bluuebunny/crossref_metadata_2025 huggingface dataset:
Assessment of Clinical Risk Factors for Symptomatic Venous Thromboembolism in Common Cancers: A VERITY Registry Study
Authors: Paneesha Shankaranarayana , McManus Aidan , Arya Roopen , Scriven Nicholas , Nokes Tim , Farren Timothy , Bacon Sue , Nieland Anthea , O’Shaughnessy Denise , Rose Peter
Published: October 2019

The association between venous thromboembolism (VTE) and cancer is well-recognised, but the thrombosis risk factor profile of patients with cancer-associated VTE is poorly characterised; it is unclear if additional risk factors contribute to the risk of thrombosis beyond the cancer itself, or if the risk factor profile is tumour-specific. Our aim was to compare the thrombosis risk factor profiles of cancer patients with or without symptomatic VTE enrolled in VERITY, an ongoing UK prospective VTE registry. The VERITY registry records data on patients with VTE and those in whom the diagnosis of VTE is excluded. Between Jun 05 and Mar 08, 49044 patient entries were made. Individual case data for patients with cancer were extracted. Using available risk factor data, univariate analysis of 9 established risk factors for VTE (medical in-patient history/immobilisation >3 days within last 4 weeks; major surgery in the last 4 weeks; hormonal risk factor; previous history of VTE; family history of VTE; known thrombophilia; intravenous drug abuse; current smoking; and leg paralysis), comparing VTE and non-VTE cancer cases, was performed for the 4 most common cancers using SPSS. To account for the potential impact of age and sex on VTE risk, age-adjusted values were calculated for breast and prostate cancer, and age- and sex-adjusted values for colorectal and lung. A nominal level of 5% statistical significance was assumed. Of 2825 cancer cases, 1382 had an objectively confirmed diagnosis of VTE and in 1443 the diagnosis of VTE was excluded. Breast (n=498), prostate (n=374), colorectal (n=343) and lung cancer (n=275) accounted for 53% of cancer cases. Univariate associations between risk factors and symptomatic VTE were found only for prostate cancer: history of VTE (odds ratio [OR] = 3.48; 95% CI, 2.01, 6.02; p < 0.00001), family history of VTE (OR = 2.56; 95% CI, 1.02, 6.44; p = 0.046), hormonal risk factor (OR = 2.22; 95% CI, 1.00, 4.92; p = 0.049). In colorectal cancer, smoking was less likely in VTE cases (OR = 0.54; 95% CI, 0.34, 0.87; p = 0.012). Adjusting for age (and sex), univariate associations between risk factors and symptomatic VTE were again found only for prostate cancer: history of VTE (OR = 3.23; 95% CI, 1.56, 6.68; p = 0.002), with smoking less likely in age-adjusted VTE cases (OR = 0.50; 95% CI, 0.28, 0.91; p = 0.022). Our analysis of a registry population found few associations between known thrombosis risk factors and symptomatic VTE in patients with common cancers, suggesting these factors impact little on thromboembolic risk in these cancers.
Changes of Platelet and Endothelial Microparticles in Patients with Venous Thromboembolism
Authors: Oh Doyeun , Jang Mun Ju , Lee Sun Joo , Bang Soo-Mee , Park Seonyang , Yun-Choi HS
Published: October 2019

Background: Microparticles (MP) are small membrane vesicles released from a variety of cells including platelet, leukocyte, endothelial cell which mediate intravascular coagulation. In this study, the plasma levels of total microparticle (TMP), platelet microparticle (PMP) and endothelial microparticle (EMP) in patients with venous thromboembolism (VTE) were investigated to test their clinical utilities.

Methods: Subjects were divided to 3 groups consisted of 119 patients with VTE, 72 cancer without VTE and 93 healthy controls. MP were assayed by flow-cytometry using non-washed, frozen platelet-poor plasma samples. TMP, PMP and EMP were identified using antibodies against Annexin V, CD41a, and CD144 respectively.

Results: The mean levels of plasma TMP, PMP, but not EMP in VTE without caner group (n=77) were significantly increased compared with those in healthy controls (n=93); 1972.9/μl vs 917.0/μl, p=0.001; 1496.6/μl, 586.5/μl, p=0.001; 108.0/μl vs 97.8/μl, p=0.321). In the VTE without cancer group, the mean levels of plasma TMP, PMP, but not EMP in newly diagnosed VTE group (n=59) were significantly increased compared with previously diagnosed VTE group (n=18); 2242.5/μl vs 1089.1/μl, p=0.008; 1728.6/μl vs 735.9/μl, p=0.005; 101.8/μl vs 84.6/μl, p=0.885). Mean peak thrombin generations in VTE samples (n=9) were increased in platelet poor plasma (385.2 nM vs 163.1 nM, p=0.002) but not in microparticle free plasma (MFP) (86.2 nM vs 22.6 nM, p=NS) as compared with those of controls (n=10) suggesting the thombin generation is originated from MP of plasma. But, the mean levels of plasma TMP, PMP, but not EMP in VTE with caner group (n=42) were not increased compared with those in cancer controls without VTE (n=72); 2158.1/μl vs 1644.2/μl, p=0.094; 1637.2/μl vs 1246.8/μl, p=0.168; 86.0/μl vs86.0/μl, p=0.996). The plasma levels of B-TG were well correlated with those of PMP. The levels of plasma TMP, PMP, and EMP did not show any correlation with the level of D-dimer.

Conclusion: The plasma levels of TMP and PMP are significantly increased in VTE patients and the levels are normalized after anticoagulation, suggesting MP may be used as surrogate markers for monitoring of VTE.
IL-3 Coordination of the Myeloblast Transcriptome by Modulating mRNA Stability
Authors: Ernst Jason , Bar-Joseph Ziv , Ghanem Louis , Steinman Richard A.
Published: October 2019

The growth factor interleukin-3 (IL-3) promotes the survival and growth of multipotent hematopoietic progenitors and stimulates myelopoiesis. It has also been reported to oppose terminal granulopoiesis and to support leukemic cell growth through autocrine or paracrine mechanisms. While most investigations of the action of hematopoietic cytokines such as IL-3 have focused on cytokine signal transduction leading to transcriptional activation, IL3 has been reported to downregulate proapoptotic Bim in factor-dependent lymphocytes (Matsui et. al., Mol. Cell, 2007). We hypothesized that IL-3 could support the myeloblast phenotype and oppose differentiation by stabilizing transcripts involved in proliferation or differentiation blockade. Kinetic microarray analysis of actinomycin chase experiments in IL-3-dependent 32Dcl3 myeloblasts demonstrated that IL-3 caused stabilization of hundreds of transcripts in pathways relevant to myeloblast function. Control of transcript decay by IL-3 did not require new transcription. Among the most IL-3 stabilized transcripts, 8 out of the top 22 gene ontology categories at a corrected significance level <0.001 were related to cell cycling, and 5 were related to RNA processing. Northern Blotting validated stabilization of transcripts by IL-3, including transcripts associated with proliferation and leukemic transformation (pik3cd, myb, pim-1), hematopoietic development (cited2), differentiation control (cdkn1a) and RNA processing (BRF1, BRF2). A domain was localized in the 3′-utr of IL-6 that mediated IL-3 responsiveness. Proteins known to modulate mRNA stability bound to this domain and to other IL-3 controlled transcripts. The v-Abl oncoprotein (that can support cytokine-independent proliferation of 32Dcl3 myeloblasts) was also shown to stabilize several of the IL-3 targeted transcripts. These findings support a new model of IL-3 action through coordinated mRNA stability control and suggest that leukemic oncoproteins can hijack the ability of IL3 to regulate the decay rate of critical transcripts in myeloblasts.
Evidence for the Interaction Between VWF Carboxyl-Terminal Domains and ADAMTS13 Domains Distal to Its Spacer Domain
Authors: Zanardelli Sara , Chion Chan Kwo , Laffan Michael , Lenting Peter J , Groot Evelyn , Lane David
Published: October 2019

ADAMTS13 plasma metalloprotease regulates the multimeric size of von Willebrand factor (VWF) by cleaving the Y1605-M1606 bond in the A2 domain. Several studies have characterised high affinity binding sites in the A2 domain, probably interacting with the ADAMTS13 spacer and proximal domains. We have previously provided preliminary evidence for an additional ADAMTS13 binding site distal to the A domains, by measuring a ~7-fold increase in affinity for ADAMTS13 of plate-immobilised VWFA1CK (residues 1260-2813) (KD 11 nM) compared with VWFA1A2A3 (residues 1260-1874) (KD 78 nM) (Zanardelli et al, JTH, volume 5 Supplement I, XXI ISTH congress 2007, abstract number O-W-018). We subsequently expressed a series of VWF carboxyl-terminal fragments, VWFD4CK (residues 1874-2813), VWFA3CK (residues 1671-2813), VWFD4 (residues 1874-2299) and VWFB1CK (residues 2296-2813). Using an equilibrium binding assay, we assessed their binding to full-length ADAMTS13 and truncated ADAMTS13 proteins MDTCS (residues 34-685) and del(TSP5-CUB) (residues 34-894). KD values calculated for VWFD4CK and VWFA3CK binding to full-length ADAMTS13 were 134 and 108 nM, respectively. This confirmed the presence of an ADAMTS13 binding site outside VWF A2 domain, in the region D4CK. It also excluded a major role in the interaction for the VWF A3 domain. However, isolated VWFD4 and VWFB1CK did not bind significantly, suggesting that integrity of the VWF C-terminal region is also required for the binding. Furthermore, MDTCS bound to VWFA1CK and VWFA1A2A3 with similar affinity (KD 120 and 82 nM, respectively) and no significant interaction occurred with VWFD4CK. Similar results were found for the binding of del(TSP5-CUB) to the VWF fragments (VWFA1CK KD 85 nM, VWFA1A2A3 KD 65 nM and VWFD4CK no binding). Together these results suggested a multiple interaction between the carboxyl-terminal region of VWF (within D4CK) and the distal domains of ADAMTS13 (TSP5–8 and CUB domains). The VWF carboxyl-terminal binding site may be important for initial anchoring of ADAMTS13, allowing the subsequent binding between the spacer domain and VWF A2 domain.
Evaluation of Moyamoya Disease in Sickle Cell Anemia Patients After Encephaloduroarteriosynangiosis
Authors: Earl Janice , Sun Peter , Martin Kenneth , Quirolo Keith C. , Vichinsky Elliott , Hoppe Carolyn
Published: October 2019

Abstract 1059

Background:

Moyamoya disease is a progressive cerebrovascular complication of sickle cell anemia characterized by collateral arterial formation in response to progressive stenosis of the large intracranial arteries. Despite chronic transfusions, moyamoya is associated with a high risk of stroke recurrence. Encephaloduroarteriosynangiosis (EDAS) is a surgical technique used to reestablish or augment cerebral perfusion in patients with moyamoya disease. However, evidence for its efficacy in SCA patients is limited. We initiated a prospective study to determine whether EDAS is safe and increases blood flow to ischemic areas of brain in SCA patients with moyamoya.

Methods/Results:

SCA patients who had complete or partial obstruction of the dICA with progression to moyamoya disease were eligible for EDAS. Progressive cognitive decline, dystonia and persistent seizures were also factors influencing eligibility. Seven SCA patients (age: median 16yrs, range 4–23yrs; sex: 5F/2M ) with moyamoya disease underwent encephaloduroarteriosynangiosis (EDAS) procedures between 3/2007 and 9/2010. All 7 patients were regularly transfused for a history of stroke or MRA-documented ICA stenosis. Complete or partial obstruction of the dICA with moyamoya changes was detected on MRA scans and confirmed by cerebral angiography. Bilateral (n = 2) or unilateral (n = 5) encephaloduroarteriosynangiosis procedures in which the superficial temporal artery is transplanted through a small craniotomy with dural and arachnoid opening and sutured to the pia were performed. Perfusion MR-weighted imaging was performed pre- and post-operatively to evaluate changes in relative cerebral blood volume and time to peak enhancement.

Results:

All patients have remained free of neurovascular complications and have had no new ischemic changes on MRI with an average follow-up of 26 months. All patients subjectively reported improvement in overall well-being. A decrease in the frequency of headaches (n=1), resolution of seizures (n=2) and improved gait (n=1) were also observed. Collateral anastomoses between external and internal carotid arteries were established by MRA in all patients. All 4 patients who had pre and post-procedure MRA perfusion imaging showed a reduction in the delay of time to peak enhancement in the affected territory after EDAS.

Conclusion:

Our results indicate that EDAS is a safe and effective surgical procedure to re-establish blood flow to ischemic brain tissue in patients with SCA who develop moyamoya disease. MRA perfusion imaging may be a useful tool to evaluate improved perfusion status after EDAS. Further examination of neuropsychologic correlates, such as executive function, with EDAS procedures in these patients is presently underway.

Disclosures:

Vichinsky: Novartis: Research Funding; Apotex: Research Funding.
AK2 Regulates Hematopoietic Cell Survival, Proliferation and Differentiation Along the T-Cell and Neutrophil Lineages
Authors: Six Emmanuelle M. , Lagresle-Peyrou Chantal , Chappedelaine Corinne Demerens-de , Trimouillas Justine , Rieux-Laucat Frédéric , Cassinat Bruno , Chomienne Christine , Fischer Alain , Cavazzana-Calvo Marina
Published: October 2019

Abstract 1315

AK2 deficiency is responsible for Reticular Dysgenesis (RD), a rare form of human Severe Combined Immunodeficiency which is characterized by the absence of blood neutrophils and T lymphocytes. AK2 which is located in the mitochondria interspace, plays an important role in energy metabolism and in efficient energy transfer through conversion of ADP into ATP and AMP and regulation of cellular adenine nucleotides homeostasis.

We modelled the RD pathology using a lentiviral-mediated RNA interference strategy to knock-down AK2 expression. Human CD34+ progenitors invalidated for AK2 are deeply affected in their proliferative ability during the process of T-cell differentiation. Between 3 to 7 days after initiation of T-cell differentiation on the OP9-Delta1 stroma, we observed an increased cell apoptosis through disruption of the mitochondrial membrane potential, associated with a 2-fold decreased in the percentage of proliferative cells. In the presence of shAK2, the apparition of CD7+ T-cell precursors was profoundly reduced - as compared to the shControl - showing that the process of differentiation itself was affected.

We also demonstrated that AK2 knock-down inhibits neutrophil differentiation using both G-CSF-mediated differentiation of CD34+ progenitors and ATRA-mediated differentiation of the HL60 cell line. In the HL60 system, we showed that the survival defect induced by the shAK2 could be rescued by expression of the anti-apoptotic protein Bcl-2, while this latter was not sufficient to restore a normal differentiation process.

These data suggest a novel mechanism in which AK2 regulates not only the process of differentiation but also survival and proliferation of hematopoietic cell lineages.

Disclosures:

No relevant conflicts of interest to declare.
Mitochondrial Dysfunction As a Potential Source of Reactive Oxygen Species in Cellular Models of Shwachman-Diamond Syndrome
Authors: Henson Adrianna , Moore Joseph B , Liu Johnson M. , Ellis Steven
Published: October 2019

Abstract 1343

Shwachman-Diamond Syndrome (SDS) is an inherited bone marrow failure syndrome linked to defects in ribosome synthesis. The heterogeneous array of clinical findings associated with this disease state most commonly includes exocrine pancreas insufficiency, neutropenia, and metaphyseal chondroplasia. Patients also show a predisposition for progression to myelodysplastic syndromes and acute myelogenous leukemia. Mutations in the gene SBDS are known to be responsible for most cases of SDS. Initial studies of the yeast ortholog of SBDS, Sdo1, revealed that this family of proteins is involved in the maturation of 60S subunits. Other studies have suggested that SBDS is a multifunctional protein affecting processes other than ribosome synthesis. Most recently it has been shown that reactive oxygen species are dysregulated in TF-1 erythroleukemic cells depleted of SBDS leading to increased cell death (Pediatr Blood Cancer. 2010 Dec 1;55(6): 1138–44). In an effort to elucidate potential sources of increased reactive oxygen species we investigated mitochondrial function in yeast and human models of SDS. Yeast cells lacking Sdo1 fail to grow on media containing only respiratory carbon sources, indicative of a defect in mitochondrial energy metabolism. Related studies in human TF-1 cells revealed that cells depleted of SBDS exhibit reduced oxygen consumption relative to controls. Given that the largest producer of reactive oxygen species is the mitochondrial electron transport chain, perturbation of respiratory function in cells depleted of SBDS family members could be a potential source of elevated reactive oxygen species. To investigate the potential molecular mechanisms underlying these respiratory deficient phenotypes we carried out a proteomic analysis comparing yeast cells depleted of Sdo1 with controls. Our data reveal that cells lacking Sdo1 overexpress Por1, an ortholog of human VDAC1. VDAC1 is a voltage dependent anion channel of the mitochondrial outer membrane that is thought to be an essential component of the mitochondrial permeability pore. Both over and under expression of VDAC1 have been shown to disrupt mitochondrial function and lead to enhanced apoptosis. Current efforts are focused on possible changes in VDAC1 expression and the role they play in the respiratory deficient phenotype in human SDS models. These studies continue to shed further insight into the molecular mechanisms underlying SDS pathophysiology.

Disclosures:

No relevant conflicts of interest to declare.
Modulation of Anthracycline-Induced Cytotoxicity by Targeting the Prenylated Prenylome in Myeloid Leukemia Cells
Authors: Morgan Michael A , Onono Fredrick O , Spielmann Peter H , Subramanian Thangaiah , Scherr Michaela , Venturinin Letizia , Dallmann Iris , Ganser Arnold , Reuter Christoph W.M.
Published: October 2019

Abstract 1418

Background:

Deregulation of Ras/ERK signaling in myeloid leukemias makes this pathway an interesting target for drug development.

Design and Methods:

Myeloid leukemia cell lines (HL60, KG1a, THP-1, K562) were screened for idarubicin-induced apoptosis, cell cycle progression, cell-cycle dependent MAP kinase kinase (MEK-1/2) activation, and Top2 expression. Cell-cycle dependent activation of MEK/ERK signaling was blocked using farnesyltransferase inhibitor (FTI) BMS-214,662 and dual prenyltransferase inhibitor (DPI) L-778,123 to disrupt Ras signaling. Analysis of synergy was done by the method of Chou and Talalay using the CompuSyn program. Analysis of the prenylated proteome was performed using a tagging-via-substrate approach. Knock-down of K-Ras and Cdc42 protein levels was done by RNA interference (RNAi) using lentiviral shRNA constructs.

Results:

Idarubicin caused a G2/M cell-cycle arrest characterized by elevated diphosphorylated MEK-1/2 and Top2a expression levels. Although cells were most sensitive to idarubicin, DPI L-778,123 and FTI BMS-214,662 also potently inhibited leukemia cell growth with IC50 values in the micromolar and sub-micromolar range. The FTI/DPIs elicited distinct effects on Ras signaling, protein prenylation, cell cycling, Top2 expression and apoptosis. Combining these FTI/DPIs with idarubicin synergistically inhibited proliferation of leukemia cell lines (KG1a, THP-1 and K562), but the L-778,123+idarubicin combination exhibited synergistic growth inhibition over a greater range of drug concentrations. Both BMS-214,662 and L-778,123 effectively inhibited prenylation of the majority of larger molecular weight proteins (e.g. Lamins, NAP1L1, HDJ-2), and some smaller molecular weight proteins (e.g. H-Ras, N-Ras). Additionally, only DPI L-778,123 blocked prenylation of smaller molecular weight proteins such as K-Ras and Cdc42. Interestingly, combined FTI/DPI treatment synergistically inhibited cell proliferation, induced apoptosis and nearly completely blocked protein prenylation in all cell lines tested. However, RhoB and RhoC remained prenylated even upon combined FTI/DPI treatment. Inhibition of K-Ras expression by RNA interference or blockade of its post-translational prenylation led to increased BMS-214,662-induced apoptosis.

Conclusion:

Our results suggest that nearly complete inhibition of protein prenylation using an FTI+DPI combination is the most effective method to induce apoptosis and to block anthracycline induced activation of ERK signaling.

Disclosures:

Reuter: Sanofi: Honoraria; Amgen: Honoraria.
Homologous Recombination of Wild TYPE JAK2, A NOVEL EARLY STEP In the DEVELOPMENT of Myeloproliferative Neoplasm
Authors: Vilaine Mathias , Olcaydu Damla , Harutyunyan Ashot , Bergeman Jonathan , Mourad Tiab , Ramée Jean-François , Chen Jian-Min , Kralovics Robert , Hermouet Sylvie
Published: October 2019

Abstract 2805

Background:

Adequate expression and function of Jak2 in hematopoietic progenitors is critical for normal myelopoiesis. The JAK2 46/1 (GGCC) haplotype, a congenital particularity, predisposes to myeloproliferative neoplasm (MPN) both independently and through mutation of the JAK2 gene. The JAK2 V617F mutation and acquired homozygous status for JAK2 V617F are frequent in MPN. JAK2 V617F homozygosity is currently explained acquisition of the JAK2 V617F mutation followed by mitotic homologous recombination (HR) of JAK2 occurred between wild-type and mutant alleles, leading to uniparental disomy (UPD) of chromosome 9p (9pUPD). Here we report the cases of 2 PV patients (Na1061 and Na1253) with acquired homozygous status for the JAK2 46/1 haplotype yet their granulocytes carried <20% JAK2 V617F.

Aim:

To determine whether HR of JAK2 can precede the V617F mutation in MPN.

Methods:

Granulocyte DNA and CD3+ lymphocyte DNA were examined in parallel with qPCR assays specific for the wild type and 46/1 haplotypes using rs12343867, a JAK2 intron 14 marker, as well as 4 other single nucleotide polymorphisms (SNP) on chromosome 9p. 9pUPD clonality and length were determined using SNP array studies.

Results:

For both patients, lymphocytes were heterozygous for the 46/1 haplotype, confirming that granulocyte 46/1 homozygosity was acquired. Direct sequencing of the JAK2 and GNE genes and SNP array analyses revealed homologous recombination of part of the JAK2 gene (exons 6–19, patient Na1061) and of the complete 46/1 JAK2 haplotype (patient Na1253). Furthermore, for both patients, full length sequencing of JAK2 cDNA revealed no additional mutation. In both cases, HR of wild-type JAK2 was associated with growth advantage and high expression of recombined JAK2. For both patients, further SNP array analyses revealed partial 9pUPD concerning <30% cells, which correlated with %JAK2 V617F and was consistent with 9pUPD having occurred after JAK2 V617F (Figure 1).

The distortion of SNP allelic differences was higher at the telomeric end than in the centromeric region of chr. 9p. This indicated 2 distinct partial 9pUPDs for Na1061 and 1 partial 9pUPD for Na1253.

Conclusion:

Homologous recombination involving wild type JAK2 can precede JAK2 mutation and 9pUPD in MPN. Thus multiple paths and diverse alterations of the JAK2 gene can lead to MPN in individuals carrying the JAK2 GGCC haplotype. We propose a new model with JAK2 HR as early event, followed or not by JAK2 mutation, or/and JAK2 mutation(s) facilitating subsequent recombination resulting in 9pUPD and JAK2 V617F homozygosity.

Disclosures:

No relevant conflicts of interest to declare.
JAK2 V617F, PRMT5, and GATA-1 Form a Regulatory Loop That Controls Autophagy Via Effects on ATG3 Gene Expression
Authors: Harr Michael W , Zhao Xinyang , Klein Mary , Liu Fan , Hatlen Megan , Nimer Stephen
Published: October 2019

Abstract 2822

The JAK2 V617F mutation is the most prevalent genetic abnormality in Philadelphia chromosome negative myeloproliferative neoplasia. We recently discovered that JAK2 V617F phosphorylates and inactivates PRMT5, a type II arginine methyltransferase that promotes transcriptional repression. To evaluate the PRMT5 dependent and independent effects of JAK2 V617F, we performed microarray analysis of human erythroleukemia cells (homozygous for JAK2 V617F) treated with a JAK2 inhibitor or transduced with PRMT5 shRNA. We discovered 5 autophagy-related genes (ATG3, ATG7, ATG2B, ATG5, and ATG16L1) that were positively regulated by JAK2 V617F, but negatively regulated by PRMT5. PRMT5 was bound to the promoters of these genes and binding was enhanced when PRMT5 was re-activated by a JAK2 inhibitor. Sequence analysis of ATG promoters identified at least one potential GATA-1 binding site in each ATG gene. Yet, knock-down of GATA-1 decreased the expression of ATG3 (by approximately 50%), but not the other ATG genes. ChIP analysis identified binding of both GATA-1 and PRMT5 to the ATG3 promoter, which suggests that ATG3 is transcriptionally induced by GATA-1 when PRMT5 is inactivated by JAK2 V617F. But when PRMT5 was active, it was bound to the GATA-1 promoter and ATG3 expression was downregulated. As JAK2 V617F (but not wild type) induces ATG3 expression and autophagy in AML cells and ATG3 levels are elevated in CD34+ cells from polycythemia vera patients, we conclude that JAK2 V617F promotes autophagy by inactivating PRMT5 and promoting GATA-1-dependent transcription of ATG3. Targeting autophagy with JAK2 inhibitors or other agents may be efficacious in the treatment of myeloproliferative neoplasia.

Disclosures:

No relevant conflicts of interest to declare.
Targeting Bruton's Tyrosine Kinase As a Novel Approach to Inhibit Osteoclast Function in Multiple Myeloma
Authors: Eda Homare , Santo Loredana , Cirstea Diana D. , Pozzi Samantha , Canavese Miriam , Scullen Tyler A. , Vallet Sonia , Evans Erica , Singh Juswinder , Raje Noopur S.
Published: October 2019

Abstract 2882

Bone disease is a hallmark of multiple myeloma (MM) and targeting osteoclasts (OC) to alleviate bone destruction is a component of the standard of care for MM. The activation of Bruton's tyrosine kinase (Btk), a member of the Tec family of tyrosine kinases, regulates B-cell activation and development and plays an important role in antibody production. Interestingly, Btk activation also occurs downstream of RANK signaling in OCs and its activation stimulates essential calcium signaling which plays an important role in OC function. Given this dual role of BTK in both B cell activation and osteoclastogenesis, we studied its role in the context of multiple myeloma (MM). Accordingly, we examined the efficacy of a potent and specific inhibitor of Btk (AVL-292) in OCs derived from MM patient monocytes.

AVL-292 is a highly selective, covalent Btk inhibitor that potently silences Btk enzymatic activity (IC50 < 0.5nM) and inhibits primary B cell proliferation and activation (EC50 ∼ 10nM). The compound showed high selectivity towards Btk when tested in a broad kinase panel of 62 kinases and importantly did not show significant inhibition against other kinases involved in BCR signaling (Syk, Lyn). OC derived from MM patient monocytes were assayed with or without AVL-292 for OC maturation by TRAP staining and functional activity by resorptive pit formation assay. OC function was inhibited in the presence of AVL-292 as determined by a decrease in pit formation. To delineate the mechanism of action of AVL-292 against OC function, the RANK signaling proteins were detected by western blotting and intracellular Ca2+ concentration was measured by fluorescence. AVL-292 inhibited phosphorylation of the Btk substrate, PLC γ2 in OCs. This was associated with an inhibition of intracellular Ca2+ release by AVL-292 which otherwise increased with RANKL stimulation in OCs. Although AVL-292 did not demonstrate direct cytotoxicity or inhibition of proliferation of MM cells, ongoing studies are confirming its activity in the context of co-cultures with accessory cells like OCs. These data demonstrate that the novel BTK inhibitor AVL-292 inhibits OC function through inhibition of Ca2+ mobilization through RANK signaling. These results suggest inhibition of Btk with AVL-292 has therapeutic potential for the treatment of myeloma related bone disease.

Disclosures:

Evans: Avila Therapeutics: Employment, Equity Ownership. Singh:Avila Therapeutics: Employment, Equity Ownership. Raje:Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Acetylon: Research Funding.
Multiple Myeloma Sequencing Reveals Subclonality and Timing of Genetic Alterations
Authors: Lohr Jens , Stojanov Petar , Lawrence Michael S , Auclair Daniel , Carter Scott , Sougnez Carrie , Gould Joshua , Cruz-Gordillo Peter , Chapman Michael A , Getz Gad , Golub Todd R.
Published: October 2019

Abstract 2897

Multiple myeloma is considered to be a homogenous disease within a given patient, and current classification systems and treatment algorithms are based on this assumption. We have asked if there is genetic heterogeneity of multiple myeloma within a patient and if this heterogeneity can be quantified.

To address this question, we have used massively parallel whole exome and whole genome sequencing of tumors and matched normal controls of 64 patients with multiple myeloma. We present an analytic strategy to distinguish potential driver mutations based on their clonality. We demonstrate that in some patients there are many somatic mutations that are only present in a subclonal fraction of the malignant plasma cells, and the subclonal fraction comprises up to 50%. These mutations are therefore less likely to confer a selective clonal advantage and are less attractive therapeutic targets because they only affect a small fraction of the myeloma cells. As an example, we found KRAS to be one of the most prevalent mutated genes in multiple myeloma, and KRAS mutations are significantly more likely clonal than subclonal, while other mutations in other genes are predominantly subclonal.

We also used this approach to investigate how copy number variations are related to somatic mutations, i.e. to define the temporal sequence of these events. This question is particularly relevant for hyperdiploidy in multiple myeloma, since this is associated with trisomies of odd numbered chromosomes. However, these trisomies do not occur with the same frequency in all odd numbered chromosomes and some hyperdiploid samples are also associated with trisomies of various even numbered chromosomes. It is unclear if these trisomies occur as a single catastrophic event, or rather in a sequential fashion. By assuming a constant rate of somatic mutations and utilizing this rate as a “timer” for chromosomal duplications we demonstrate that trisomies of odd-numbered chromosomes appear to occur early in a distinct order, whereas trisomies of even-numbered chromosomes and chromosome 1q occur late.

Our analyses allow us to determine which somatic mutations occurred before chromosomal duplication and may therefore give insight in the time course of pathogenic genetic alterations in multiple myeloma. Our work may also play an important role in prioritizing somatic mutations for therapeutic targeting in multiple myeloma.

Disclosures:

No relevant conflicts of interest to declare.
CD8+ CD26++ Lymphocytes Are T Memory Cells with T Cell Repopulating Capacity and Are Depleted in the Course of Haematopoietic Progenitor Cell Aphaeresis
Authors: Hildebrandt Martin , Daemen Kerstin , Stevanovic-Meyer Maja , Falk Christine
Published: October 2019

Abstract 2989

The glycoprotein dipeptidyl peptidase IV (DPP IV, CD26) possesses an enzymatic activity which is crucial for the turnover of chemokines including CCL5/RANTES, CXCL12/SDF-1a, CCL8/MCP-2 and CCL11/Eotaxin. CD26 is primarily expressed on subsets of human lymphocytes as well as on murine splenocytes, thymocytes and decidual lymphocytes. A subpopulation of memory T cells shows a high surface expression of CD26 that has been shown to be related to resolved viral infections, production of interleukin-2, and tissue invasion. Furthermore, CD26 affects the turnover of adenosine, since human CD26 anchors adenosine deaminase (ADA) to the lymphocyte surface.

We had shown previously that, in the course of haematopoietic progenitor cell (HPC) aphaeresis, a distinct subset of CD8+CD26++ lymphocytes was enriched in the aphaeresis products to a varying extent. High numbers of these cells in HPC autografts were associated with a poor clinical outcome after autologous HPC transplantation. A phenotypic characterization of CD26++ cells was performed with samples of peripheral blood from healthy donors using multicolor flow cytometric analysis.

The characterization of CD26++/CD8+ cells identified these cells as a homogeneous population with a distinct T memory cell phenotype (CD45RO+, CD161++, IL18Ralpha++, CCR7−), consistent with a population of T cells with high rhodamine efflux, chemoresistance and T cell repopulating capacity described before. Conversely, gating of CD45RO+, CD161++, IL18Ralpha++, CCR7− cells yielded a population of cells that were exclusively CD8+ and CD26++.

We conclude that CD8+ CD26++ cells define T memory cells with chemoresistance and repopulating capacity as defined by the phenotype: CD45RO+ CD161++ IL18Ralpha++ CCR7−. The observation that the enrichment of these cells in the course of progenitor cell apheresis is associated with poor clinical outcome after PBSCT could be explained by a depletion of these cells from the peripheral blood which could be hazardous. Technical improvements of the apheresis procedure are feasible. Furthermore, the characterization of T memory cells with repopulating capacity as CD8+ CD26++ allows a simple approach for their isolation fur further processing. The quantification of CD8+ CD26++ cells has now been integrated into the routine immunomonitoring to further assess this facet of post-transplantation immune reconstitution and possible correlations with the clinical course after allogeneic stem cell transplantation.

Disclosures:

No relevant conflicts of interest to declare.
Yttrium-90-Ibritumomab Tiuxetan (Zevalin) in Combination with a Fludarabine-Based Reduced Conditioning Regimen (RIC) Followed by Allogenic Stem Cell Transplantation for Advanced/Aggressive Non-Hodgkin's Lymphoma (NHL): A Prospective, Multicentre, Open Phase II Study
Authors: Bouabdallah Krimo , Furst Sabine , Bouabdallah Reda , Vigouroux Stephane , Tabrizi Reza , Melot Cyril , Gouill Steven Le , Fegueux Nathalie , Tournilhac Olivier , Mohty Mohamad , Blaise Didier , Milpied noel-Jean
Published: October 2019

Abstract 3027

Patients (pts) with advanced/aggressive NHL who relapse after autologous stem cell transplantation (auto-sct) or who fail to respond to front-line chemotherapy have a very bad prognosis with a poor survival. We performed a prospective and multicentre study (Clinical Trials.gov, NCT 00607854) to evaluate the tolerability and the benefit of Zevalin added to a fludarabine-based RIC followed by allogenic stem cell transplantation (allo-sct) in pts with a poor prognosis lymphoma.

Between January 2008 and October 2010, 30 pts under 65 years old with NHL in relapse after at least 2 previous regimen with or without auto-sct underwent an allo-sct with this preparative regimen. The underlying disease was DLBCL (10 pts), MCL (9 pts) and low grade lymphoma (FL=9 and MZL=2). Patients had to be in CR or PR after the last salvage regimen (Cheson criteria) and to have a HLA-compatible (10/10) match-related (MRD) or unrelated (MUD) donor or 9/10 mismatched UD. GVH prophylaxis was based on CsA alone or in combination with Methotrexate in case of mismatch. The primary objective of the study was TRM at day 100.

All the pts are evaluable Twenty two were male (73%) and the median age was 57 (32–64). Twenty nine pts (97%) had prior auto-sct. Twenty pts were transplanted from a sibling donor, 8 from MUD and 2 pts had a C or DQ mismatch. At time of allo-sct, 18 pts were in CR and 12 pts in PR. Median time between diagnosis and allo-sct and between auto-sct and allo-sct was 36 mo and 18 mo respectively. Four pts died from a-GVH (n=2) at d 40 and 70, multi-organ failure (n=1) at d 117 and disease progression (n=1) at d 114 respectively. The TRM up to day 100 was 7% (n = 2). The median follow-up is 16 mo (9–40). All pts had a rapid and sustained engraftment. Twenty five pts are alive in CR. One pt died in CR (suicide) one year after transplant. The actuarial 3-year event-free and overall survival are 83% (± 6%). Estimated EFS at 2 years for the 3 different subgroups of diagnosis are 100% for FL and MZL, 89% (± 10%) for MCL and 70% (± 14%) for DLBCL.

We conclude that Yttrium-90-ibritumomab tiuxetan (Zevalin) in combination with a fludarabine-based regimen is safe with a very low TRM rate. The results we report here are in pts with a very bad prognosis are very encouraging with a very high response rate and survival.

Disclosures:

Off Label Use: Zevalin not licensed in France in this indication. Zevalin has been kindly provided by BayerHealthcare company for the study.
The Results of Patients with b Thalassemia Major Undergoing Peripheral Blood Stem Cell From Alternative Donor: New Conditioning Regimen for Thalassemia
Authors: Li Chunfu
Published: October 2019

Abstract 3034

Background:

Hematopoietic stem cell transplantation (HSCT) remains the only potentially curative treatment for thalassemia patients, However, most candidates for HSCT do not have a suitable family donor. In order to evaluate whether HSCT from an HLA–well -matched alternative donor can offer a probability of cure comparable to that of HSCT from sibling donor. The new conditioning regimen and peripheral blood stem cell transplant (PBSCT) from alternative donor (NF-08-thalassemia protocol) were used for b thalassemia patients. The improved outcome was summarized here.

Objective:

To evaluate the overall survival (OS), thalassemia-free survival (TFS), rejection (RE) and transplant related mortality (TRM) of NF-08-thalassemia protocol.

Patients and Methods: Sixty-one thalassemia patients, 42 male and 19 female (median age, 7 years; age range, 3–15 years), were transplanted from 56 (32 patients were 8/8 and 24 patients 7/8 locus matched at HLA-A, B, Cw and DRB1) unrelated and 5 (1 patient 8/8 and 4 patients 7/8 antigen matched) parents donors from Oct.2008 to Jan.2011(median follow-up time, 14 months; range, 6–31months). NF-08-thalassemia protocol included Cyclosphosphamide (d-10 to d-9), Busulfan (d-7 to d-5), Thiotepa (d-4), Fludarabine (d-8 to d-4) and ATG (d-3 to d-1). PBSCs from unrelated (56 cases) and parents (5 cases) donor were used as only source of stem cells in all patients. GVHD prophylaxis included Cs A, MMF and sMTX.

Results:

OS, DFS and TRM in the cohort of 61 patients were 91.8%, 90.2% and 8.2%, respectively. Only one rejected his graft two months after transplantation. Acute GVHD III∼IV was 8.2% (5 patients) and none suffered from extensive chronic GVHD. Three patients died of acute GVHD and two died of the infection after transplantation.

Discussion:

In comparing this alternative donor HSCT with sibling donor HSCT (30 patients, using the same protocol, meanwhile), OS, TFS, RE and TRM were 91.8% vs. 85.9% (P=0.401), 90.2% vs. 79.2% (p=0.147), 1.6% vs. 6.9% (p=0.197) and 8.2%vs.14.1%, respectively. No significant difference was found.

The results of alternative donor HSCT for b thalassemia patients are comparable with sibling donor transplant with using NF-08-thalassemia protocol.

Disclosures:

Li: Qi fa Liu: Workmates.
Total Therapy 6: Initial Results of Dose-Dense Strategy for Previously Treated Multiple Myeloma
Authors: Usmani Saad , Mitchell Alan , Nair Bijay , Waheed Sarah , van Rhee Frits , Alsayed Yazan , Petty Nathan M , Steward Doug , Panozzo Susan , Hoering Antje , Crowley John , Barlogie Bart
Published: October 2019

Abstract 3081

Background:

We have reported extensively on applying dose-intense total therapy approach in newly diagnosed multiple myeloma (MM), yielding long CR durations in ∼80% GEP-defined low-risk myeloma patients. Herein, we present for the first time the initial results of a phase II trial employing a dose-dense approach in previously treated MM. Patients & Methods: Patients received 5 cycles of combination chemotherapy (Figure 1) followed by maintenance with bortezomib, lenalidomide and dexamethasone until relapse or disease progression by IMWG 2006 criteria. Overall survival and progression free survival were estimated using the Kaplan-Meier method. Survival distributions were compared using the log-rank test. Results: 63 patients with previously treated MM were enrolled. The median follow-up from enrollment was 15.6 months. Baseline characteristics included age >=65yr in 37%, ISS stage II/III was seen in 41%/19% of patients, cytogenetic abnormalities (CA) in 37%, and GEP-70 high risk-MM (HRMM) in 26% of patients with available gene expression data. 11 patients (17%) had >2 prior lines of therapy including regimens that contained bortezomib (91%), thalidomide(45%), lenalidomide(81%), melphalan(27%) and steroids(100%). 14 patients (22%) discontinued therapy primarily due to progression, death or toxicity. Cumulative incidence of PR/VGPR/CR at 18 months was 69.4%, 54.8% and 37.0% respectively. Of the 60 patients with at least 3 months of follow-up, 33% had stable disease as the best overall response. The time to achieving PR or better at 6 months was more rapid in HR-MM vs LRMM (63.6% vs. 50%, p=0.008). Hematologic toxicities occurred in all patients (100%), whereas the most common non-hematologic toxicities counting all toxicities (>grades 3) included metabolic/electrolyte disturbances (95%) followed by infections (31%), hepatobiliary (20%) and gastrointestinal (16%) toxicities. Overall survival (OS) and progression free survival (PFS) at 12 months was 90% and 87%, respectively (Figure 2). The OS (96% vs 60%, p=0.0002) and PFS (92% vs 53%, p<0.0001) at 12 months was markedly superior in GEP-70 defined low-risk MM (LRMM) compared with high-risk MM (HRMM). ISS staging and GEP-defined p53 deletion did not impact OS/PFS at 12 months. Conclusions: Dose-dense strategy is highly effective in previously treated LRMM. Even though time to achieving best response was quicker in HRMM, response duration was shorter thus augmenting the significance of sustaining best response rather than depth of response in HR-MM.

Disclosures:

No relevant conflicts of interest to declare.
Prognostic Factors of Reducing Relapse in Patients Undergoing Allogeneic Hematopoietic Cell Transplantation From Related Donor for Acute Myeloid Leukemia: The Japan Society for Hematopoietic Cell Transplantation (JSHCT) AML Working Group
Authors: Yano Shingo , Yokoyama Hiroki , Nishimura Rimei , Fukuda Takahiro , Ogawa Hiroyasu , Ohashi Kazuteru , Kanamori Heiwa , Tanaka Junji , Sakamaki Hisashi , Atsuta Yoshiko , Takami Akiyoshi
Published: October 2019

Abstract 3085

Background:

A llogeneic hematopoietic cell transplantation (allo-HCT) is a curative treatment option for patients with acute myeloid leukemia (AML), however a major obstacle to success is represented by the relapse of the disease. We retrospectively analyzed prognostic factors to determine whether there are clinical features that predict relapse after transplantation.

Patients and Methods:

Clinical data were collected from the Transplant Registry Unified Management Program (TRUMP) in Japan. An individual patient data-based analysis was performed on patients with AML who achieved or continued complete remission (CR) after related allo-HCT between 1991 and 2009. A logistic regression model was used to analyze associations between relapse and clinical factors. Patients with CR1 or CR2 were defined as having a standard-risk disease, and patients with ≤CR3 or non-CR were defined as having a high-risk disease. Conditioning regimens included ≤ 8Gy in fractionated doses of total body irradiation, busulfan dose < 9mg/kg, melphalan dose ≤ 140 mg/m2 defined as a reduced-intensity conditioning (RIC) regimen.

Results:

We analyzed a cohort of 1, 960 patients with AML. The median age at allo-HCT was 42 years (range, 16 to 74 years). Among the 1, 960 patients, 778 patients (38%) relapsed after allo-HCT. The probabilities of survival at 5-year for all patients, standard-risk patients, and high-risk patients were 49%, 61%, and 31%, respectively. In a multivariate analysis, significant factors for no relapse were standard-risk disease at the time of allo-HCT (HR 0.366, 95%CI 0.279–0.480, p<0.0001), presence of chronic GVHD (HR 0.442, 95%CI 0.338–0.579, p <0.0001), and CMV infection (HR 0.452, 95%CI 0.230–0.886, p =0.021). Intensity of conditioning regimen (RIC vs myeloablative), disease subtype of AML, HLA matching (matched donor vs mismatched donor), source of stem cells (peripheral blood vs bone marrow), and presence of acute GVHD did not affect relapse rates.

Conclusion:

These results indicate that in patients with standard-risk AML at the time of HCT, chronic GVHD or CMV infection after HCT were crucial for reducing relapse. Immune modulation mediated by chronic GVHD or CMV infection may have played a role in preventing disease relapse. The use of donor lymphocyte infusion before relapse may reduce the recurrence for selected patients who do not develop chronic GVHD.

Disclosures:

No relevant conflicts of interest to declare.