bluuebunny/crossref_metadata_2025_split huggingface dataset:
Prognostic Score Based on Clinical and Biochemical Surrogates of Disease Status in DLBCL Trated with Chemo-Immunotherapy
Authors: Marcheselli Luigi , Bari Alessia , Pozzi Samantha , Marcheselli Raffaella , Cox M. Christina , Baldini Luca , Luminari Stefano , Sacchi Stefano 
Published: October 2019

Abstract
Introduction.
In the past years there were many attempts to replace or improve the IPI score for overall survival evaluation (OS) in diffuse large B cell lymphoma (DLBCL). We focused our attention on prognostic factors, studied in the past or in recent years, that could be used as surrogates of the disease status. We considered the serum albumin (SA) level as surrogate of age, comorbidity and disease severity; LDH as surrogate of cell activity and disease stage; absolute monocyte count (AMC) as surrogate of inflammatory status and absolute lymphocyte count (ALC) as surrogate of immune status of patients.
Patients and method
We collected data of 322 patients comprehensive of IPI factor, SA, AMC and ALC recorded in the "Gruppo Itliano Studio Linfomi" (GISL) database from 2003 to 2012. The score was obtained summing the factors SA <3.7 g/dL, LDH >UNL, AMC>630 /uL and ALC <840 /uL, any with weight one. The factors were not the results of statistical procedure but were chosen on the basis of clinical and pathophysiological considerations, about the role played by these factors in the DLBCL. The OS was estimated using the Kaplan-Meier method; the discriminate ability of the score was checked by means of log-rank test and using the ROC curve, sensibility and specificity at 3-years of follow-up.
Results
All the 322 patients included in the study were treated with R-CHOP and R-CHOP like regimens. The median age at diagnosis was 67 years (range 22-86) and 54% were males; SA <3.7 was identified in 44% of the patients, LDH>UNL in 51%, AMC >630 in 33% and ALC <840 in 23%. The sum of those four factors defined a score with three levels of risk: low (0-1, n=69, 21%), intermediate (2, n=94, 29%) and high (3-4, n=159, 49%). The OS at 5-yrs was 96%, 76% and 46% for score 0-1, 2 and 3-4, respectively The log-rank between score 0-1 and 2 was 11.2 (p=0.0008) and 16.9 between score 2 and 3-4 (p<0.0001). The ROC curve at 5-yrs was 72.1%, with 75.8% of sensitivity and 68.5% of specificity. The score showed a moderate accordance with IPI coded 0-1, 2 and 3/5 (k-statistic 0.37).
Conclusion
This approach showed that it is possible to define a simple prognostic score taking into account simple factors, easily available in every hospital, and related to the disease status. This selection was based on the clinical and pathophysiological knowledge accumulated over the years and, also, avoiding complicated statistical procedures for the selections of the prognostic factors. As it appears in the graph (Fig.1) , patients can be clearly stratified in three groups with deeply different survival outcome, based only on biochemical factors.
Figure 1 Figure 1.

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

Abstract
Background: Clonal heterogeneity is a hallmark of many solid and hematologic malignancies. Clonal heterogeneity may foster clonal evolution and affect personalized care strategies in multiple myeloma(MM). In order to grasp this ubiquitous phenomenon and its effect on MM patients’ prognosis, we report on sequential analysis by quantitative multicolor-fluorescence in situ hybridization (QM-FISH) in single cell of myeloma patients who were longitudinally followed.
Methods: Bacterial artificial chromosomes (BAC) clones that contain target genes including TP53, Rb1, CKS1B, CYLD and cIAP, which belonged to secondary genetic variation of MM and were searched in the data base UCSC Genome Bioinformatics. Multicolor FISH probes were prepared by linking fluorescein labeled dUTP and dCTP to target genes by nick translation. We performed QM-FISH in single cell of 42 specimen from 17 myeloma patients who were longitudinally followed and compared the difference between myeloma patients at initial diagnosis and progression of disease.
Results: Sequential samples revealed that MM may follow three clonal evolution patterns in the course of disease progression. In these 17 patients, myeloma clone in 5 patients had no changes over the time, 6 patients acquired one or more molecular abnormalities and only one predominant clone over the course, 6 patients had more than 1 clone in the course and all clones competing and tiding, or acquired one or more molecular abnormalities resulted in new clones emerging and all involved clones waxing and waning. Furthermore, we analyzed clone architecture in samples at initial diagnosis and 15/17(88.2%) cases had multiple subclones architecture at initial diagnosis. 3 patients of the first group (3/5, 60%) had more than 1 subclone, and all patients of the latter two groups had multiple subclones at initial time. Clonal heterogeneity may contribute to clonal evolution
Conclusions: The outcome of tracking clonal heterogeneity and evolutional paths by QM-FISH is effective and feasible. Myeloma patients may follow 1 of 3 pathways to progress. Clonal heterogeneitymay foster lonal evolution in the course of disease progression.

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

Abstract
Generation of hematopoietic stem cells (HSCs) from pluripotent stem cells, in vitro, holds great promise for future somatic gene and cell therapy. So far, HSCs capable of long-term multilineage reconstitution in mice have only been obtained when the homeodomain transcription factor HOXB4 was ectopically expressed during pluripotent stem cell differentiation (Kyba et al. Cell 109(1): 29-37, 2002; Pilat et al. Proc Natl Acad Sci USA 102(34): 12101-12106, 2005; Lesinski et al. Stem Cells Transl Med 1(8): 581-591, 2012). However, the primary "target" cell of HOXB4 during hematopoietic development, in vitro, is not yet known. Its identification is a prerequisite for unambiguously identifying the molecular circuits driving HSC development, at least in vitro.
To pin down this cell, we retrovirally expressed HOXB4 or a Tamoxifen-inducible HOXB4-ERT2 fusion protein in different reporter and knock-out mouse embryonic stem cell (ESC) lines. For these experiments, ESCs were differentiated for 6 days as embryoid bodies (EBs), dissociated and subsequently cocultured on OP9 stroma cells in medium supplemented with 100 ng/ml mSCF, 40 ng/ml mTPO, 100 ng/ml hFlt3L and 40 ng/ml hVEGF (STFV) for further 3 days. Use of a Runx1(-/-) ESC-line containing a doxycycline-inducible Runx1 coding sequence (“iRunx1”; kindly provided by G. Lacaud, Manchester) uncovered that HOXB4 acts during formation of the hemogenic endothelium (HE) from which HSCs arise. Without Runx1 induction, which arrests hematopoietic development at the HE-stage, ectopic HOXB4 expression mediated an approximately 30-fold increase in the number of circular endothelial, bona fide HE-sheets being Flk1+VE-Cadherin+Tie2+ (mean values: control: 11+/-4.8 n=7; HOXB4: 301+/-47 n=7; P<0.0001, unpaired, 2-sided Student´s t-test) and expressing Sox17 and Lmo2. This observation suggested an expansion of HE progenitors, detectable from day 5 of EB differentiation on. Determination of their frequencies within the VE-Cadherin+ population revealed a HOXB4-mediated increase from 1:360 cells (control) up to 1:15 cells (HOXB4; 95% C.I. = 1:12-1:21). After additional Runx1 induction, the endothelial cells morphologically underwent an Endothelial-to-Hematopoietic Transition (EHT) as verified at the single cell level by time-lapse microscopy. Concomitantly, they upregulated transcription of Gfi1, Gfi1b and Pu.1, initiated surface expression of the pan-hematopoietic marker CD45 and generated hematopoietic colony forming cells (CFC), thus proving their identity as real hemogenic endothelial cells.
Taken together, our results strongly suggest that HOXB4 first and foremost promotes hematopoiesis by substantially increasing the number of hemogenic endothelium progenitors during mouse pluripotent stem cell differentiation.

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

Abstract
While most leukemic cells are initially sensitive to chemo- and radiotherapy, leukemic stem cells (LSC) persist and are therefore considered to be the basis for disease relapse in acute myeloid leukemia (AML) (Nat Biotechnol, 25: 1315-1321, 2007). The discovery of epigenetic mutations being the cause of a number of cancers has increased interest in their therapeutic potential (Nat Rev Genet, 7:21-33, 2006; Cell, 128: 683-692, 2007). Although the clinical importance of epigenetic dysregulation has been recognised in various cancers, including MLL-rearranged AML, this topic is currently a novel area of study.
Our microarray analysis comparing genes differentially expressed between LSC and normal hematopoietic stem cells (HSC) identified JMJD1c, a histone demethylase, as a potential LSC-specific target. An analysis of a comprehensive patient outcome database showed that high levels of JMJD1c were associated with significantly poorer survival in patients with AML (P = 0.0029), making it clinically relevant and emphasising the therapeutic potential of JMJD1c. Furthermore, the JMJD1c mRNA level was significantly higher in MLL-rearranged than in non-MLL AML patient xenograft samples.
Western blot and quantitative PCR confirmed the microarray results, where JMJD1c was significantly upregulated in MLL-AF9 LSC compared to Hoxa9/Meis1a derived LSC and HSC. Overexpression of JMJD1c in Hoxa9/Meis1a pre-LSC significantly increased the clonogenic activity and proliferation compared to control cells. Subsequent microarray experiment identified Hox genes as downstream targets of this epigenetic regulator (fold change > 2, P < 0.05), suggesting the regulatory role of JMJD1c in the self-renewal pathway. Consistent with in vitro observations, there was a significant increase in disease progression as shown by leukemia onset in mice with Hoxa9/Meisa1 pre-LSC transduced with JMJD1c cDNA compared to control (P < 0.0001; at least 12 mice per cohort). Stable knockdown of JMJD1c in MLL-AF9 cells using shRNA significantly impaired the proliferation, clonogenic activity and induced cell differentiation in vitro. In vivo survival studies in mice showed that JMJD1c knockdown severely impaired the maintenance of MLL-AF9 induced leukemia (P < 0.0001; 12 mice per cohort).
In conclusion, our studies have identified JMJD1c as an important driver in leukemia maintenance and strongly suggest that JMJD1c possesses an oncogenic role in MLL-AF9 leukemogenesis. Targeting this oncogene may enable us to directly target LSC as a strategy for overcoming drug resistance in AML patients.

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

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

Disclosures
Eaton: Syros Pharmaceuticals: Employment, Equity Ownership. Lopez:Syros Pharmaceuticals: Employment, Equity Ownership. Ke:Syros Pharmaceuticals: Employment, Equity Ownership. Fritz:Syros Pharmaceuticals: Employment, Equity Ownership. Olson:Syros Pharmaceuticals: Employment, Equity Ownership. Loven:Syros Pharmaceuticals: Employment, Equity Ownership.
Spontaneous Mutations of Bcor and Jak1/2 genes Lead to an Aggressive Leukemia of B-1 Progenitor B Cells
Authors: Gough Sheryl M , Goldberg Liat , Pineda Marbin , Walker Robert L , Zhu Yuelin J , Bilke Sven , Meltzer Paul S , Aplan Peter D 
Published: October 2019

Abstract
NUP98 gene fusions, generated by non-random chromosomal translocations, are associated with a wide spectrum of high risk hematologic malignancies and have been shown to alter normal hematopoietic stem and progenitor cell (HSPC) gene expression programs. A recurrent t(11;17)(p15;p13) translocation in patients with AML leads to the production of a NUP98–PHF23 (NP23) fusion gene. The consequent NP23 fusion protein retains the PHD domain, known to bind H3K4me3, and is thought to have aberrant chromatin regulation properties. We have generated a transgenic mouse model of the NUP98-PHF23 gene fusion which develops a range of hematologic malignancies, most commonly pre-T LBL and AML. However, approximately 10% of NP23 mice develop an aggressive B-1 progenitor acute lymphoblastic leukemia (pro B-1 ALL).
B-1 and B-2 lymphocytes have distinct developmental pathways and are thought to represent arms of the innate and adaptive immune systems, respectively. Mature B-2 lymphocytes predominate in the peripheral circulation, and are characterized by expression of B220; whereas B-1 lymphocytes are more prevalent in the pleural and peritoneal cavities, and do not express B220. Murine B cell malignancies typically stain positive for B220, and represent transformed B-2 cells. In the present study, NP23 progenitor ALLs displayed an immunophenotype (Lin-B220- CD19+ AA4.1+) that was identical to that of the recently described B-1 progenitor cell. All B-1 progenitor ALLs exhibited clonal rearrangements of the IgH gene locus. Specifically, these rearrangements involve favored usage of 3’ VH regions, similar to observations with fetal B-1 progenitor cells, further supporting the notion that these are leukemias of B-1 progenitors. Using whole exome sequencing, we found acquired mutations in the BCL6 interacting corepressor (Bcor) gene in 5 out of 7 B-1 progenitor leukemias. The mutations were all frame shift or nonsense mutations, and were located within a 9 bp “hot spot” in Bcor exon 8. In addition, 4 of 7 cases had somatic mutations of Janus kinase 1 (Jak1) or 2 (Jak2), and 7/7 cases showed hyperphosphorylation of Stat3 or Stat5, consistent with the contention that the Jak1/2 mutations are activating mutations, and leading to a hypothesis that the NP23 pro B-1 ALLs which do not harbor Jak1/2 mutations may have acquired an unidentified mutation in the Jak-Stat pathway. Of note, Jak1/2 mutations have previously been identified in a subset of high-risk pediatric B-cell precursor ALL patients. The striking correlation between Bcor and Jak1/2 mutations, occurring specifically in a subset of NP23 leukemias, implies that these three mutations (NP23, Bcor, and Jak1/2) collaborate and provide the oncogenic setting for B-1 progenitor transformation.

Disclosures
No relevant conflicts of interest to declare.
GNA13, a Novel Component of the GPR84/Beta-Catenin Signaling Axis in AML Stem Cells, Regulates the Maintenance of MLLAF9 Leukemogenesis
Authors: Dietrich Philipp A , Norris Murray D , Wang Jenny Yingzi 
Published: October 2019

Abstract
Beta-catenin is required for the establishment of leukemic stem cells (LSC) in mixed lineage leukemia (MLL)-rearranged acute myeloid leukemia (AML). Targeted inhibition of beta-catenin signaling has been hampered by the lack of pathway components amenable to pharmacological inhibition. Our recent study has identified a new beta-catenin regulator, GPR84, a member of the G protein-coupled receptor family that represents a highly tractable class of drug targets. Since no GPR84-specific anti-cancer compound has been developed to date, we aim to further delineate the GPR84/beta-catenin axis in order to inhibit other components of this signaling pathway pharmacologically.
Preliminary data from our lab has implicated GNA13 as a potential downstream target gene of GPR84. In this study we sought to further examine the involvement of GNA13 in the GPR84/beta-catenin signaling axis. Functional studies showed that shRNA-mediated ablation of GNA13 significantly decreased the capacity of MLLAF9-LSC to form colonies in vitro (P=0.0184). We further demonstrated that GNA13 inhibition in LSC induced a significant G1 cell cycle arrest in vitro (P=0.0192). Subsequent in vivo studies showed that GNA13 downregulation in MLLAF9-LSC significantly delayed the onset of AML (P=0.0016) and significantly decreased LSC proliferation (P=0.0066). Hence GNA13 inhibition mimics the previously described GPR84-deficient phenotype in MLLAF9-LSC (Blood 2013;122:3781). In contrast, the GPR84-deficient phenotype could be rescued in MLLAF9-LSC by GNA13 overexpression in vitro (P=0.0130) and in vivo (P=0.0012). Further analysis showed that there was a significant increase in the mitotic index of GPR84-deficient LSC transduced with GNA13 cDNA in vitro, evidenced by an increase in G2/M-phase accumulation (P=0.0030) and p-histone H3 (P=0.0040). In addition, our data demonstrated a significant increase in BrdU incorporation of GPR84-deficient LSC overexpressing GNA13 in vivo (P<0.0001).
In conclusion, our studies have identified GNA13, a novel component of the GPR84/beta-catenin axis. Since this pathway is pivotal for MLLAF9 AML maintenance by sustaining LSC self-renewal, targeting the GPR84/GNA13/beta-catenin signaling axis may provide a novel and promising strategy for improving the therapy and outcome of AML patients.

Disclosures
No relevant conflicts of interest to declare.
Influence of Dihydrofolate Reducatse (DHFR) Gene Polymorphisms on Outcome of Methotrexate Maintenance Therapy in Patients with Acute Lymphoblastic Leukemia
Authors: Biswajit Dubashi , Kodidela Sunitha , Pradhan Suresh Chandra , Basu Debdatta , Prasad Ravi , Gurusamy Umamaheswaran 
Published: October 2019

Abstract
Objectives: 1)To determine the influence of DHFR gene polymorphisms on outcome of methotrexate-based maintenance therapy in patients with acute lymphoblastic leukemia (ALL) 2)To compare the relapse free survival between DHFR genotype 3)To establish the normal allele and genotype frequencies of the DHFR polymorphisms in the South Indian population Methods: A total of thirty one patients with ALL were included for the study. A total of 85 healthy volunteers from the South India were recruited to establish the allelic frequencies of the A-317G & C-680A variants of DHFR gene. DNA was extracted by phenol chloroform method and genotyping was done by allelic discrimination method. Patients were followed up for two years for the outcome. Results: Homozygous mutant genotype carriers (GG) were at two-fold increased risk of relapse when compared to the other genotypes (AA & AG) [Relative risk= 2.6; 95% CI, 1.260 to 5.365, P<0.05]. Relapse-free survival rate was lower in GG genotype carriers when compared to wild and hetero genotypes [Log-rank test, p<0.05]. The genotype frequencies of A-317G variant in healthy volunteers were- AA=51.76%, AG=37.64%, GG=10.58% and C-608A variant genotype frequency were-CC=0%, AC=20.98%, AA=79.01%. Conclusion: GG variant of DHFR A-317G is associated with increased relapse and lower relapse-free survival in ALL patients.

Disclosures
No relevant conflicts of interest to declare.
Ectopic Expression of Lin28 Biases Myeloid Differentiation to Promote an Immature Mast Cell State and Is Implicated in Aggressive Mastocytosis
Authors: Wang Leo D. , Nguyen Phi , Rowe Robert G , Nageswara Rao Tata , Daley George Q. , Wagers Amy J. 
Published: October 2019

Abstract
Dysregulated mast cell development leads to systemic mastocytosis, a clinically variable but often devastating family of hematologic disorders. Lin28 is a heterochronic gene and pluripotency factor implicated in many types of malignancy, and prior studies suggest that Lin28 expression can restore a fetal hematopoietic program in adult mice. However, the role of Lin28 in hematologic malignancy remains controversial. In our study, we induced expression of Lin28 in adult mice using a doxycycline-responsive transgenic system. Lin28 induction caused marked mast cell accumulation in target organs such as the skin and peritoneal cavity. In vitro assays revealed a skewing of myeloid commitment in Lin28-expressing hematopoietic progenitors, with increased levels of Lin28 in common myeloid progenitors and basophil-mast cell progenitors altering gene expression patterns to favor cell fate choices that enhance mast cell specification. In addition, Lin28-induced mast cells appeared phenotypically and functionally immature, and in vitro assays suggested a slowing of mast cell terminal differentiation in the context of Lin28 upregulation. Finally, interrogation of human mast cell leukemia samples revealed upregulation of LIN28 in abnormal mast cells from patients with aggressive systemic mastocytosis (ASM). This work identifies Lin28 as a novel regulator of innate immune function and a new protein of interest in mast cell disease.

Disclosures
No relevant conflicts of interest to declare.