: October 2019

In spite of an explosion of data regarding mutations associated with childhood ALL, to date these key genetic changes rarely have been the driver of therapy. Clinical parameters at presentation (WBC, age, T- vs. B-lineage, etc.) have dictated initial risk stratification and induction approaches, followed by risk-adapted therapy based upon leukemic response measured by minimal residual disease (MRD, either PCR- or flow cytometry-based). With minor variations, rapid disappearance of peripheral MRD, followed by significant clearance from the marrow after induction, and most importantly, the level of MRD after consolidation have allowed clear distinctions in outcomes that have driven intensification or de-intensification of therapy resulting in improved outcomes. Although specific gene mutations have been associated with risk, MRD has further identified better risk patients within genetic subgroups. For patients noted to be very high risk who are candidates for hematopoietic cell transplantation (HCT), the presence of MRD both pre- and post-transplant has been associated with increased risk of relapse; the risk being modified by level of MRD, whether or not GVHD occurs after HCT, and timing after HCT when MRD is measured. In lower risk patients being treated with chemotherapy and higher risk patients eligible for HCT, more sensitive approaches to flow cytometry and PCR, as well as next-generation sequencing (NGS) MRD approaches (sensitive to 1/10^7 cells) are currently being tested. It is not clear yet whether NGS-MRD offers substantial improvements in patients treated with chemotherapy, as broad-based testing is underway; the latest comparative outcomes will be presented. There is evidence of a striking improvement in our ability to define patients who will do very will after transplant (not relapse), and preliminary evidence that post-HCT NGS MRD testing is more sensitive that other methodologies in defining risk of relapse after transplant. As the latest information about the ability of different approaches to MRD is shown in this session, we will also present how response to therapy based upon MRD interacts with various genetic subtypes (Ph+ ALL, extreme hypodiploidy, etc.). Even in subclasses that are considered very high risk based solely upon genetics, measurement of MRD can define higher and lower risk groups. Going forward, as more and different types of patients are subcategorized and treated with targeted agents based upon specific mutations, it is likely MRD response will continue to be important in mapping intensity of approach and defining children at highest risk of relapse who might benefit from HCT or other cellular therapeutic approaches.

Disclosures

Pulsipher: Novartis: Membership on an entity's Board of Directors or advisory committees.
The Role of Hepcidin in Health And Disease
Authors: Muckenthaler Martina U.
Published: October 2019

Imbalances of iron homeostasis account for some of the most common human diseases. Pathologies result from both, iron deficiency or overload and frequently affect the hepcidin/ferroportin regulatory system that balances systemic iron metabolism. The small hepatic peptide hormone hepcidin orchestrates systemic iron fluxes and controls plasma iron levels by binding to the iron exporter ferroportin on the surface of iron releasing cells, triggering its degradation and hence reducing iron transfer to transferrin. Hepcidin thus maintains transferrin saturation at physiological levels assuring adequate iron supplies to all cell types. My presentation will focus on mechanisms that control hepcidin and ferroportin expression as well as on pathologies that arise when this key regulatory circuitry underlying systemic iron homeostasis is disrupted.

Disclosures

No relevant conflicts of interest to declare.
Vascular and Endothelial Biology of Aging
Authors: Faraci Frank M.
Published: October 2019

Although aging is one of the greatest risk factors for vascular disease, very little is known regarding mechanisms that control the progression of vascular aging at the level of the endothelial cell. Endothelial dysfunction - a critical element of carotid artery and cerebrovascular disease - progresses with age, contributing to hypoperfusion, increased risk for ischemic stroke, and cognitive decline. Studies from several laboratories support the concept that age-induced endothelial dysfunction may occur earlier and be larger in magnitude in the cerebral circulation than in blood vessels outside of the brain. Thus, the circulationof the brain may be particularly sensitive to age-induced endothelial dysfunction. In relation to underlying mechanisms, angiotensin II type 1 receptors, NADPH oxidase, and oxidative stress appear to play a key role in age-related vascular dysfunction. The nuclear receptor peroxisome proliferator-activated receptor-g (PPARg) exerts protective effects in the vasculature when pharmacologically activated. We recently examined the hypothesis that endothelial PPARg protects against vascular aging. We studied carotid arteries from adult and old transgenic mice with endothelial specific expression of a human dominant negative mutation in PPARg driven by the vascular cadherin promoter (designated E-V290M), along with non-transgenic littermates. Endothelial function was similar in arteries from adult non-transgenic and E-V290M mice as well as old non-transgenic mice. In contrast, there was a marked reduction in endothelial function in old E-V290M mice. This augmented endothelial dysfunction was not altered by inhibition of cyclooxygenase, but was restored to normal by a superoxide scavenger, an inhibitor of NADPH oxidase, or inhibition of Rho kinase. Oxidant and inflammatory related mechanisms often interact. Vascular expression of interleukin-6, another mediator of vascular disease, was increased 1.6-fold in old non-transgenic mice, but almost 9-fold in old E-V290M mice. Expression of CDKN2A, a molecular marker of senescence, was ~two-fold greater in old E-V290M mice compared to controls. These findings provide the first evidence that senescence and age-related vascular dysfunction is accelerated following cell-specific interference with endothelial PPARg through mechanisms that involve oxidative stress, inflammation, and Rho kinase. This critical role for endothelial PPARg has implications for vascular pathophysiology as well as therapeutic approaches for age-induced large and small vessel disease.

Disclosures

No relevant conflicts of interest to declare.
Fresh Frozen Plasma Transfusion Ameliorates INR and aPTT but Does Not Increase Thrombomodulin-Corrected Thrombin Generation in Patients with Cirrhosis
Authors: Rassi Amanda Bruder , Rocha Tania RFR , Farias Alberto Q , Mariano Livia Caroline Barbosa , D'Amico Elbio Antonio
Published: October 2019

Introduction: In vitro studies have shown that transfusion of fresh frozen plasma (FFP) has limited efficacy in correcting coagulopathy in cirrhosis. This study aims to assess the effect of FFP transfusion on endogenous thrombin potential (ETP) in patients with cirrhosis with abnormal coagulation test results. Methods: Using a stringent protocol for control of pre-analytic variables, blood samples were collected before and up to 8 hours after FFP transfusion. All samples were tested for INR, aPTT and ETP. The ETP assay was performed in platelet-poor plasma, using an automated fluorometer (CAT, Netherlands), using the technique developed by Hemker and modified by Tripodi. ETP was defined as the area under ROC curve and results were expressed as the ratio of ETP with thrombomodulin addition to ETP without (used to mimic in vivo conditions when patients are transfused). Results: 42 patients were included (male n=28; Child-B n= 11; Child-C n=31). Patients underwent FFP transfusion prior to high-risk invasive procedures (n=25), for treatment of bleeding (n=10) or for both reasons (n=6). The mean dose of FFP was 11.3 ml/kg (95% CI 9.16 - 20.46). After transfusion, INR decreased from 2.6±1,4 to 2.09±0.5 (p<0.0001) and aPTT from 1.47±0.5 to 1.27±0.26 (p<0.0001). At baseline, normal or above normal values of ETP ratio (³0.66) were found in 37 (88%) patients, and mean values remained largely unchanged after FFP transfusion (0.75±0.22; median 0.82 before to 0.76±0.16; median 0.81 after transfusion; p=0.56). The results for INR, aPTT and ETP are shown in figure 1. During the one-week follow-up period, 4 patients (9,5%) had acute reactions to transfusion, including fluid overload and allergic reactions. Of the 31 patients that underwent invasive procedures, four had severe bleeding. Discussion: FFP transfusion at standard doses ameliorated INR and aPTT, but did not increase thrombin generation corrected by thrombomodulin. That might be explained by the fact the former tests detect only 5% of the amount of thrombin generated. Besides, they are performed without the addition of thrombomodulin, an endothelial protein with anticoagulant effects, to which patients with cirrhosis have shown to be resistant. Patients were exposed to the risks of transfusions, without a clear benefit in the improvement in thrombin generation and decreasing the risk of bleeding. Our results call into question the common use of INR and aPTT for guiding the transfusion policy of FFP when treating coagulopathy in cirrhosis.

Figure 1: Results showing comparison before and after FFP transfusion

for (A) INR, (B) aPTT and (C) ETP ratio with and without thrombomodulin.

Disclosures

No relevant conflicts of interest to declare.