bluuebunny/crossref_metadata_2025 huggingface dataset:
MicroRNA Profiling of Human Acute Myeloid Leukemia and Normal Hematopoietic Stem/Progenitor Cells Reveals a Leukemia Stem Cell Signature.
Authors: Park Christopher Y. , Wang Yulei , Prohaska Susan , Tseng Diane , Weissman Irving L.
Published: October 2019

While numerous studies have contributed important insights into the molecular origins of human acute myeloid leukemia (AML), many may not accurately reflect molecular pathways critical to AML development or maintenance because they ignore the inherent heterogeneity among AML blasts. One subset of blasts - leukemia stem cells (LSCs) - exhibits the unique ability to self-renew and to engraft disease in immunodeficient mouse hosts, suggesting that their elimination is critical to developing curative therapies. In addition, there is little information regarding the role of microRNAs (miRNAs) in regulating gene expression or biologic function in AML. In order to assess the potential contribution of miRNAs to AML LSC biology, we have evaluated the expression profile of 315 mature miRNAs in FACS-purified AML LSC and compared it to both non-LSC blasts as well as normal human bone marrow (BM) derived hematopoietic stem cells (HSC) and committed progenitors using a multiplexed TaqMan-based real-time PCR strategy. SAM analysis with stringent criteria (at least 25% samples with Ct <30, FDR <1%) reveals that AML LSC and non-LSC blasts are more similar to one another than to normal HSC or committed progenitors. Among the BM populations tested, AML LSC and non-LSC populations are most similar to the granulocyte-macrophage progenitor (GMP). A set of miRNAs distinguishes AML LSC and non-LSC from normal HSC and committed progenitors, including 35 miRNAs that are under-expressed and 33 miRNAs that are over-expressed in both AML fractions versus the normal populations; many of these differentially expressed miRNAs show a range of expression exceeding 3 orders of magnitude. Supervised clustering analysis of AML LSC and non-LSC blasts reveals an LSC signature composed of 89 miRNAs, with nearly all differentially expressed miRNAs (86/89) exhibiting lower expression levels in AML LSC than non-LSC blasts. Finally, supervised clustering identifies a “stem-cell” signature composed of 17 miRNAs that are over-expressed in AML LSC and HSC versus committed progenitors. This group of miRNAs does not include miRNAs previously described as being highly expressed in embryonic stem cells. Together, these studies represent the first direct comparison of miRNA expression in a human cancer stem cell to its normal counterpart, thereby identifying miRNAs that may regulate AML LSC and/or normal HSC/committed progenitor function. Initial functional studies in vivo using LNA knockdown strategies indicate that a subset of miRNAs highly expressed in HSC and LSC is important in regulating normal HSC function. We are currently expanding these studies to test the role of these miRNAs in maintaining engrafted AMLs in the xenotransplant setting.
The MLL Recombinome of Adult ALL - Results from the GMALL Study Group.
Authors: Burmeister Thomas , Meyer Claus , Schwartz Stefan , Hubert Daniela , Molkentin Mara , Kowarz Eric , Goekbuget Nicola , Hoelzer Dieter , Marschalek Rolf , Thiel Eckhard
Published: October 2019

The pro B and the CD10-negative pre B immunophenotypes are found in approximately 12% and 2–3% of adult acute lymphoblastic leukemia cases, respectively. Both are characterized by a high prevalence of MLL aberrations. MLL fusion genes define a high risk group of ALL patients and they have proven useful as target structures for assessing minimal residual disease. It has been a matter of debate whether MLL aberrations are of prognostic significance within the group of pro B ALL patients. To better characterize the MLL recombinome in adult ALL we have prospectively and retrospectively investigated BCR-ABL-negative pro B (N=152) and CD10-negative pre-B ALL (N=31) patient samples obtained within the framework of the GMALL study group between 2001 and 2007. We used RT-PCR to detect MLL-AF4, MLL-ENL and MLL-AF9 and other MLL fusion genes. An inverse long PCR method was used to identify the exact chromosomal breakpoints in the MLL gene or unknown MLL fusions.

Results: RT-PCR analysis revealed a MLL-AF4 fusion in 20/31 (64.5%) and a MLL-ENL fusion in 2/31 (6.5%) of CD10-negative pre-B cases. A MLL-AF4 fusion was detected in 101/151 (67.3%) and a MLL-ENL fusion in 9/151 (6.0%) of pro B cases. Additionally, two pro B patients showed a MLL-AF9 and MLL-CXXC6 fusion, respectively. The relative frequency of MLL aberrations was higher (50/68 = 73.5%) in those pro B patients with immunophenotypic coexpression of myeloid markers than in those without myeloid coexpression (41/84 = 48.8%). The chromosomal breakpoints in the MLL gene and the respective partner gene were determined on the genomic level in 91 patients. The distribution of chromosomal breakpoints in the MLL gene was the following: 1 in intron 7, 1 in intron 8, 33 in exon/intron 9, 27 in exon/intron 10, 28 in intron/exon 11, 1 in intron 12. These data illustrate the spectrum and relative frequency of MLL fusion genes in adult pro B and CD10-negative pre B ALL. The chromosomal breakpoint data provide insight into the molecular mechanisms leading to MLL aberrations in adult ALL. Chromosomal breakpoint sequences are powerful molecular markers for assessing MRD in individual patients as has been shown in the context of the GMALL MRD evaluation trials.
Three-Way Translocations and Insertions Are the Molecular Basis for Complex MLL Rearrangements.
Authors: Meyer Claus , Kowarz Eric , Hofmann Julia , Dingermann Theo , Klingebiel Thomas , Marschalek Rolf
Published: October 2019

The human MLL gene on 11q23 is frequently involved in chromosomal rearrangements including almost all human chromosomes. These rearrangements are associated with high-risk acute leukemias. Today more than 60 MLL fusion partner genes which represent the MLL recombinome have been characterized at the molecular level. Interestingly, more than 20% of all MLL translocations are highly complex. Here we distinguish between two types of complex rearrangements: Type I: A complex rearrangement is a genetic prerequisite due to the transcriptional orientation of the involved genes, for example t(10;11) with MLL and AF10, respectively; Type II: A complex rearrangement is not prerequisite, although identified in leukemia patients. More than 500 MLL rearrangements have been analyzed at the Diagnostic Center of Acute Leukemia so far and here, we present data on 60 complex translocations that were resolved at the molecular level. Three-way chromosomal translocations, insertions and combinations thereof are the molecular basis for most of these complex MLL rearrangements which in most cases create 3 or more fusions. However, type I is mainly based on insertions and type II is based on 3 way translocations. Within these complex rearrangements, the 5′ part of the MLL gene is in general fused in frame to one of the most frequent partner genes. But the 3′ part of the MLL gene is fused in 36 cases (60%) to a novel gene such as, for example, APBB1IP or CMAH. We defined this heterogeneous group of novel translocation partner genes as “reciprocal MLL fusion genes” as they had not been identified as fusion partners to the 5′ part of the MLL gene before. These fusions are only in frame in four cases; the remaining fusions are out of frame or the genes have an opposite transcriptional orientation. I.e. these genes show genetic haplo-insufficiency and thus may also cooperate in promoting leukemogenesis. For the remaining 40% the 3′ part of the MLL gene is fused to a chromosomal area which does not encode any gene. In many complex aberrations additional genes like for example RREB1 or OS9 are fused mostly out of frame to other genes or non-coding chromosomal areas of these complex rearrangements. These genes are also haplo-insufficient and may also contribute to the leukemogenic process. These results demonstrate that LDI-PCR is a potent tool to identify highly complex and cryptic rearrangements as both MLL fusion alleles are analyzed at the same time and reveal immediately if a balanced or more complex rearrangement is present. Whether the pathobiology of type II complex rearrangements is different in comparison to their not complex counterparts has still to be proven. Supported in part by grants Ma 1876/7-1, /8-1 and /9-1 from the DFG, grant N1KR-S12T13 from the BMBF and grant 102362 from the Deutsche Krebshilfe.
PAX5/TEL Causes Down Modulation of B-Lineage Specific Genes, and Enhances Migration to CXCL12 in preBI Cells.
Authors: Fazio Grazia , Palmi Chiara , Rolink Antonius G. , Cazzaniga Giovanni , Biondi Andrea
Published: October 2019

PAX5 is a transcription factor essential for B-cell development. Recently, it has been found as frequent target of abnormalities in childhood ALL (30% of B-cell precursor ALL cases), showing monoallelic loss, point mutations or chromosomal translocations. The role of these lesions is still poorly understood. We previously cloned the PAX5/TEL fusion gene in a patient affected by B-cell precursor ALL with t(9;12) translocation. We investigated the functional roles of PAX5/TEL protein in vitro, in murine wild type preBI cells, primary cells derived from a wild type mouse and positive for B220, cKIT and CD19 antigens.We demonstrated that the PAX5/TEL protein acts as a transcriptional repressor, down regulating not only CD19, but also other B-lineage specific genes, such as BLNK and MB-1. In addition, PAX5/TEL down regulates FLT3, B220 and μ heavy chain expression, and it does not activate MCSFR. Moreover in PAX5−/− preBI cells, PAX5/TEL did not restore CD19 expression. Comprehensively, these findings suggest that the fusion protein functions as a dominant repressor of transcription on many PAX5-target genes. It is known that CXCL12/SDF1 is a growth factor promoting B-cell progenitor proliferation, acting as a chemo attractant to the bone marrow. In several hematopoietic malignancies, tumor cells express CXCR4, and that the CXCL12-CXCR4 axis may influence the biology of tumor, favoring the metastasis process and the tumor proliferation. Indeed, we demonstrated that PAX5/TEL enhances cell migration towards CXCL12, with over-expression of CXCR4. These phenomena could indicate that leukemic cells carrying PAX5/TEL are able to access to niches that are normally restricted to progenitor cells, and thereby reside in a microenvironment that favours their growth and survival, although this finding must be proved in vivo. Together with previous evidences on the PAX5/TEL capacity to overcome IL7 withdrawal and to interfere with TGFbeta1 pathway, we conclude that PAX5/TEL induces resistance to apoptosis and interferes with the processes of B-cell differentiation and migration. Taken together, these phenomena likely represent key events in the process of B-cell transformation.
Cytogenetic and Molecular Genetic Aberrations in Bone Marrow Mesenchymal Stroma Cells from Patients with Myelodysplastic Syndrome and Acute Myeloid Leukemia.
Authors: Blau Olga , Hofmann Wolf-Karsten , Blau Igor W. , Baldus Claudia D. , Thiel Gundula , Nowak Verena , Schuemann Elke , Sindram Annette , Kuehne Korinna , Thiel Eckhard
Published: October 2019

Introduction : The biology of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) is heterogeneous. Ineffective hematopoiesis results from complex interactions between hematopoietic cells (HC) and the hematopoietic microenvironment. Bone marrow mesenchymal stroma cells (BMSC) are key components of the hematopoietic microenvironment. The question of whether BMSC from patients with hematological disorders have cytogenetic abnormalities is discussed controversially.

Methods: We performed standard cytogenetic analyses (G-banding), FISH, M-FISH, and FLT3 mutation examinations of both HC and BMSC from 56 patients (30 MDS and 26 AML) and 9 healthy individuals. For BMSC selection, mononuclear cells were isolated from fresh bone marrow aspirates at the time of initial diagnosis and were further expanded in cell culture.

Results: Clonal cytogenetic aberrations were observed in HC from 14 (46%) MDS and 14 (53%) AML patients. Cytogenetic analyses of BMSC were successfully performed in 50 of the 56 cases. Structural chromosomal aberrations, including t(1;7); t(1;3); t(1;10); t(4;7); t(7;9); t(7;19); i(1q); inv(X); del(1q); del(2q); del(3p); del(4p); del(11q); del(13q); del(17p), and others were detectable in BMSC from 42% of patients. The breakpoints of chromosomes in BMSC were typical for leukemia aberrations. Six patients showed clonal chromosomal markers: t(1;7), t(1;10), t(7;9), inv(X), del(17p), and monosomy 4. Interestingly, cytogenetic markers in BMSC differed to the aberrations in HC from the same individual. No deletions or monosomy of chromosomes 5 or 7 in BMSC (FISH, 500 cells) were found in BMSC, even in those patients, who showed these aberrations in HC. M-FISH confirmed chromosomal aberrations in BMSC. We have found cytogenetic abnormalities in BMSC from patients presenting with cytogenetic alterations in their HC as well as from those with normal karyotype. We did not find structural chromosomal alterations in BMSC cultures of healthy bone marrow donors. Fourteen percent of AML patients showed FLT3 mutations in HC, but no FLT3 mutations were found in BMSC.

Conclusion: We showed that BMSC from AML and especially from MDS patients are characterized by genetic instability. The breakpoints of chromosomes in BMSC were typical for leukemia aberrations. The fact that BMSC showed typical chromosomal changes may suggest enhanced genetic susceptibility and potential involvement in the pathophysiology of MDS. Characterization of BMSC may help us to better understand the biology of this disease.
CTCF and Nucleophosmin Participate in Long Distance Cyclin D1 Deregulation in Mantle Cell Lymphoma.
Authors: Epner Elliot M. , Huang Jing , Liu Hui , Wang Jin , Liang Shugiang , Fleming William H.
Published: October 2019

Cyclin D1 expression in B cells is deregulated by long distance chromosome translocations involving the immunoglobulin heavy chain (IgH) locus in mantle cell lymphoma (MCL). Ge. Using chromatin immunoprecipitation (ChIP) assays, we show that the cyclin D1 (CCND1) loci in MCL cell lines and MCL patient samples are packaged in chromatin containing CCCTC binding factor (CTCF) and nucleophosmin (NPM). Using allelic chromatin immunoprecipitation (ChIP) assays, we demonstrate that both the translocated and nontranslocated cyclin D1 alleles bind both CTCF and NPM, while RNA polymerase II is only bound to the translocated allele. Sequential ChIP assays demonstrated CTCF and NPM binding at the cyclin D1 loci in MCL cells. By immunoFISH, we demonstrate that the translocated and nontranslocated cyclin D1 loci colocalize at the nucleolus. CTCF and NPM are bound at the IgH 3′ regulatory elements only in MCL cell lines. NPM shRNA effects a specific growth arrest in these cells. These data demonstrate a role for CTCF and NPM in long distance cyclin D1 deregulation in mantle cell lymphoma.
A Novel Mechanism of Ig and Myc Translocations in AID-Deficient Mice.
Authors: Kovalchuk Alexander L. , Mushinski Elizabeth , Burkholder Brad , Qi Chen-Feng , Li Zhaoyang , Shin Dong-Mi , Morse Herbert , Potter Michael
Published: October 2019

Consistent with the role of activation induced cytidine deaminase (AID) as a major “catalyst” of aberrant translocations between the Ig switch regions and c-myc, AID-sufficient Bcl-xL transgenic mice rapidly develop transplantable plasmacytomas with classical T(12;15) translocations. Unexpectedly, we found that Bcl-xL transgenic BALB/cAn mice deficient for AID (designated pBxAicda−/− mice) also developed plasma cell tumors but with a lower frequency (24% vs. 62%) and with a longer mean latency (108 d vs. 36 d) than AID-sufficient controls. Six out of nine of primary tumors were shown by interphase FISH to contain a T(12;15) translocation and one other had a T(6;15). pBxAicda−/− tumors did not transplant well because they were presumably in early stages of neoplastic development or had not progressed to full malignancy including association with ascites. Nevertheless, two tumors (4885 and 4961) were successfully transplanted and established as stable cell lines. They exhibited mature plasma cell phenotype (CD45−, CD138+, PC-1+, CD19−, CD23−) and secreted IgM. Gene expression profiling showed no significant difference from control plasma cell tumors of AID-sufficient mice. Detailed molecular and cytogenetic analysis of 4885 uncovered an unusual unbalanced T(12;15) translocation with IgH Cμ and Pvt-1 in a head to tail orientation at the breakpoint, resulting in elevated c-myc expression as detected by qPCR. In contrast, 4961, a T(12;15) negative cell line, had elevated N-myc expression as a result of paracentric inversion of Chr. 12. These rearrangements had no direct association with RAG activity. We conclude that rapid development of malignant plasma cell tumors with reciprocal T(12;15) does require AID, and that in AID deficiency a novel less efficient mechanism can be utilized to bring c-myc and Ig genes into juxtaposition.
A Genome-Wide Map of Acquired Uniparental Disomy in Acute Myeloid Leukemia.
Authors: Gupta Manu , Raghavan Manoj , Gale Rosemary E. , Chelala Claude , Allen Christopher , Molloy Gael , Chaplin Tracy , Linch David C. , Cazier Jean-Baptiste , Young Bryan
Published: October 2019

The recent discovery of acquired uniparental disomy (aUPD) in acute myeloid leukemia (AML) has been linked to homozygosity for mutations in certain genes (Raghavan et al, Cancer Res. 2005, Fitzgibbon et al, Cancer Res. 2005). Although this phenomenon, which is undetectable by conventional cytogenetics, has been confirmed in subsequent small-scale studies, its extent and frequency remains uncertain. To determine the frequency and distribution of aUPD, DNA samples from 455 young adult AML patients entered in the UK Medical Research Council AML10 trial were analyzed using Mapping 10K 2.0 single nucleotide polymorphism (SNP) arrays (Affymetrix Inc.). Genomic DNA from blood samples of ten non-leukemic individuals was used as control to estimate the copy number values (control set I). We defined aUPD as 50 consecutive homozygous markers but allowed 2 heterozygous calls to accommodate contaminating normal tissue. Using this criterion a false positive rate of 3.3% was calculated from an available data of 90 independent controls (control set I). Overall, 120 regions of UPD were observed in 79 AML cases (17%), 87% of which involved at least one breakpoint, i.e. resulted from mitotic recombination, and 13% were whole chromosome aUPDs arising from chromosomal non-disjunction. They were the sole aberration, as detected by SNP arrays, in 61 samples (13%), and 84% of these had only a single region of aUPD. There was a non-random distribution across chromosomes; 13q (n=18 cases), 11p (n=8) and 11q (n=9) were the most frequently affected. Other chromosomes with regions of recurrent aUPD were 2p (n=7), 2q (n=6), 1p (n=5), 19q (n=4), 17q12–q21.2 (n=4), 21q (n=4), 9p (n=3), Xq (n=3), 6p (n=2), and 17p (n=2). Acquired UPDs were observed across all cytogenetic risk groups: in 25% of adverse risk patients, 11% of favorable risk, 19% of normal karyotype and 10% of the remaining intermediate risk patients. Samples with aUPD13q (5% of samples) belonged exclusively to the intermediate risk group. Chromosome 13 was the only chromosome to show recurrent whole chromosome aUPD. Fifteen samples with aUPD13q covered the region of the FLT3 gene at 13q12.2; all 15 had a FLT3-internal tandem duplication (ITD) and all cases with a high FLT3-ITD mutant level > 50% of total had 13q aUPD. Gains and losses were observed in 12% and 14% of the samples respectively. As expected, gains on chromosome 8 and losses on chromosomes 5 and 7 were common, confirming the general utility of this approach. No homozygous losses were observed. Comparison of arrays with cytogenetic analysis showed that additional information (aUPDs and/or copy number changes) was obtained in 23% of cases with a normal karyotype and 38% of cases without available cytogenetics. This study highlights the importance of aUPD in the development of AML and pinpoints regions that may contain novel mutational targets.
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

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 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 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 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 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 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 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 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 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.
Kininogen Deficiency Protects From Ischemic Neurodegeneration in Mice by Reducing Thrombosis, Blood-Brain-Barrier Damage and Inflammation
Authors: Langhauser Friederike , Gob Eva , Kraft Peter , Geis Christian , Schmitt Joachim , Brede Marc , Göbel Kirsten , Helluy Xavier , Pham Mirko , Bendszus Martin , Jakob Peter , Stoll Guido , Meuth Sven G , Nieswandt Bernhard , McCrae Keith R. , Kleinschnitz Christoph
Published: October 2019

Abstract 104

Background and objective:

High molecular weight kininogen (HK) is an abundant plasma protein that serves as an important component of the intrinsic pathway of coagulation. Recent studies have suggested an important role for the intrinsic coagulation pathway in pathologic thrombosis. We have previously described the production of kininogen deficient mice (Kng1−/−), and the prolongation of Rose-Bengal induced carotid artery occlusion in these animals with no effect on the bleeding time. Kininogen also plays a central role in enhancing vascular permeability through the release of the nonapeptide bradykinin following cleavage by plasma kallikrein, and has been implicated in mediating acute inflammatory responses. Since thrombosis and inflammation are hallmarks of ischemic stroke still unamenable to therapeutic interventions, we hypothesized that the severity of acute ischemic stroke would be ameliorated in kininogen deficient mice.

Results:

We investigated the consequences of kininogen deficiency in models of ischemic stroke. Kng−/− mice of either sex subjected to transient middle cerebral artery occlusion (tMCAO) developed dramatically smaller brain infarctions and less severe neurological deficits without an increase in infarct associated hemorrhage. This protective effect was preserved at later stages of infarction as well as in elderly mice, but was lost after replacement of exogenous kininogen in the deficient mice. The improved outcomes were attributable to markedly reduced thrombus formation in ischemic vessels of mKng1−/− mice, improved cerebral blood flow, and less severe blood-brain barrier damage and edema formation. Moreover, the ingress of inflammatory cells (monocytes and neutrophils), and levels of IL-1 were significantly decreased in the absence of kininogen, consistent with a reduced inflammatory response following ischemic injury. The survival of mice subjected to tMCAO was markedly improved compared to control animals.

Conclusion:

Mice deficient in kininogen were markedly protected from acute ischemic stroke in the transient middle cerebral artery occlusion model. Protection was mediated through decreased thrombus formation, diminished vascular permeability, and a dampened inflammatory response. The mechanisms underlying these altered responses are currently under investigation. However, since kininogen appears to be instrumental in pathologic thrombus formation and inflammation, but entirely dispensable for normal hemostasis, kininogen inhibition may offer a safe and selective strategy for combating the effects of stroke and other thromboembolic diseases.

Disclosures:

No relevant conflicts of interest to declare.