bluuebunny/crossref_metadata_2025_split huggingface dataset:
Epigenetics of Hematopoiesis, Stem Cell Reprogramming, and Cancer
Authors: Feinberg Andrew P. 
Published: October 2019

Abstract
Since the discovery of altered DNA methylation in cancer in 1982, most studies of cancer epigenetics have focused on epimutations which could serve as surrogates of mutation. In the past decade, we and our collaborators have been leading efforts to develop whole-genome approaches to epigenetic analysis of human disease, that include novel approaches to array-based analysis and whole-genome bisulfite sequencing. This has led to the first whole-genome bisulfite sequencing methylation map of the cancer genome. Surprising results have been the discovery of CpG island shores and large hypomethylated blocks corresponding to nuclear lamina/heterochromatin regions, and accounting for the vast majority of epigenetic alterations in cancer. These results point to the possibility that at least solid tumors represent epigenetically a single process with a common molecular characteristic; namely increased epigenetic plasticity that allows selection of the tumor cells at the expense of the host. This view can explain most of the hallmarks of cancer, as well as offering a strategy for powerful new approaches to risk detection and treatment. This whole-genome epigenetic approach has also revealed that the classical lineage structure of hematopoiesis is very well reflected in DNA methylation, but that there are very surprising dynamic changes in this process, with waves of both increasing and decreasing methylation changes. This work has also revealed a molecular basis for phenotypic memory in induced pluripotent stem cells, as well as a strong connection between reprogramming differentially methylated regions, or R-DMRs, and tissue-specific differentially methylated regions, or T-DMRs.

Disclosures:
No relevant conflicts of interest to declare.
Insights from Familial Leukemia and Myelodysplastic Syndromes
Authors: Horwitz Marshall 
Published: October 2019

Abstract
Most malignant hematopoietic disorders arise spontaneously. Nevertheless, hereditary factors substantively contribute to risk for myelodysplastic syndrome (MDS) and leukemia, and their molecular genetic identification can help unravel pathogenesis. In some instances, germline mutations causing Mendelian forms of MDS and leukemia are similar to those occurring somatically in sporadic cases, indicating their potential to function as drivers of malignancy. In other circumstances, genes involved in hereditary predisposition to MDS and leukemia are infrequently implicated in acquired cases, suggesting they may contribute to parallel pathways of malignant transformation. Hereditary mutations in genes encoding transcription factors directing hematopoietic differentiation, including RUNX1, CEBPA, and GATA2 in MDS and AML, and PAX5 in acute lymphoblastic leukemia, indicate that disturbed hematopoietic stem cell dynamics may initiate leukemogenesis. On the other hand, for at least some leukemia-predisposing syndromes – including congenital neutropenia when caused by mutations in the gene, ELANE, encoding neutrophil elastase – terminal effectors of hematopoietic differentiation can lead to malignancy, implying that contributions to malignant transformation may go beyond the stem cell. The molecular genetic basis of hereditary leukemic disorders and how they mesh with current concepts of disease mechanism and other insights ensuing from cancer genome sequencing will be discussed.

Disclosures:
Horwitz: United States Government: Consultancy, United States ex rel. Horwitz v. Amgen Inc., Civil Action No. C07-0248 (W.D. Wash.) Other.
Adoptive Transfer of T-Cell-Receptor Engineered Human T Cells Specifically Reduces Viral Titers in HLA-Transgenic NSG Mice Infected with a Humanized Cytomegalovirus
Authors: Thomas Simone , Lemmermann Niels , Klobuch Sebastian , Podlech Jürgen , Theobald Matthias , Reddehase Matthias J , Herr Wolfgang 
Published: October 2019

Abstract
Reactivation of latent human cytomegalovirus (HCMV) infection is a frequent complication in patients after allogeneic hematopoietic stem cell transplantation (HSCT). Preclinical research in murine models as well as clinical phase I/II trials have shown that the adoptive transfer of virus-specific CD8+ T cells is a therapeutic option for preventing HCMV disease. However, the feasibility of HCMV-specific immunotherapy is currently limited in clinical routine due to technical restrictions. It has also limitations, if the donor is HCMV-seronegative or carries only low numbers of HCMV-specific memory T cells. In this situation, grafting non-reactive T cells by virus-antigen specific T-cell receptors (TCR) may be an alternative means to transfer HCMV-specific T-cell function into HSCT recipients. Nevertheless, improvement of clinical protocols is needed before HCMV-specific cell therapy can be implemented in general clinical practice.
Here we describe a novel preclinical mouse model that will help to evaluate new HCMV-immunotherapy options. Due to the strict species specificity of CMV, we constructed a recombinant murine CMV (mCMV) coding for the HLA-A2 restricted pp65495-503 (NLV) peptide epitope of HCMV. This peptide is expressed with flanking amino acids within the mCMV-IE2-protein in the context of experimental infection with mCMV-IE2/NLV. The NLV peptide was found to be presented by infected murine fibroblasts from NOD/SCID/IL-2Rγc-/--HLA-A2-transgenic (NSG-A2) mice as shown by T cell specific recognition in ELISPOT and cytotoxicity assays. Interestingly, this presentation proved to be susceptible to the inhibitory function of mCMV immune evasion proteins. NSG-A2 mice were then used to evaluate antiviral control by HCMV-NLV-specific TCR (TCRNLV)-engineered human T cells. After infection with mCMV-IE2-NLV, the NSG-A2 mice showed high viral titers in liver, spleen, lungs, and salivary glands as well as a dramatic virus-induced morbidity and mortality. In contrast, after intravenous injection of TCRNLV-transduced human CD4+/CD8+ T cells, viral infection was reduced in all tested organs, and mice showed improved survival. These findings suggest that the NLV peptide is processed and presented by stromal and parenchymal cells of the NSG-A2 mice and promise validity of the model for preclinical evaluation of adoptive transfer protocols, for instance cell therapy with TCR-engineered T cells alone or in combination with therapeutic vaccination.

Disclosures
No relevant conflicts of interest to declare.
Consolidative Autologous Stem Cell Transplant for Double Hit Lymphoma
Authors: Chen Andy I , Okada Craig , Gay Nate , Reiss Phil , Spurgeon Stephen , Fan Guang , Braziel Rita M. , Maziarz Richard T. 
Published: October 2019

Abstract
Double hit lymphoma (DHL), defined as B cell lymphoma with a c-MYC rearrangement plus a IGH/BCL2 rearrangement, confers a poor prognosis. Outcomes with R-CHOP are dismal with reported 2 year EFS <30% in multiple series. To improve upon these results, Burkitt type regimens such as R-HyperCVAD and dose adjusted (DA) R-EPOCH have been attempted, but 2 year EFS remains < 50% in early reports. In addition, the SWOG-9704 study found a benefit for consolidative autologous stem cell transplant (autoSCT) in high risk DLBCL, likely including some DHL, although this reported benefit has not been confirmed in three other randomized European studies.
Since 2010 the treatment algorithm at Oregon Health & Science University for fit patients with DHL has been DA-R-EPOCH followed by consolidative autoSCT in responding patients. Here we report the first 16 patients treated with consolidative autoSCT. At diagnosis, the median age was 58, and the median IPI was 3. All patients had rearrangements of c-MYC and IGH/BCL2. One of the c-MYC rearrangements was a classical t(8;14); the translocation partners in the other cases were unknown. Three patients also had a ‘triple hit’ with an additional BCL6 rearrangement. Fourteen patients received DA-R-EPOCH with intrathecal prophylaxis for induction. One received R-CHOP at another institution, and one received R-HyperCVAD. 15 of 16 patients were in CR after induction. Stem cell mobilization was performed with G-CSF +/- plerixafor, and BEAM was utilized as the high dose conditioning regimen.
With a median follow-up of 18 months, the estimated 2 year PFS is 91 %, and the 2 year OS is 91 % in the 16 patients who underwent consolidative autoSCT. There were no deaths from non-relapse mortality, and the only relapse was 6 months post-transplant. All other patients undergoing autoSCT remain alive and in remission.
An additional 15 patients also underwent induction with DA-R-EPOCH but did not proceed to autoSCT. Four patients who had intended to undergo autoSCT were refractory to induction. Of the remaining 11 patients, 9 did not pursue autoSCT because of age and/or comorbidities, and 1 each declined out of personal preference or insurance denial. Four of these 11 have relapsed or died.
Our results suggest that consolidative autoSCT, especially after DA-R-EPOCH induction, is an effective treatment strategy for DHL in fit patients and should be explored in prospective studies.

Disclosures
No relevant conflicts of interest to declare.
Gemcitabine in Combination with Oxaliplatin (GEMOX) As a Salvage Regimen in Patients with Relapsed/Refractory Hodgkin's Lymphoma
Authors: Ozdemir Evren , Aslan Alma , Turker Alev , Barista Ibrahim , Kars Ayse 
Published: October 2019

Abstract
This study's objective was to evaluate the efficacy and safety of gemcitabine in combination with oxaliplatin (GEMOX) as a salvage regimen in patients with relapsed or refractory Hodgkin's lymphoma. Twenty-five patients were enrolled. All patients had received ≥ 2 prior chemotherapy regimens, had an ECOG performance status ≤ 2 and had adequate organ function. Patients received intravenous gemcitabine (1000 mg/m2) and oxaliplatin (100 mg/m2) on days 1 and 15, every 4 weeks. The median age was 29 years (range, 18-64) and 16 (68%) were male. Twenty-one (84%) had primary refractory disease (n=13) or relapsed within 12 months after initial therapy (n=8). All had previous platinum-based salvage chemotherapy (ICE, 23; DHAP, 2). Ten patients (40%) had relapsed/refractory disease following autologous stem cell transplantation (SCT). None had previous brentuximab vedotin treatment. Twenty-one (84%) patients were refractory or progressive on the last treatment. Median number of previous lines of chemotherapy was 2 (range, 2-4). Median number of GEMOX cycles administered to the patients was 3 (range, 2-6). Treatment response was evaluated with PET-CT before and 2-3 cycles after treatment, and those patients who demonstrated a response continued to receive a maximum of 6 courses of GEMOX or bridged to SCT. Of 25 patients, 2 (8%) had complete response, 9 (36%) had partial response and the remaining patients had refractory/progressive disease with an overall response rate of 44%. Seven of the 10 patients who had relapsed/refractory disease after autologous SCT achieved a response (CR, 2; PR, 5). The median time to progression for responding patients was 3 months (range, 1-40 months). One patient is disease free for 40 months. Three patients were successfully bridged to SCT (autologous, 2; allogeneic, 1). Main toxicity was hematological. Grade ≥ 3 hematologic toxicity occurred in 10 patients: thrombocytopenia (36%), neutropenia (16%) and anemia (8%). Among these, 7 had previous autologous SCT. One patient had grade 4 neutropenia and thrombocytopenia. Treatment cycle postponed in 6 patients without dose reduction because of hematological toxicity. Seven patients (28%) needed G-CSF support. One patient developed febrile neutropenia. No treatment-related deaths occurred. GEMOX was shown to be an effective salvage regimen in patients with relapsed/refractory Hodgkin's lymphoma, producing an overall response rate of 44%. It is an active regimen in patients who had relapsed/refractory disease after autologous SCT. Although, the median PFS time was short, some patients can be bridged to SCT and some can get long-term PFS. Hematological toxicity was common, especially in patients with previous autologous SCT.

Disclosures
Off Label Use: Gemcitabine in Hodgkin's Lymphoma Oxaliplatin in Hodgkin's Lymphoma.
Targeting Myddosome Self-Assembly in Waldenstrom's Macroglobulinemia
Authors: Liu Xia , Hunter Zachary , Xu Lian , Chen Jie , Chen Jiaji , Tsakmaklis Nicholas , Patterson Christopher J , Castillo Jorge J , Buhrlage Sara , Gray Nathanael , Treon Steven P , Yang Guang 
Published: October 2019

Abstract
Background: MYD88 mutations are present in over 95% of patients with Waldenstrom's Macroglobulinemia (WM), and promote Myddosome self-assembly that triggers NFκB-dependent survival through BTK and IRAK1/IRAK4 (Blood 122(7):1222-32). While current therapeutic strategies are aimed at blocking these downstream kinases, peptidomimetics that interfere with Myddosome self-assembly may offer a more targeted approach for blocking aberrant MYD88 signaling.
Methods: We expressed by lentiviral transduction mini-peptides of MYD88 Toll/Interleukin-1 Receptor (TIR) or Death Domain (DD) sequences in mutated MYD88 WM and wild-type MYD88 control cells (Figure 1). We used phospho-flow analysis to evaluate for changes in pBTK, pIRAK1/IRAK4, and pNFKB, and determined cell growth and survival by Alamar Blue Assay, Annexin V, and cleaved Caspase 3 staining.
Results: Transduction of TIR or DD mini-peptides in mutated MYD88 WM cells but not wild-type MYD88 control cells reduced NFKB activation and tumor cell growth, and prompted Annexin V and/or cleaved Caspase 3 staining. TIR interfering mini-peptides impacted BTK but not IRAK1/IRAK4 activation, whereas DD interfering mini-peptides showed an opposite effect.
Conclusions: The findings demonstrate differences in BTK versus IRAK1/IRAK4 directed NFKB signaling in response to Myddosome self-assembly in MYD88 mutated WM cells. The feasibility of directly targeting MYD88 homodimerization to block aberrant MYD88 signaling was also recognized, and suitable peptide sequences for the development of peptidomimetics that interfere with Myddosome self-assembly and signaling were identified. The findings provide a framework for direct targeting of the Myddosome in MYD88 mutated WM disease.
Figure 1. Figure 1.

Disclosures
No relevant conflicts of interest to declare.
Mirna Regulation of Blood Group AB0 Genes
Authors: Kronstein-Wiedemann Romy , Nowakowska Paulina , Milanov Peter , Seifried Erhard , Tonn Torsten 
Published: October 2019

Abstract
In clinical transfusion and transplantation medicine, AB0 is the most important blood group system. A and B antigens are synthesized by glycosyltransferases A and B encoded by co-dominantly inherited A and B genes. The 0 phenotype derives from a single nucleotide deletion; the resulting stop codon in the exon 7 of glycosyltransferase A leads to a lack of glycosylation activity to A and/or B. The DNA structure of the three major alleles of the human blood group AB0 system was first described in 1990. Besides the 4 major AB0 blood groups (A, B, AB, 0), weak expression variants commonly occur. Beside introns and exons, two regions were found to be critical for transcriptional activity of AB0 genes: a promotor sequence upstream of the translation start site and a CBF/NF-Y-binding enhancer element. However, the molecular basis for the weak expression levels of AB0 blood group receptors is not fully understood. Here we asked whether microRNA´s may play a role in the regulation of blood group A and B antigens. MiRNA's are small, non-protein encoding RNA sequences that bind to the 3' untranslated region (UTR) of target mRNAs resulting in translation repression or mRNA degradation. By distinct complementary approaches, including gene array analysis and overexpression of glycosyltransferase specific micro-RNAs (miRNAs) in primary hematopoietic stem cells, we identify here that miR-331-3p and miR-1908-5p directly target glycosyltransferase A and B. Consistently, overexpression of miR-331-3p and -1908-5p in hematopoietic stem cells (HSCs) leads to a 30-50% reduction of blood group A antigens per cell in differentiated RBCs. Downregulation of endogenous miR-1908-5p but not miR-331-3p leads to increased amount of blood group A antigen per red blood cell compared to control cells. This discrepancy between the effects of downregulation of miR-1908-5p and of miR-331-3p is likely explained by the constitutive expression levels of both miRNAs during erythropoiesis. In particular, miR-1908-5p is expressed throughout erythroid differentiation, while miR-331 displays low expression, thus, further downregulation of miR-331-3p could not further affect blood group A antigen levels. Furthermore miR-331-3p and -1908-5p were enhanced in red blood cells of Aweak variants. Sequencing of the 3'UTR of 6 Aweak variants revealed the presence of more miRNA binding sites for miR-1908-5p compared to normal controls.
These data identify microRNAs as regulators of blood group glycosyltransferase expression, thereby extending our understanding of blood group regulation in carriers of weak blood group variants. Regulating blood groups by microRNAs could pave the way for the development of universal blood.

Disclosures
No relevant conflicts of interest to declare.
Mechanism Governing Heme Synthesis Reveals a GATA Factor-Heme Circuit That Controls Differentiation
Authors: Bresnick Emery H , Tanimura Nobuyuki , Miller Eli , Igarashi Kazuhiko , Yang David T , Burstyn Judith , Dewey Colin 
Published: October 2019

Abstract
Metal ion-containing macromolecules have fundamental roles in essentially all biological processes throughout the evolutionary tree. For example, iron-containing heme is a cofactor in enzyme catalysis and electron transfer and an essential hemoglobin constituent. To meet the intense demand for hemoglobin assembly in red blood cells, the cell type-specific factor GATA-1 activates transcription of Alas2, encoding the rate-limiting enzyme in heme biosynthesis, 5-aminolevulinic acid synthase-2 (ALAS-2). Using genetic editing to unravel mechanisms governingheme biosynthesis, we discovered a GATA factor- and heme-dependent circuit that establishes the erythroid cell transcriptome. CRISPR/Cas9-mediated ablation of two Alas2 intronic cis-elements strongly reduced GATA-1-induced Alas2 transcription and heme biosynthesis. Surprisingly, the cis-element ablation impaired GATA-1 regulation of other vital constituents of the erythroid cell transcriptome, including components mediating autophagy and nutrient transport. Bypassing ALAS-2 function in Alas2 cis-element-mutant cells by providing its catalytic product 5-aminolevulinic acid rescued heme biosynthesis and a sector of the GATA-1-dependent genetic network. A component of the gene cohort deregulated in heme deficiency could not be reactivated by rescuing heme biosynthesis, and we are testing whether irreversible changes in their chromatin states upon heme deficiency confer stable repression. Heme amplified GATA-1 function by downregulating the heme-sensing transcriptional repressor Bach1 and via a Bach1-insensitive mechanism. Based on this dual mechanism, we developed a paradigm in which a fundamentally critical process in hematology, heme biosynthesis, is intricately linked to the actions of a quintessential regulator of a cell type-specific transcriptional program that controls cellular differentiation. As known mechanistic insights into GATA factor mechanisms do not predict whether a GATA-1 target gene will be heme- or Bach1-sensitive, these studies revealed a new mode of GATA factor function that controls erythroid cell development and function. We will discuss how this mechanism is being dissected in biologically important murine and human systems.

Disclosures
No relevant conflicts of interest to declare.
Congenital Neutrophilia Arising from a T618I Germline Mutation in the CSF3R Gene
Authors: McMahon Daniel P , Lance Amanda , Price Andrea E. , Gerber Jonathan M. , Baxter Sarah A , Druhan Lawrence J , Steuerwald Nury M , Avalos Belinda R 
Published: October 2019

Abstract
Recently, somatic mutations in the granulocyte colony-stimulating factor receptor (CSF3R) have been identified in the majority of patients with chronic neutrophilic leukemia (CNL). The T618I mutation is the most common of these mutations, and it has been suggested that this specific mutation be added to the current WHO criteria for the diagnosis of CNL. In vitro studies of cells transfected with the T618I mutation have demonstrated that the mutant receptor confers a hyperproliferative phenotype.
We report here a case of congenital neutrophlia with an associated germline T618I CSF3R mutation. The patient, currently a 10 year old female, has had an elevated but stable leukocytosis (WBC 24,000 - 35,000) since birth, comprised of approximately 80% neutrophils. She has been healthy, with no increase in frequency of infections. Bone marrow studies performed at 5 months and 10 years of age have shown the marrow to be hypercellular, with normal karyotype/FISH (including BCR/ABL negative) and no dysplastic features. Additionally, no mutations have been detected in JAK2, MPL, CALR, PDGFA/B, and FGFR1.
DNA was extracted from whole blood and buccal cells collected from the patient, and using next generation sequencing a T618I mutation in CSF3R was identified in both her buccal and blood cells, confirmed by Sanger sequencing. The T618I mutation was present at an approximately 50% allele frequency in both tissues. Using a combination of next generation and Sanger sequencing, the patient's DNA was also analyzed for the presence of mutations in ASXL1, SETBP1, and TET2; as these have been reported in combination with the T618I mutation in many cases of CNL. However, no such additional mutations were detected in our patient.
Congenital neutrophilia arising from another activating mutation in CSF3R has previously been reported in a family from France (Plo et al., J Exp Med 2009). The mutation identified in that family was reported to be at amino acid position 617 (T617N) (numbering from the methionine 1, which includes the leader sequence, this correlates to a T640N mutation). Similar to the T618I mutation in our patient, none of the 12 affected family members (ages 8-80 years) progressed to an acute leukemia. Collectively, these observations suggest that activating CSF3R mutations, including the T618I mutation, may result in chronic neutrophilic leukemia but that subsequent disease progression may be due to other, cooperating mutations.

Disclosures
No relevant conflicts of interest to declare.