bluuebunny/crossref_metadata_2025_split huggingface dataset:
Are Mast Cells a Biologic Predictor of Outcome in Follicular Lymphoma?
Authors: Howard Matthew T , Pohlman Brad , Jin Tao , Hsi Eric 
Published: October 2019

Abstract
Background: The tumor microenvironment is important in the biology of follicular lymphoma (FL). In addition to macrophages, which have been shown to be associated with outcome in FL, mast cells (MCs) have the ability to influence the microenvironment through secretion of numerous biologically active molecules such as growth factors and cytokines/chemokines. Increased numbers of tumor-infiltrating MCs have been suggested as an unfavorable prognostic marker that also supercedes the predictive value of lymphoma-associated macrophages (LAM) in FL patients treated with immunochemotherapy. This study examines the effects of tumor-infiltrating MCs in FL patients at Cleveland Clinic.
Design: Tissue microarrays were constructed from 94 newly diagnosed cases of FL from 09/01/1985 to 12/13/2002 that had been previously characterized for CD68+ LAMs and demonstrated that extrafollicular (EF) LAMs (>16.8/high power field) were a poor prognostic marker (Kelley T et al 2007). Immunohistochemistry for tryptase was performed on these microarrays to enumerate MCs per high powered (400x) field. In each core, all available lymphomatous tissue was evaluated (mean of 8.13 hpf/case, range 1–9). The cell counts were compared to the available clinical follow-up data, using overall survival as the measurable outcome.
Results: The FL patients (44 women, 50 men) had a mean age at diagnosis of 59 years and a mean follow-up of 95.8 months. The patients were treated heterogeneously; the great majority did not receive immunochemotherapy as initial treatment. MC data was available in 91 patients. The mean MC count was 4.14 MCs/hpf (range 0–17.33). As shown previously, > 16.8 EF LAMs/hpf was a predictor of poor overall survival; hazard ratio of 2.28, 95% CI: 1.22–4.24, P = .009. In the entire cohort, MCs were not associated with overall survival. However, in the subgroup with low LAM (CD68 <16.8/hpf), increased MCs (>3.3/hpf) was associated with better overall survival, HR = 0.40, 95% CI: 0.16–0.99, P = .049.
Conclusion: In our data set, increased LAMs in FL are associated with poor overall survival. Overall, MCs do not appear to predict outcome; however, in the subgroup of patients with low LAMs, increased MCs are associated with a favorable overall survival. This suggests a complex interaction between cellular constituents of the tumor microenvironment that affects patient outcome. The application of immunochemotherapy or other immunomodulatory therapies may alter these interactions and outcomes. Further work is needed to better understand these interactions and to validate biomarkers in therapy specific contexts.
Identification of ERG Specific Target Genes by Genome-Wide Screening in T-Lymphoblastic Leukemia
Authors: Mochmann Liliana H , Neumann Konrad , Bock Juliane , Tanchez Jutta Ortiz , Bohne Arend , Thiel Eckhard , Hofmann Wolf K , Baldus Claudia D 
Published: October 2019

Abstract
The Ets related gene, ERG, encodes a transcription factor with a vital role in hematopoiesis. Recent findings have shown that ERG knockout mice require a minimum of one functional allele to ensure embryonic blood development and adult stem cell maintenance. Moreover, it was earlier reported that enforced expression of ERG induced oncogenic transformation in 3T3 cells. Overexpression of ERG, observed in a subset of acute T-lymphoblastic and acute myeloid leukemia patients, was associated with an inferior outcome. However, the impact of ERG contributing to this unfavourable phenotype has yet to be determined, as downstream targets of ERG in leukemia remain unknown. Herein, we conducted a genome-wide analysis of ERG target genes in T-lymphoblastic leukemia. Chromatin immunoprecipitation-on-chip array (ChIP-on-chip) analyses were performed using two ERG specific antibodies for the enrichment of ERG-bound DNA templates in T-lymphoblastic leukemia cells (Jurkat) with input DNA or IgG precipitated DNA as controls. Enriched DNA templates and control DNA were differentially labelled and co-hybridized to high resolution promoter chip arrays with 50–75mer probes (770,000) representing 29,000 annotated human transcripts (NimbleGen). Based on two independent ChIP-on-chip assays, bioinformatic analysis (ACME) yielded statistically significant enriched peaks (using a sliding window of 1000 bp, and a P-value < 0.0001) identifying promoter regions of 365 potential ERG target genes. From these genes, clustering by functional annotation was performed using the DAVID database and subsequently genes related to leukemia were further selected for quantitative PCR validation. The design of promoter primers included the highly conserved ETS GGAA DNA binding site. Genes with greater than two-fold enrichment (ERG ChIP versus control) included WNT2 (17-fold), OLIG2 (14-fold), WNT11 (7-fold), CCND1 (5-fold), WNT9A (4-fold), CD7 (3-fold), EPO (3-fold), ERBB4 (3-fold), RPBJL (3-fold), TRADD (3-fold), PIWIL1 (2-fold), TNFRSF25 (2-fold), TWIST1 (2-fold), and HDAC4 (2-fold). Interestingly, enriched target genes involved in developmental processes (WNT2, WNT9A, WNT11, TWIST1, PIWIL1, ERBB4, and OLIG2) have shown oncogenic potential when mutated or overexpressed. Thus, we hypothesize that overexpression of ERG may contribute to T-cell leukemogenesis by the deregulation of these oncogenic targets. Further disclosure of ERG directed downstream pathways may contribute to the design of specific treatment strategies (such as WNT inhibitors) with particular effectiveness in ERG deregulated leukemia.
Lipid and Protein Substrates of PI3K in Cytokine Receptor Survival Signalling: Deregulation in Leukemia
Authors: Thomas Daniel , Woodcock Joanna , Powell Jason A , Barry Emma F , Lopez Angel F , Guthridge Mark A 
Published: October 2019

Abstract
New therapeutic approaches to acute myeloid leukemia (AML) must ultimately target cell survival pathways in leukemic cells in order to be effective. We have identified a serine residue (Ser585) in the cytoplasmic domain of the common GM-CSF and IL-3 receptor beta subunit which is phosphorylated in response to sub-picomolar concentrations of growth factor and is involved in signalling cytokine-mediated survival via 14-3-3 zeta phosphoserine adaptor. While Serine 585 is tightly controlled in non-transformed haematopoietic cells from normal donors, Serine 585 is constitutively phosphorylated in AML blasts suggesting a role in AML cell survival and a novel target for anti-leukaemic therapy. We attempted to isolate Ser585 kinase activity from leukemic blasts and characterise this activity in response to serine/threonine kinase inhibitors in biochemical and biological assays.
Results: Cell extracts from primary AML blasts (>99% blasts by flow/morphology) obtained from adult patients were fractionated and assayed for intrinsic serine 585 peptide (13-mer) kinase activity via 32P gamma-ATP in vitro kinase assay. A single peak of Ser585 kinase activity was isolated and tested against a panel of serine/threonine kinase inhibitors. Kinase activity was selectively sensitive to LY294002, wortmannin and quercelin suggesting a role for the PI3K family of kinases in activating this residue. Ser585 kinase activity was also directly present in both p85 and p110 alpha PI3K immunoprecipitates from AML blasts and leukemic cell lines tested on both Ser585 peptide and recombinant beta cytoplasmic domain protein substrates. Serine 585 phosphorylation induced by sub-picomolar concentrations of GM-CSF in TF1.8 cells was inhibited by three different isoform selective p110 alpha inhibitors used at low nanomolar ranges consistent with reported IC50s. These results suggest a novel role for protein kinase rather than lipid kinase activity of PI3K alpha subunit in low dose cytokine signalling. We also show induction of serine phosphorylation of p85 PI3K regulatory subunit on Ser608 by GM-CSF, a previously reported protein substrate of PI3K. Furthermore, p110 alpha and delta inhibitors abrogate GM-CSF dependent survival of murine lineage negative bone marrow progenitor cells and also exert apoptotic activity on flow-sorted CD34+CD38−CD123+ sub-populations of primary AML blasts.
Conclusions: Inhibition of Ser585 phosphorylation by targetting PI3K protein kinase activity by isoform selective inhibitors represents a novel approach toward the eradication of residual leukemic stem cells.
Factor H Binds to Von Willebrand Factor (VWF) and Regulates Its Cleavage by ADAMTS-13
Authors: Cruz Miguel A. , Gushiken Francisca C. , Rumbaut Rolando , Patel Kavita N , Turner Nancy , Nolasco Leticia , Han Hyojeong , Pickering Matthew , Dong Jing-fei , Moake Joel L. , Afshar-Kharghan Vahid 
Published: October 2019

Abstract
Factor H is a plasma protein that regulates activation of the alternative complement system. Mutations in the factor H gene are associated with a rare form of thrombotic microangiopathy, known as atypical hemolytic uremic syndrome. Hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP) are characterized by systemic (TTP) or renal (HUS) microvascular thrombosis. The clinical presentations of TTP and HUS have some common clinical features, including the presence of thrombocytopenia, intravascular hemolysis, and mechanical injury to red cells. We investigated whether factor H has any interaction with proteins involved in the pathogenesis of thrombotic thrombocytopenic purpura, namely von Willebrand Factor (VWF) and ADAMTS-13. We found that factor H binds to the A1 and A2 domains of VWF, and inhibits cleavage of VWF by ADAMTS-13 under static and flowing conditions. Factor H deficient mice had a delayed thrombous formation after vessel wall injury, and showed a lower mortality rate and a higher platelet count after Shiga toxin/lipopolysaccharide challenge compared to their wild-type littermates. We concluded that factor H, in addition to its complement regulatory activity, might regulates cleavage of VWF by ADAMTS-13. Presence of an abnormal factor H that is unable to control complement activity on cell surfaces but still capable of inhibiting cleavage of VWF might contribute to the pathogenesis of thrombotic microangiopathies.
Activity of SYK and PLCγ2 Predict Apoptotic Response of Chronic Lymphocytic Leukemia Cells to SRC Tyrosine Kinase Inhibitor Dasatinib.
Authors: Wang Y. Lynn , Song Zibo , Lu Pin , Furman Richard R , Leonard John P. , Martin Peter , Tyrell Lauren , Lee Francis Y. , Knowles Daniel M. , Coleman Morton 
Published: October 2019

Abstract
Abstract 1249
Poster Board I-271

Purpose
B-cell receptor (BCR) signaling plays an important role in the pathogenesis of chronic lymphocytic leukemia (CLL). However, blocking BCR signaling with dasatinib, an inhibitor of SRC kinase, produced variable results in pre-clinical and clinical studies. We aim to define the molecular mechanisms underlying the differential dasatinib sensitivity and to uncover more effective therapeutic targets in CLL.

Experimental Design
Fresh CLL B cells were treated with dasatinib and cell viability was followed. The CLL cases were then divided into good and poor responders. The cellular response was correlated with the activities of BCR signaling molecules as well as with molecular and cytogenetic prognostic factors.

Results and Conclusions
Among 44 CLL cases, dasatinib treatment reduced cell viability by 2-90%, with an average reduction of 48% on day four of culture. The drug induced CLL cell death via the intrinsic apoptotic pathway mediated by reactive oxygen species. Unexpectedly, phosphorylation of SRC family kinases was inhibited by dasatinib in good as well as poor responders. As opposed to SRC family kinases, activities of two downstream molecules, SYK and PLCγ2, correlate well with the apoptotic response of CLL cells to dasatinib. Thus, SYK inhibition predicts cellular response to dasatinib. SYK, together with PLCγ2, may serve as potential biomarkers to predict dasatinib therapeutic response in patients. From the pathogenic perspective, our study suggests the existence of alternative mechanisms or pathways that activate SYK independent of SRC kinase activities. The study further implicates that SYK might serve as a more effective therapeutic target in CLL treatment.

Disclosures
Lee: Bristol-Myers Squibb Research & Development: Employment. Coleman:Bristol-Myers Squibb Research & Development: Consultancy.
Glanzmann Mutations : New Allelic Variants and Role of the β3 Lys253 in the αIIb/β3 Interaction Highlighted by the Lys253Met Substitution.
Authors: Jallu Vincent , De Brevern Alexandre , Panzer Simon , Torchet Marie-Francoise , Kaplan-Gouet Cecile 
Published: October 2019

Abstract
Abstract 1312
Poster Board I-336

Introduction
Glanzmann thrombasthenia (GT) is an autosomal recessive inherited bleeding disorder characterized by an impaired platelet aggregation. GT results from defects of the platelet fibrinogen receptor αIIbβ3. GT mutations provide useful tools for structure-function relationship studies of αIIbβ3.

Patient and methods
Genomic DNA from 6 patients has been amplified for αIIb and β3 promoters and exons sequences. PCR products were directly sequenced. Potential RNA processing alterations have been studied in silico by using Genscan, NNSPLICE and ESEFinder online tools. When no RNA splicing anomaly was predicted, the effect of single point mutation on αIIbβ3 expression has been studied by using transiently transfected Cos cells. Finally, structural consequences of amino acid substitutions has been studied using the published model of αIIbβ3 (code PDB 2VDL) and structural modelling.

Results
7 new mutations have been characterized. 1 deletion / insertion, 2 single point mutations inducing stop codon and 1 resulting in splicing site disruption were identified. The 3 last identified single point mutations were not predicted to affect normal RNA processing but has been shown to prevent normal expression of mutant αIIbβ3 at the surface of Cos cells. The p.Meth118Arg and p.Gly221Asp substitutions that induce both important steric hindrance and charge modifications, are located inside the β-I domain of β3. So they should deeply alter the proper folding of the β-I domain, preventing the complex expression at the platelet surface. On the other hand, the p.Lys253Met protrudes from the β-I domain toward the αIIb β-propeller. A structural model of the Met253 β-I mutant has been done. An estimation of the direct electrostatic and desolvation free energies of interaction between the β-I domain surface and the αIIb β-propeller indicated that rather than the presence of a methionine, it is the lost of the Lys253 which is responsible for the complex expression defect.

Conclusion
Seven new GT mutations have been identified and the p.Lys253Met substitution helped to define a key role of the Lys253 in the αIIb β-propeller / β3 β-I domains interaction.

Disclosures
No relevant conflicts of interest to declare.
Barriers to Reduced-Intensity Conditioning (RIC) Transplant in Patients with Chronic Lymphocytic Leukemia.
Authors: Jaglowski Samantha M. , Lin Thomas S. , Elder Patrick , Scholl Diane , Byrd John C. , Devine Steven M. , Andritsos Leslie 
Published: October 2019

Abstract
Abstract 1374
Poster Board I-396

Introduction:
Allogeneic stem cell transplantation is the only potentially curative therapeutic option for patients with CLL. However, in spite of recent improvements in transplant-related mortality due to the introduction of reduced-intensity conditioning (RIC), only a proportion of patients go on to receive a stem cell transplant. The aim of this study was to quantify how many patients are evaluated for transplant versus how many go on to transplant, and to examine the reasons why transplantation was not performed.

Patients and methods:
A list of all patients with CLL who have been referred to the Ohio State University for a transplant evaluation from January 2003 to the present was obtained. Two electronic databases, E-Results and TransChart, were reviewed to determine if a consultation was performed, if patients were subsequently transplanted, and if not, why.

Results:
The outcomes of the referrals are summarized in Table 1. From January 2003 to the present, 205 patients with CLL were referred to our center for a transplant evaluation. Of those, 34 (16.6%) underwent transplantation. For patients who did not receive a transplant, it was not indicated at the time of consult according to current standard of care in 37 patients (18%), and in 29 (14.1%), transplant was indicated but patients were unable to obtain a remission in order to proceed. The differences between patients who went on to transplant and those who could not obtain remission were analyzed more closely. The median age in the transplant group was 55, and the median age in the group that could not obtain remission was 58. Both groups received a median 2 lines of treatment prior to transplant evaluation. The cytogenetic abnormalities of the groups are summarized in Table 2.

Conclusions:
The majority of patients who did not undergo transplantation did not have an indication for transplant, reflecting previous standards of care prior to the understanding of the poor prognosis of patients with high risk karyotype and fludarabine-refractory CLL. In contradistinction, an inability to obtain a remission in order to proceed to transplantation was the second most common reason transplantation was deferred. These data reflect the difficulty in some cases of determining the appropriate timing for allogeneic transplantation in CLL and the risk of delaying a transplant referral in patients with fludarabine refractory disease. Appropriate timing of transplant referral and choice of effective salvage therapy are critical to the successful long-term management of patients with fludarabine-refractory CLL.

Disclosures:
No relevant conflicts of interest to declare.
MPL Mutation Profile in JAK2 Mutation-Negative Patients with Myeloproliferative Neoplasms.
Authors: Ma Wanlong , Zhang XI , Wang Xiuqiang , Zhang Zhong , Yeh Chen-Hsiung , Sferruzza Anthony , Uyeji Jennifer , Albitar Maher 
Published: October 2019

Abstract
Abstract 1891
Poster Board I-914
Myeloproliferative neoplasms (MPNs) are multipotent hematopoietic stem cell neoplasms characterized by excess production of various blood cells. Mutations in the thrombopoietin receptor gene (MPL) have been reported in JAK2 V617F-negative patients with MPNs. We evaluated the prevalence of MPL mutations relative to those of JAK2 mutations at V617 or in exons 12 through 15 in a large number of patients with suspected MPNs. A total of 2790 patients with suspected MPNs referred to our institution were first screened for JAK2 V617F and exon 12–15 mutations. Patients with no JAK2 mutations detected were then tested for MPL mutations in exons 10 and 11 by means of a sensitive MPL reverse-transcription-PCR-based assay with direct bidirectional sequencing. All JAK2 and MPL mutation assays were performed on plasma RNA, rather than DNA isolated from blood or bone marrow cells. Of the 2790 patients, 529 (18.96%) had a V617F mutation; 12 (0.43%) had small insertion/deletions in exon 12; and 7 (0.25%) had other JAK2 mutations, including point mutations in exons 13–15 and an exon 14 splice mutation. MPL mutations were identified in 66 of the 2242 (2.94%) JAK2 mutation-negative patients (2.37% of all tested patients). W515L was the dominant MPL mutant detected in 46 patients (70%) including two patients with homozygous W515L mutation. The other W515 variants (W515K, W515R, W515S, W515G, W515A, W515*) comprised 16% (N=11) of the MPL mutations. The remaining MPL mutations (n =9, 14%) were detected at other locations in exon 10 and 11. Two of these were novel exon 10 deletion/insertion mutations and two were unreported exon 11 point mutations. The exon 10 T496-A497ALVI (4AA) homozygous insertion was detected in one patient with a confirmed diagnosis of idiopathic myelofibrosis and the W515-P518 del/ins (KT) mutation was detected in another patient with unspecified MPN. The two novel MPL exon 11 point mutations were D545G and D545N. The S505N mutation (n=2) and V507I (n=1), R514K (n=1), and A519V (n=1) were also detected. Furthermore, three unreported silent polymorphisms/mutations (T496, L543, and D534) were detected. In conclusion, our results demonstrate that for every 100 V617F mutations detected in patients with MPNs, there are 2.3 JAK2 exon 12 mutations, 1.3 JAK2 exon 13–15 mutations, and 12.5 MPL mutations. Thus, MPL mutation detection should be performed on all JAK2 V617F-negative patients with suspected MPNs. In addition, our findings indicate that >20% of MPL mutations would have been missed if only W515L and W515K were analyzed; thus, sequencing of both exon 10 and 11 may be beneficial.

Disclosures:
No relevant conflicts of interest to declare.
The Role of JAK3 mutations in Adult T-Cell Leukemia/Lymphoma.
Authors: Elliott Natalina E , Cleveland Susan , Grann Victor , Janik John E. , Dave Utpal P. 
Published: October 2019

Abstract
Abstract 1940
Poster Board I-963
Adult T-cell leukemia/lymphoma (ATLL) is an aggressive and incurable cancer caused by infection from a retrovirus, Human T-cell Leukemia Virus type-1 (HTLV-1). HTLV-1 infects T cells and induces cell transformation through the expression of viral oncogenes such as tax. In newly infected cells, tax activates the expression of Interleukin-2 and the alpha subunit of its receptor (IL2RA) resulting in autocrine signaling that drives proliferation. In ATLL cells, tax is frequently deleted or silenced by methylation but there is constitutive activation of pathways downstream of IL-2 and related cytokines. These data led us to hypothesize that somatic mutations in this cytokine pathway may play a role in ATLL tumor progression. Encouraged by the recent discovery of activating mutations in JAK3 in diverse hematologic malignancies, we sequenced the exons of JAK3 in a small cohort of ATLL patients and found three missense mutations in the amino terminal FERM domain which were not found in Single Nucleotide Polymorphism databases. IL-2 signaling recapitulated in Human Embryonic Kidney 293T cells showed that these missense mutations caused increased tyrosine phosphorylation of the JAK3 substrate, STAT5A. Furthermore, enforced expression of these mutant JAK3s caused cytokine independent growth of BaF3 cells. The FERM domain of JAK3 is similar to the FERM domain of Focal Adhesion Kinase (FAK), which has a solved crystal structure. Interestingly, the FAK FERM domain contacts and autoinhibits its own kinase domain. Our homology-based model suggests that the JAK3 missense mutations disrupt the secondary structure needed for this intramolecular contact thereby perturbing autoregulation of JAK3 kinase activity. Our findings suggest that JAK3 gain of function may play an important role in ATLL disease progression.

Disclosures:
No relevant conflicts of interest to declare.