bluuebunny/crossref_metadata_2025_split huggingface dataset:
Collaboration between Activating Mutations in JAK2 and Trisomy 21 in the Acute Lymphoblastic Leukemias of Down Syndrome (DS).
Authors: Ganmore Ithamar , Bercovich Dan , Scott Linda M. , Green Anthony R. , Cazzaniga Giovanni , Biondi Andrea , Basso Giuseppe , Cario Gunnar , Schrappe Martin , Stanulla Martin , Strehl Sabine , Haas Oskar A. , Binder Vera , Borkhardt Arndt , Trka Jan , Bielorei Bella , Avigad Smadar , Stark Batia , Bourquin Jean-Pierre , Smith Owen , Kempski Helena , Izraeli Shai 
Published: October 2019

Abstract
Additional copies of chromosome (chr) 21 are the most common chromosomal aneuploidy in childhood leukemia. Patients with DS have a markedly increased risk for both AML and ALL. Thus trisomy 21 is leukemogenic. DS-AML is uniquely characterized by an acquired mutation in the chr X gene GATA1. The existence of a similar unique collaborating mutation in DS-ALL has been postulated but remained elusive. To identify such mutations, we performed a large mutational screen in 81 diagnostic samples of B cell precursor DS-ALL, stored in central labs of 9 European childhood ALL protocols (representing more than 8000 samples of childhood ALL). Mutations in JAK2 were identified in 16 (19.7%) DS-ALL patients. The mutations are different from those observed in myeloproliferative disorders. In fifteen patients a point mutation resulted in substitution of the conserved Arginine at position 683. In one patient an in-frame insertion caused displacement of the same amino-acid. The mutations cause constitutive activation of JAK2 and are predicted to disrupt a critical interaction between the pseudokinase and the SH2 domains. The mutations are acquired as they do not exist in remission samples. The mutated RNA is expressed. They are unique to DS-ALL as screening of over 300 non DS-ALL, as well as DS-AMKL, has revealed only one instance with a similar mutation. Strikingly, that patient had an ALL with an iso21q abnormality! The clinical presentation and survival data of DS-ALL patients with and without JAK2 mutation is currently analyzed and will be presented in the meeting. Our data demonstrate that novel mutations of JAK2 cooperate with trisomy 21 in at least 20% of ALLs in Down Syndrome. Thus it seems that, like DS-AML, at least 20% of DS-ALLs are different from sporadic childhood leukemias and characterized by unique acquired mutations in genes located outside chr 21 (GATA1 on the chr X and JAK2 on chr 9). Beyond the obvious therapeutic implications, these observations raise the hypothesis that JAK2 is important in B cell development and that constitutive activation of JAK2 in B cell precursors provides a survival advantage in the presence of a germline trisomy 21.
NOTCH1/FBXW7 Mutations, but Not Low ERG/BAALC Expression, Identify a Major Subgroup of Adult T-ALL with a Favorable Outcome: a GRAALL Study.
Authors: Abdelali Raouf Ben , Buzyn Agnes , Asnafi Vahid , Noir Sandrine Le , Ifrah Norbert , Dombret Herve , Macintyre Elizabeth 
Published: October 2019

Abstract
Abstract 1568
Poster Board I-592
Several somatic genetic abnormalities have been identified in T-ALL but therapeutic stratification still relies on clinical markers. NOTCH1 and/or FBXW7 (N/F) mutations both lead to activation of the NOTCH1 pathway and are amongst the most frequent mutations in T-ALL. We identified N/F mutations as a good prognostic marker, found in 70% of patients within the French LALA-94 and GRAALL-2003 prospective adult T-ALL trials, whereas the German GMALL group reported that low ERG and BAALC (E/B) transcript expression also predicts a highly favorable outcome in 41% of adult T-ALL (Baldus et al. J Clin Oncol. 2007 25(24):3739-45). In order to compare the predictive prognosis value of N/F mutations versus low E/B expression, we analyzed 189 adult T-ALL for both, including 107 previously reported cases and 82 unreported cases included within the ongoing GRAALL-2005 trial up to the first planned interim analysis.
E/B were quantified by real time RT-PCR and the comparative cycle threshold (Ct) method was used to determine the relative expression levels for ERG and BAALC to ABL. T-ALL patients were classified as low E/B expressers (E/Blow) or high E/B expressers (E/Bhigh) as previously reported by Baldus et al. Despite the use of ABL rather than GPI as internal control, we observed the same incidence of E/Blow. 41% (n=77) of T-ALLs were classified as E/Blowand 59% (n=112) as E/Bhigh. 68% (n=128) of cases were mutated for NOTCH1 and/or FBXW7 (N/Fmut) and 32% (n=61) were germline for both (N/FGL). As expected E/Bhigh correlated with an immature phenotype (p<0.0001). There was no association between E/B expression level and F/Nmut (p=0.5) and no significant correlation for either N/F or E/B status with age and leucocytosis at diagnosis.
There was no difference in overall survival (OS) and event free survival (EFS) between E/Bhigh and E/Blow (4-years OS: 57% v 55 %; 4-years EFS: 46% v 47%). In contrast, N/Fmut compared with N/FGL had a better OS (67% v 36%; P= .002) and EFS (56% v 32%; P=.008). When the LALA and GRAALL protocols were analyzed separately, N/Fmut retained their significant favorable outcome, including in the GRAALL-2003 and GRAALL-2005 trials, which give overall good results for T-ALL. E/B expression did not impact on prognosis in either the GRAALL trials (E/Bhigh OS =68% EFS= 64% v E/Blow OS= 62% EFS=50%) or the LALA trials (E/Bhigh OS =46% EFS= 30% v E/Blow OS= 39% EFS=29%) individually.
Taken overall, our data demonstrate that N/Fmut, but not low E/B expression, identify a major subgroup (68%) of adult T-ALL with a highly favorable outcome that could justify individual therapeutic stratification for T-ALL. N/F mutation status is also better suited to individual patient stratification than the continuous spectrum of ERG and BAALC transcript expression.

Disclosures
No relevant conflicts of interest to declare.
Spectral Analysis of Platelet Oscillations in Cyclic Thrombocytopenia.
Authors: Potts Jayson M , Moore Jane C , Kelton John G. , Leber Brian , Arnold Donald M. 
Published: October 2019

Abstract
Abstract 4459

Introduction
Cyclic thrombocytopenia (CT) is a rare autoimmune condition characterized by predictable oscillations in platelet count levels. Periodic platelet cycles in CT have not been well characterized.

Methods
We studied three patients with wide platelet count fluctuations to identify patterns consistent with CT. Medical charts were reviewed and platelet levels and treatments were abstracted. Platelet counts were plotted over time for each patient. Spectral analyses were completed using the method of Lomb and Scargle to determine if platelet count fluctuations were random or had a stable period of oscillation. Where stable periods were identified (fixed time from peak to peak) the likelihood of those periods being random was calculated using the false-alarm probability.

Results
Regular periods of platelet oscillation were identified for each patient (Table 1). Periods varied between 23 and 42 days and were not random. Patient I had a 2-year remission induced by immune suppressive medications during which time the platelet count was normal and stable. Platelet count oscillation amplitudes were 350 ×109/L both at presentation of CT and at relapse. Gradual building and decaying of oscillations were noted at the onset of relapse, and remission, respectively.

Patient II
Matching a sine curve to the platelet data demonstrated that platelet nadirs of <20 ×109/L could be predicted to within 4 days for every cycle up to one year in advance. Patient III had typical CT oscillations for 6 years, but thereafter platelet fluctuations became random and no regular period was observed. Treatment with eltrombopag, a thrombopoietin (TPO) mimetic agent for 5 days resulted in a peak platelet count peak in excess of 1200 ×109/L in this patient.

Conclusion
In three patients with CT, we identified regular periodic oscillations in platelet counts that were non-random. Some patients have predictable cycles that may allow timed delivery of treatments such as TPO-mimetic agents. These findings may provide insight into the nature of the autoantibody in CT.

Disclosures:
Arnold: Hoffman-LaRoche: Research Funding; Amgen: Consultancy, Honoraria.
Smif (Smad4 Interacting Factor) Is a Negative Regulator of T Cell Activation and Autoimmunity In Vivo.
Authors: Leibig Wibke , Kuhn Sebastian , Patzelt Thomas , Mugler Claudia , Ren-Yuan Bai , Quintanilla-Fend Leticia , Förster Irmgard , Peschel Christian , Ruland Jürgen , Duyster Justus 
Published: October 2019

Abstract
Abstract 712
Transforming growth factor β (TGFβ) plays a critical role in regulating cellular processes like proliferation, extracellular matrix production, vasculogenesis and angiogenesis as well as immunomodulation. TGFβ is a pluripotent cytokine with a pronounced immunosuppressive effect by controlling proliferation, differentiation and activation of immune cells. TGFβ binding to its receptor leads to the phosphorylation of R-Smads. R-Smads again form a heteromeric complex with the cytosolic common Smad4. This Smad complex, together with additional cofactors, translocate into the nucleus, where they control the transcription of TGFβ target genes. Smif was originally identified in our lab as an interaction partner of Smad4. Functional analysis revealed a stimulatory effect in regulating TGFβ-dependent genes like the early target gene JunB. After TGFβ stimulation, Smif tranlocates, together with Smad4, into the nucleus, where Smif acts as a coactivator. To investigate the role of Smif in mammals, we generated a Smif knockout mouse. To this end exon 2 of Smif was replaced by GFP and an inverted neomycin selection cassette. Smif-deficient mice were viable but exhibit a shortened life span. On the average, these mice die at 12 month of age due to multifocal inflammatory disease. Overall pathological analysis of diseased mice revealed extensive lymphocytic infiltrates in multiple organs. Moreover, Smif-deficiency caused immune complex induced glomerulonephritis associated with proteinuria. In line with these findings, autoantibodies could be detected in the serum of Smif knockout mice. Interestingly, we identified T cells and not B-cells as the important target in Smif-deficient mice. T cells lacking Smif were spontaneously activated. In addition, TGFβ was not able to block T cell proliferation of CD4+ cells in vitro, whereas B cells isolated from Smif knockout spleens behave as wildtype. Transcription of TGFβ responsive reporter constructs was greatly reduced in Smif knockout Mefs and could be rescued by the reexpression of functional Smif. Taken all together, the observed autoimmune phenotype found in Smif-deficient mice is at least partially caused by overactivated T cells due to downregulation of the inhibitory TGFβ pathway.

Disclosures:
No relevant conflicts of interest to declare.
A p53-Dependent, DNA Damage Response Mediates Ineffective Erythropoiesis Caused by Deregulated Cyclin E Activity.
Authors: Xu Yanfei , Minella Alexander C. 
Published: October 2019

Abstract
Abstract 74
Cyclin E is a critical regulator of cell cycle progression that is frequently found over-expressed in human cancers. We previously described a knock-in mouse model that permitted study of the physiologic consequences of cyclin E deregulation, via abrogation of the ubiquitin-proteasome pathway controlled by the Fbw7 tumor suppressor. We found that erythroid progenitor cells of cyclin ET74A T393A knock-in mice exhibit abnormally increased proliferation, increased apoptosis, impaired maturation, and dysplastic morphologies. To investigate further the molecular mechanisms by which deregulated cyclin E activity impairs normal erythropoiesis, we have now compared gene expression profiles of CD71high/Ter119high erythroid precursors from the cyclin E knock-in and wild-type, littermate control bone marrows. Most prominent among the gene expression abnormalities identified in the cyclin E knock-in erythroid cells were up-regulated expression of multiple target genes of the p53 tumor suppressor, indicative of p53 pathway activation. We confirmed these findings using both quantitative real-time RT-PCR and western blot, the latter of which identified increased phosphorylation on p53 at serine 15, which is phosphorylated by kinases activating the DNA damage response. These findings were previously identified in cultured primary fibroblasts expressing high cyclin E by ectopic expression, and our data now demonstrate that impaired cyclin E degradation activates p53 in a cell type-specific context in vivo. To test directly the importance of these gene expression abnormalities to the erythroid maturation defects in cyclin E knock-in cells, we utilized an in vitro system for erythroid differentiation of primary bone marrow progenitor cells. We first recapitulated the erythroid differentiation defect identified in vivo in cultured cyclin ET74A T393A knock-in bone marrow cells. Then, to determine the functional importance of p53 activation in cyclin E knock-in erythroid progenitors, we used small hairpin RNAs to knock-down p53 expression in hematopoietic progenitors. We found that reduced p53 expression rescued the erythroid differentiation defects in cyclin E knock-in progenitor cells, while not having a significant effect in wild-type cells. Together, our findings suggest that oncogenic activation of p53 in bone marrow progenitor cells can directly impair erythroid differentiation.

Disclosures:
No relevant conflicts of interest to declare.
Screening Small Molecule Inhibitors of VLA-5 to Block the Interaction Between Acute Lymphoblastic Leukemia with Ph+ and Their Microenvironment.
Authors: Hu Zhongbo , Li Xiaomiao , Ostrov David , Slayton William 
Published: October 2019

Abstract
Abstract 1027
Integrin VLA-5 (α5β1, CD49e/CD29) plays an important role in hematopoietic cells functioning as well as in promoting tumor angiogenesis and tumor metastasis. Molecules targeting VLA-5 can be rapidly developed into anti-inflammatory and anti-tumor pharmaceuticals. VLA-5 is highly expressed on Ph+ leukemia cells and VLA-5 inhibitory antibodies can significantly inhibit the adhesion of Ph+ leukemia cells to human fibronectin. We generated an atomic homology model of VLA-5 based on the crystal structure of the extracellular segment of integrin αVβ3 in complex with a cyclic peptide presenting the Arg-Gly-Asp sequence and utilized this structure-based approach to identify VLA-5 binding drug-like small molecules. We selected the Arg-Gly-Asp binding residues and the epitopes of VLA-5 antibody as the target for small molecule binding using SPHERE_SELECT in DOCK6. The grid-based scoring system was used for scoring with the non-bonded force field energy function. The 100 highest scoring small molecules were assayed in an in vitro adhesion assay using leukemia cell lines and solid phase assay. This approach identified several leading small-molecule compounds, V10, V20, V37 and L4. Their IC50 are respectively 22.5μM, 23.7μM, 32.0μM and 28.9μM. These compounds can inhibit the adhesion of VLA-5 expressing Philadelphia chromosome positive leukemia to both human fibronectin and bone marrow stromal cells. Compounds V10 and V20 also significantly inhibited the growth of Ph+ leukemia cells. These compounds can enhance the effect of imatinib and dasatinib to kill Ph+ leukemia cells when cultured contacting with bone marrow stromal cells. We are currently testing the synergistic effect of these compounds with tyrosine kinase inhibitors to treat the Ph+ acute lymphoblastic leukemia in NOD/SCID animal model.

Disclosures:
No relevant conflicts of interest to declare.
High Thrombin Generation Measured In the Presence of Activated Protein C Is Associated with An Increased Risk of Recurrence Among Women 18–64 Years After a First Event of VTE
Authors: Sonnevi Kristina , Tchaikovski Svetlana N , Bremme Katarina , Holmstrom Margareta , Rosing Jan , Larfars Gerd 
Published: October 2019

Abstract
Abstract 154

Background:
Patients with venous thrombosis (VTE) have a high risk of a recurrence after discontinuation of anticoagulants. Both prolonged anticoagulant treatment with increased risk for bleeding and a recurrence reduces quality of life. In women younger than 65 years old the recurrence rate is relatively low. However, identification of the patients at risk of a recurrence remains difficult.

Objective:
The aim of our study was to investigate whether a global coagulation test such as thrombin generation can be used as a marker for the increased risk of a recurrence of VTE among women of 18–65 years old after a first event of deep venous thrombosis (DVT) or pulmonary embolism (PE).

Methods:
139 women with an objectively documented first event of DVT or PE were enrolled into the study at diagnosis. Plasma was collected at least 4 weeks after discontinuation of anticoagulant treatment. Patients with cancer or pregnancy within 3 months after the first event were excluded. All patients were followed up prospectively for median 47 months. Recurrences of VTE were objectively documented. No participants were taking oral estrogens treatment during the follow up. Thrombin generation was measured via calibrated automated thrombography (CAT), in the absence and in the presence of activated protein C (APC). The APC sensitivity ratio (APCsr) was calculated to estimate APC-resistance. All participants were screened for factor V Leiden and G20210A mutation.

Results:
The ETP and peak height in the presence of APC (ETP+APC, peak height+APC) were associated with a recurrence of VTE in a log-rank test. In a multivariate cox-regression patients with the ETP+APC > 169 nM*min had hazard ratios (HR) for recurrent VTE of 8.8 (p=0,008) compared to patients with the ETP+APC < 169 nM*min. Patients with the peak height+APC > 58 nM had HR of 7.2 (p=0,014). Patients with the APCsr > 3.32 had HR of 6,7 (p=0,006) for recurrence of VTE. Age, inherited thrombophilia (factor V Leiden, G20210A mutation) and BMI> 30 were included into the multivariate analysis. Conclusion: High thrombin generation in the presence of APC in women after a first event of VTE is indicative for an increased risk of a recurrence. Our data suggest that acquired APC resistance is a risk factor for recurrent VTE.

Disclosures:
No relevant conflicts of interest to declare.
IGF Binding Protein 2 Supports the Cycling of Hematopoietic Stem Cells.
Authors: Huynh HoangDinh , Zheng Junke , Zhang Chengcheng 
Published: October 2019

Abstract
Abstract 1604
Previously we identified IGFBP2 as an extrinsic factor that supports ex vivo expansion of hematopoietic stem cells (HSCs). The role of IGFBP2 in HSCs and cancer is very intriguing. IGFBP2 can bind to insulin-like growth factor (IGF) ligands and displays IGF-dependent growth inhibitory effects on many cell types. On the other hand, IGFBP2 is capable of stimulating growth of certain cancer cells, and is overexpressed in many cancer patients and its expression is correlated with cancer progression. Here we sought to study the role of IGFBP2 in regulation of the activity of normal HSCs. We showed that IGFBP2 was expressed in differentiated hematopoietic cells and bone marrow stroma but not in HSCs. Consistent with its gene expression pattern, IGFBP2-/- HSCs had similar repopulation activity as their wild-type counterparts. By contrast, when we transplanted HSCs into IGFBP2-/- or wild-type recipient mice, we found decreased in vivo repopulation of HSCs in primary and secondary transplanted IGFBP2-/- recipients, suggesting that the environmental IGFBP2 positively supports HSC activity. Further co-culture of HSCs with IGFBP2-/- or wild-type bone marrow stromal cells indicated that IGFBP2 produced by bone marrow stroma indeed supports HSC expansion. Consistently, HSCs in IGFBP2-/- mice showed decreased frequency and cell cycling, and had upregulated expression of cell cycle inhibitors of p21, p16, and p19. To determine whether IGFBP2's effect on HSCs depends on IGF signaling, we compared the repopulation of donor cells deficient for the IGF type I receptor in wild-type and IGFBP2-/- recipients. These HSCs that are defective in IGF signaling still have decreased repopulation in IGFBP2-/- recipients, suggesting that the environmental effect of IGFBP2 on HSCs is independent of IGF signaling. To identify the functional domain of IGFBP2 in regulation of HSC activity, we constructed IGFBP2 with mutated RGD domain or deleted c-terminus and used the mutant IGFBP2 proteins in ex vivo culture of HSCs. We found that the c-terminus of IGFBP2 is essential to support HSC activity. We are currently in the process of identifying the potential receptor of IGFBP2 on HSCs. In summary, we found that IGFBP2 supports the cycling of normal HSCs, and this effect is independent of IGF signaling. Our study is important in revealing the relationship among environmental cues and cell fates of stem cells and opens up a new avenue in investigation of the roles of IGFBP2 in stem cells and cancer.

Disclosures:
No relevant conflicts of interest to declare.
Outcome of Patients with Chemotherapy Refractory and Early Progressive Diffuse Large B Cell Lymphoma After R-CHOP Treatment
Authors: Hitz Felicitas , Connors Joseph M. , Gascoyne Randy D. , Hoskins Paul , Moccia Alden A. , Savage Kerry Joanne , Sehn Laurie H. , Shenkier Tamara , Klasa Richard 
Published: October 2019

Abstract
Abstract 1751

Background:
The addition of rituximab to CHOP (R-CHOP) has markedly improved the progression-free and overall survival of patients (pts) with diffuse large B cell lymphoma (DLBCL). Nevertheless, 9.5% of pts harbor lymphoma that is refractory to R-CHOP (Feugier et al) and 18–27% experience relapse after initial response (GELA and MINT trial).

Aim:
Assess outcome of pts with primary refractory and early relapsing DLBCL (within 3 months of CR/PR post completion of R-CHOP treatment).

Patients and Methods:
The BCCA Lymphoid Cancer Database identified 1126 pts diagnosed with DLBCL between December 2000 and September 2009. All ages and stages were included and primary treatment was R-CHOP in all pts.

Results:
166 pts(15%) had primary refractory or early relapsing disease, of which 93 were age >70 y and too frail to have received > 2 cycles of R-CHOP. Of the remaining 73 pts, 33 (45%) had primary refractory lymphoma and 40 (55 %) relapsed within 3 months of achieving a CR/PR. Pt characteristics (n=73): median age 57 y; 68% male; stage I (4 %), II (16 %), III (19 %), IV (60 %); PS 0,1 (41 %) PS 2–4 (59 %); LDH elevated (58 %); IPI 0 (1 %), 1 (15 %), 2 (15 %), 3 (30 %), 4 (35 %), 5 (2 %). There were no differences in the clinical characteristics of pts with primary refractory versus early relapsing disease. Of the 73 pts, 37 (50%) were without serious co-morbidity and were able to be treated with a curative intent that included multi-agent salvage chemotherapy with a plan to proceed to high-dose tx and autologous stem cell transplantation (ASCT), whereas 36 pts had palliative tx or comfort care only. 10 pts did undergo ASCT (3 primary refractory, 7 early relapsers). The median overall survival for the 73 pts was 10 months with only 6 pts alive without evidence of disease (range 14–106 months). 5 of these 6 pts had relapsed early after initial CR/PR, while only 1 patient had primary refractory disease. 3 of these 6 pts had salvage treatment without ASCT and 3 with ASCT.

Conclusion:
Chemotherapy-refractory or early relapsed disease after R-CHOP has a very poor outcome with only anecdotal survivors independent of the intended treatment approach. Biologic workup of this group of patients is urgently needed with the intention of defining the molecular underpinnings of treatment resistance.

Disclosures:
Savage: Roche: Honoraria. Sehn:Roche: Honoraria, Research Funding.