bluuebunny/crossref_metadata_2025_split huggingface dataset:
Risk Factors for Cerebral Vasculopathy in a Sickle Cell Disease Neonatal Cohort: A Prospective Study
Authors: Sommet Julie , Alberti Corinne , Couque Nathalie , Haouari Zinedine , Missud Florence , Holvoet Laurent , Ithier Ghislaine , Baruchel André , Benkerrou Malika 
Published: October 2019

Abstract
Children with sickle cell disease have a significant vascular morbidity, specifically cerebral vasculopathy (CV) that can be detected by transcranial Doppler. The aim of this study was to identify CV risk factors using longitudinal biological and clinical data including upper and lower respiratory tract evaluation in the newborn cohort of Robert Debre Pediatric Sickle Cell Referral Center. 375 patients with sickle cell anemia with a total of 2677 patient-years and a 7 years median follow-up were evaluated. Among the 59 children presenting CV, seven had a stroke. Overall, the incidence rate of CV was 2.20/100 patient-years (95% CI 1.64-2.76) and the incidence rate of stroke was 0.26/100 patient-years (95% CI 0.07-0.46). The cumulative risk of CV by the age of 11.3 years was 26.0% (95% CI 20.0%-33.3%). CV risk factors were assessed by a Cox model encompassing linear multivariable modelling of longitudinal quantitative variables. The final model retained four variables. Years with upper airways obstruction (HR=1.47, 95%CI 1.05-2.06), bronchial obstruction (HR=1.76, 95%CI 1.49-2.08), fetal hemoglobin level (HR=0.68 per 5% increase 95%CI 0.48-0.96) and reticulocyte count (HR=1.82 per 50000/mm3 increase 95%CI 1.10-3.01) were independent risk factors. An ancillary analysis performed for the 209 patients with α-thalassemia data (29 patients with CV and 180 without) found that α-thalassemia was protective in bivariate analysis but was not retained in multivariable analysis. The final Cox multivariable analysis for CV in this subgroup retained three variables. Years with upper airways obstruction and bronchial obstruction were significant risk factors for CV (with respectively HR=1.57 IC95% [1.05-2.32] and HR=1.73 IC95% [1.37-2.17]) whereas fetal hemoglobin was protective (HR=0.54 per 5% increase IC95% [0.31-0.94]). Reticulocyte count was no more retained as risk factor for CV after introducing α-thalassemia in the final multivariable model. Early evaluation and specific treatment for symptoms of asthma or nocturnal respiratory abnormalities could potentially reduce the risk of CV and stroke. They are currently under evaluation in a multicenter clinical trial with systematic nocturnal polysomnography.

Disclosures
No relevant conflicts of interest to declare.
Clinical Significance of CSF3R, SRSF2 and SETBP1 mutation in Chronic Neutrophilic Leukemia and Chronic Myelomonocytic Leukemia
Authors: Qiao Chun , Ouyang Yuan , Zhang Sujiang 
Published: October 2019

Abstract
Objective: To investigate the gene mutation and the clinical features of CSF3R, SETBP1 and SRSF2 in chronic neutrophilic leukemia (CNL) and chronic myelomonocytic leukemia (CMML) patients.
Method: Sequence analysis of CSF3R, SETBP1 and SRSF2 were performed in 10 CNL and 56 CMML patients whose clinical features were also studied.
Result: Among 10 CNL patients, 8(8/10, 80%) patients had CSF3R mutations and 7(7/8, 87.5%) of them were with CSF3R T618I. In 56 cases of patients with CMML, SRSF2 mutations were found in 14(14/56,25%), CSF3R in 4(4/56,7.1%) and SETBP1 in 3(3/56, 5.3%) patients. In CMML, compared to wild-type(wt) SRSF2 patients, SRSF2 mutated patients appeared to be more possible with SETBP1 mutations [1/14(7.1%) vs. 2/42(4.8%), P>0.05], less possible with CSF3R mutation [0/14(0%) vs. 4/42(9.5%), P<0.001]. The clinical characteristics such as age, gender, WHO category, FAB category, karyotype and blood cell counts did not reveal any difference between SRSF2 mutated and wtSRSF2 patients. Either SRSF2 mutated patients or SETBP1 mutated patients both had shorter overall survival (OS) and progression-free survival(PFS) when compared with those with wtSRSF2 (P<0.001 both) and wtSETBP1 (P<0.001 and P=0.02, respectively). No significant difference of OS and PFS between CSF3R mutated and wtCSF3R patients were observed. In multivariate analysis, SRSF2 mutation was an independent negative predictor for OS (HR, 3.307; 95% CI, 1.137 to 9.614; P=0.028) and PFS(HR, 15.431; 95% CI, 3.041 to 78.312; P = 0.001). What's more, SETBP1 mutation was also an independent negative predictor for OS(HR, 9.492; 95% CI, 1.183 to 76.128; P = 0.034).
Conclusion: The majority of patients with WHO-defined CNL have oncogenic mutations in CSF3R and the T618I mutation type is a highly sensitive and specific molecular marker of the disease. While mutations of SRSF2 are common in CMML and may be of prognostic significance. As a non-specific molecular marker, SETBP1 was found in CNL, CMML and other blood cancers, which have poor prognosis.

Disclosures
No relevant conflicts of interest to declare.
EZH2-Mediated Activation of Serine Biosynthetic Pathway Is Critical for Resistance of Clonal Cells to Stress-Related Apoptosis in Low-Grade Myelodysplastic Syndrome
Authors: Xu Feng , Chang Chunkang , Shi Wenhui , Li Xiao 
Published: October 2019

Abstract
Background
Increased cell apoptosis mediated by P53 or activated autoimmunity was frequent in low-grade MDS especially in 5q-syndrome and those with trisomy 8. However, MDS clonal cells can gradually acquired apoptosis resistance under different stressed conditions. The mechanism of apoptosis resistance is still not fully understood. The polycomb group protein EZH2 as an anti-apoptosis protein drives tumorigenesis. The association of EZH2 with apoptosis resistance of MDS clonal cells is worth further investigating.
Objective
The objective of this study is to investigate the role of EZH2 in the mechanism of apoptosis resistance of MDS.
Methods
The expression of EZH2 protein was analyzed by flow cytometry in 71 MDS patients. EZH2-targeted genes were identified through an integrated analysis of gene expression microarray analysis in MDS patients and cell line. EZH2-targeted genes were confirmed through chromatin immunoprecipitation and luciferase reporter gene assay. Cell functional and metabolic experiments were also performed.
Results
We observed upregulation of the EZH2 expression in the patients with low-grade MDS especially in the individuals with trisomy 8 although downregulation in the patients with high-grade MDS. Based on the distinct EZH2 expression pattern, we identified an EZH2-targeted gene PSAT1, a serine biosynthetic critical enzyme through an integrated analysis of gene expression microarray analysis in MDS patients and cell line. Functionally, EZH2 can directly activate PSAT1 transcription by binding to its promoter and this effect is independent of its H3K27me3 enzymatic activity in vitro. Expanded sample analysis revealed the overexpression of PSAT1 in MDS patients especially in low-grade MDS. High expression of PSAT1 was associated with severe cytopenias, iron overload and activation of T lymphocytes-mediated autoimmunity. In vitro, overexpression of PSAT1 led to a significant proliferation advantage in the MDS and leukemia cell lines. Overexpression of PSAT1 could also downregulate apoptosis sensitivity to inflammatory cytokines and cytotoxic agents in vitro. However, knockdown of PSAT1 resulted in reduced cell growth capacity and increased the apoptosis sensitivity to cytotoxic agents. Further cancer metabolic experiments showed that overexpression of PSAT1 inhibited the ROS generation and induced an increase in glucose uptake, lactate and ATP production, which may make MDS clonal cells adaptive in the stress-related apoptosis environment.
Conclusion
EZH2/PSAT1 axis-mediated serine biosynthetic pathway is activated in low-grade MDS, which may confer MDS clonal cells growth advantages and resistance to stress-related apoptosis.

Disclosures
No relevant conflicts of interest to declare.
Phosphoproteomic Profiling of the Signaling Output of FLT3-ITD and Its AC220-Resistant Mutants Reveals Profound Signaling Differences and Differential Responsiveness to Inhibition of Downstream Kinases
Authors: Doebele Carmen , Kovar Johannes , Yepes Diego , Münch Silvia , Schnütgen Frank , Bohnenberger Hanibal , Köhler Anne , Urlaub Henning , Serve Hubert , Oellerich Thomas 
Published: October 2019

Abstract
Acute myeloid leukemia (AML) is a genetically heterogeneous disease where multiple mutations coincide in hematopoietic stem and progenitor cells leading to malignant transformation. One important class of mutations alters the function of signaling intermediates such as Fms-like tyrosine kinase 3 (FLT3), thereby helping AML cells to overcome the physiological communication with their microenvironment. Activating mutations in FLT3 are found in approximately 30% of adult AML cases. Particularly common are internal tandem duplications (ITD) in the juxtamembrane domain of FLT3, which are associated with poor clinical outcome. Recently, a phase II study of the second-generation FLT3 inhibitor AC220 (quizartinib) showed a complete remission rate of 44% to 54% in relapsed/chemotherapy refractory AML. However, secondary point mutations in the FLT3 tyrosine kinase domain have been reported as common causes of acquired clinical resistance to the FLT3 inhibitor AC220.
We used quantitative mass-spectrometry-based phosphoproteomics to elucidate and compare the signaling out-put of FLT3-ITD and its AC220-resistant mutants harbouring either the F691L 'gatekeeper' substitution or the D835V activation loop mutant in AML cell models. Our comprehensive signaling analyses profiled thousands of phosphorylation events in a site-specific manner and revealed marked differences in the signaling profiles of the FLT3 mutant variants. In general, we found differential activation of signal transducer and activator of transcription 5 (STAT5) and mitogen-activated protein (MAP) kinase signaling when comparing FLT3-ITD and the AC220-resistant mutants. Interestingly, some cytosolic tyrosine kinases showed differential activation patterns. For instance, spleen tyrosine kinase (SYK) signaling was significantly enhanced downstream of FLT3-ITD-F691L and cells harbouring this mutant showed increased responsiveness to compounds targeting SYK. Our resource study shows, how point mutations conferring resistance to AC220 impact the signaling output of FLT3-ITD and uncovers pathways whose inhibition might be useful to disrupt oncogenic signaling elicited by FLT3-ITD-F691L and -D835V mutants.

Disclosures
No relevant conflicts of interest to declare.
The Tyrosine Kinase-Binding and Proline-Rich Domains of Mutant CBL Are Essential for Leukemogenesis
Authors: Nadeau Scott , An Wei , Ahmad Gulzar , Mohapatra Bhopal , Zutshi Neha , Storck Matthew , Ogawa Seishi , Band Vimla , Band Hamid 
Published: October 2019

Abstract
Mutations of the tyrosine kinase-directed ubiquitin ligase CBL are associated with myeloid malignancies, yet the molecular mechanisms by which this tumor suppressor becomes a dominant oncogene to initiate the leukemogenic process are unclear. In this study, we used systematic mutagenesis to delineate the importance of the various protein-protein interaction domains/motifs of a CBL mutant that is most frequently found in human leukemias, Y371H, in inducing leukemogenesis. We tested the impact of these secondary mutations on the ability of CBL-Y371H to impart hypersensitivity to cytokines and to upregulate the associated signaling pathways in the TF1 leukemia cell line model, and in mouse hematopoietic stem/progenitor cells from Cbl-null mice to mimic the lack of wildtype CBL expression in most of the mutant CBL bearing leukemia patients. The secondary mutations included: Cbl G306E, to abrogate the ability of the TKB domain of mutant Cbl to bind to activated tyrosine kinases; internal deletion of the proline-rich domain (AA 477-688) to abrogate interactions with SH3 domain containing partners; Cbl-Y700/731/774F triple phosphorylation site mutant predicted not to interact with the SH2 domain-containing partners; and Cbl-1-436 deletion construct lacking all C-terminal motifs. Analyses of stably expressed mutants in TF-1 cells for hypersensitivity to SCF1 demonstrated an essential role of an intact TKB domain and a particularly important role of the proline-rich domain. Transient retroviral expression in Cbl-null primary murine hematopoietic stem/progenitor cells confirmed these results. Deletion of the proline-rich domain led to substantially less tyrosine phosphorylation of the oncogenic mutant Cbl as well as of the c-Kit receptor upon SCF stimulation, and cells expressing this mutant lacked the sustained activation of Erk1/2 and Akt typically seen after SCF stimulation of the Cbl-Y371H-expressing cells. Together, our data provide conclusive evidence that interaction of leukemogenic mutant Cbl proteins with the upstream tyrosine kinase (via the TKB domain) and with partner proteins (via the proline-rich domain) provides a basic mechanism for gain of function phenotype of mutant Cbl proteins. Our studies suggest that identification of the leukemogenesis-critical partners of the proline-rich domain and targeting of the TKB-tyrosine kinase interface provide new therapeutic approaches against mutant Cbl-driven leukemias.

Disclosures
No relevant conflicts of interest to declare.
Immune Response in Acute Myeloid Leukemia with Mutated Nucleophosmin
Authors: Kuzelova Katerina , Brodská Barbora , Marková Jana , Helbig Grzegorz , Kyrcz-Krzemien Slawomira , Cetkovsky Petr 
Published: October 2019

Abstract
In our recent work, we described changes in HLA class I frequencies in AML patients with nucleophosmin (NPM) mutations. Several allelic groups were found to be depleted in patients with C-terminal NPM mutations (NPMc+), but not in patients with wild-type NPM, and these differences corresponded well to in silico prediction of NPM-derived peptides binding to HLA class I. Moreover, patients with "favorable" HLA type (i.e., expressing HLA alleles suitable for presentation of NPM-derived peptides) were found to have significantly better overall survival than those without suitable HLA alleles. Altogether, our results suggested that spontaneous anti-NPM immune response may prevent AML development and helps achieve durable cure.
In this contribution, an extended patient cohort (N = 71 patients with all available data) was divided into four groups according to HLA type (favorable/unfavorable) and Flt3-ITD status. Patients expressing suitable HLA alleles and not having internal tandem duplications in Flt3 gene (HLA+/Flt3-) had the best overall survival (Figure 1). The impact of unfavorable HLA type alone was comparable to that of Flt3-ITD positivity while the combination of both adverse factors was associated with particularly poor outcome (p = 0.0066 for trend).
On the basis of these results, we believe that NPMc+ AML patients with favorable HLA type (about 65% of all NPMc+ AML patients) could benefit from an immunomodulatory therapy. Besides inhibitory receptors (such as PD-1, CTLA-4, TIM-3), regulatory T-cells or decreased HLA expression, reduced antigen presentation by leukemic cells belongs to possible mechanisms of immune escape. Insufficiency of molecular complexes such as the proteasome or the transport-associated protein (TAP) can result in increased presentation of Class II-associated invariant chain peptide (CLIP). Indeed, increased CLIP expression on residual leukemic blasts has recently been reported to correlate with increased relapse risk in AML. Interestingly, valproic acid used in combination with standard chemotherapy has been described to significantly decrease the relapse rate in elderly AML patients with mutated NPM (but not in other AML subgroups). This could be associated with restored antigen presentation following treatment with histonedeacetylase inhibitors. In our experiments, we observed marked reduction of CLIP expression on primary NPMc+ AML blasts following in vitro treatment with suberoylanilide hydroxamic acid (SAHA, Figure 2). Both the fraction of CLIP-positive cells (open circles) and CLIP surface density on positive cells (closed circles) were reduced. HLA class I expression was normal and was not affected by SAHA treatment. Importantly, the effect on CLIP expression occurred even at subtoxic (0.5 µM) drug concentration.
Figure 1. Figure 1. Figure 2. Figure 2.

Disclosures
No relevant conflicts of interest to declare.
Molecular Evidence of Ongoing Antigen Driven Somatic Hypermutation in the Natural History of Classic and Variant Hairy Cell Leukemia
Authors: Arons Evgeny , Davies Sarah , Still Katherine , Chai Nathan , Potocka Katherine , Zhou Hong , Kreitman Robert J. 
Published: October 2019

Abstract
Classic hairy cell leukemia (HCL) is a clonal chronic B-cell malignancy representing 2% of all leukemias, with neoplastic B lymphocytes in the blood, bone marrow and organs of the classic reticuloendothelial system. HCL variant (HCLv) is less common, typically has inferior prognosis, and usually lacks several HCL-related features including BRAF V600E, CD25, and Annexin A1. The malignant cells in HCL and HCLv patients have one or in unusual cases two different functional immunoglobulin heavy chain rearrangements. Significant variability can be observed in the 3rd complementarity determining region (CDR3), comprising the variable heavy (IGHV), diversity (IGHD), and joining (IGHJ) domains. A role for continued antigen selection in the pathogenesis and evolution of HCL and HCLv has been suggested but limited evidence has been found.
We examined HCL and HCLv-derived IGHV genes by massive parallel deep sequencing in 15 HCL and 7 HCLv cases. In all studied samples, a productive monoclonal tumor-derived IGH sequence was detected. Analysis of all reads sharing clonal CDR3 motifs revealed the existence of dominant subclone expansion and multiple subclonal variants. Analysis of phylogenetic trees indicated multiple branching and therefore exposure to multiple rounds of somatic hypermutation (SHM) in the evolution of clonally-related malignant cells. To investigate a possible cause of the high SHM activity and the large number of subclones in HCL and HCLv, we studied the expression of activation induced cytidine deaminase (AID), an enzyme essential for somatic hypermutations, expressed by germinal center (GC) B-cells where SHM occurs. In 5 HCL and 3 HCLv cases we detected a high expression level of AID mRNA, including wild-type and 2 splice variants; in 10 HCL, 4 HCLv, and 3 normal peripheral blood samples, AID expression was not detected using standard end-point PCR conditions. However, more sensitive real-time quantitative PCR detected AID transcripts in virtually all tested HCL and HCLv cases, although the range of transcript levels was large between different cases and varied with individual cases over time.
The data obtained for both HCL and HCLv fit a model of tumorigenesis in which the BRAF V600E mutation (or another event in HCLv) initiates neoplastic transformation in a GC B-cell committed to terminal differentiation, but still thereafter still targeted by ongoing SHM

Disclosures
No relevant conflicts of interest to declare.
A Randomized, Phase II Study with Biomarker Analysis of Panobinostat with or without Rituximab in Relapsed Diffuse Large B Cell Lymphoma
Authors: Assouline Sarit , Crump Michael , Holm Nielsen Torsten , Alcaide Miguel , Macdonald David , Tosikyan Axel , Haliotis Tina , Greenwood Celia , Klein Oros Kathleen , Morin Ryan , Johnson Nathalie , Mann Koren Kathleen 
Published: October 2019

Abstract
INTRODUCTION: Novel therapies which consider the molecular diversity of diffuse large B cell lymphoma (DLBCL) are needed to salvage patients who fail to respond to standard chemoimmunotherapy. The majority of DLBCL contain mutations in histone modifying enzymes (HME) which suggests that histone deacetylase inhibitors (HDI) should be active. Preclinical data suggest that they also augment the effect of rituximab.
METHODS: In this randomized phase II study, we evaluated the response rate and toxicity of panobinostat, a pan-HDI, 30 mg orally 3 times a week, with or without rituximab, in 40 patients with relapsed and refractory DLBCL (median 3 prior regimens, range1-9). Tumors were sequenced for mutations known to be associated with DLBCL, and serial biopsies were analyzed for changes in biomarkers.
RESULTS: 21 patients were treated with panobinostat alone and 19 with panobinostat and rituximab. Overall, 12/40 patients (30%) responded to panobinostat (95% confidence interval 16.6%-46.5%) the addition of rituximab did not increase responses. The median duration of response was 14.5 months (95% confidence interval 9.4 to not reached) (Figure). At a median follow up of 17.5 months for responders, 6 of 12 patients have not progressed. Patients with dual expression of MYC and BCL2 (6/27) and those with FAS mutations (5/33) did not respond although responses were seen in 5/10 patients with TP53 mutations. Responses were seen in 8/24 patients with mutations in HME genes. Patients with MEF2B mutations had the greatest odds of response (P<0.05) and a higher number of HME mutations tended to increase the odds of response. Changes in histone H3 acetylation and MYC expression were observed but did not correlate with response.
CONCLUSION: Panobinostat induces durable responses in some patients with relapsed DLBCL, and accompanying biomarker analyses identified DLBCL subgroups likely to respond.
Figure 1. Progression free survival overall and for responders, N=40 patients. Figure 1. Progression free survival overall and for responders, N=40 patients.

Disclosures
Assouline: Novartis: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Janssen: Honoraria; Celgene: Consultancy. Off Label Use: panobinostat for the treatment of diffuse large B cell lymhpoma. Crump:Seattle Genetics: Consultancy; Celgene: Consultancy; Roche Canada: Consultancy. Tosikyan:Novartis: Consultancy.