bluuebunny/crossref_metadata_2025 huggingface dataset:
Concomitant Intrathecal Liposomal Cytarabine and Prednisolone Is a Safe and Well Tolerated Treatment Option in Pediatric Leukemia and Neuroblastoma Patients.
Authors: Björgvinsdóttir Helga , Bekassy Albert N. , Toporski Jacek , Heldrup Jesper
Published: October 2019

Malignant leptomeningeal dissemination poses significant treatment challenges and is difficult to eradicate. The disadvantages of conventional chemotherapy and intrathecal anticancer agents are due to limited drug penetration through the blood-brain barrier, low cytotoxic concentrations in the cerebrospinal fluid (CSF), and rapid metabolism. DepoCyte is a new liposomal, slow-release preparation of cytarabine for intrathecal use that maintains cytotoxic concentrations for as long as 14 days or more and improves the distribution of free cytarabine in the CSF.

Here, we report our experience concerning thirteen pediatric patients age 1–13 years who received more than 40 treatment doses with liposomal cytarabine either as a therapeutic or prophylactic agent in doses of either 35 mg or 50 mg (children 11 years and younger or 12 years and older, respectively). There were four patients with refractory/relapsing neuroblastoma stage IV, two of whom had confirmed central nervous system (CNS) involvement; and nine patients with CNS- and/or refractory/recurrent leukemia, including ALL, AML and infant ALL. Todate, individual patients have been treated with one to ten liposomal cytarabine doses each (medium 3,1), and one patient has received therapy over a period of one year (ten doses).

Patients were given arachnoiditis prophylaxis with either oral dexamethasone 0,15 mg/kg × 2 daily (max 4 mg per dose) for 5 days or intrathecal prednisolone in age-dependent doses (Precortalon Aquosum; 8 mg, 12,5 mg, and 15 mg for patients 1–2 years, 3–11 years, and 12 years and older, respectively). The intrathecal prednisolone that was given concomitantly with liposomal cytarabine in approximately half of the treatment doses. Was convenient and caused less side-effects than oral dexamethasone.

DepoCyte was well tolerated in most patients, but side-effects such as headache, nausea and vomiting occurred on four occasions. One patient had seizures in connection with high-dose intravenous cytarabine therapy.

We conclude, that intrathecal DepoCyte therapy in doses of 35 to 50 mg with concomitant oral or intrathecal steroids is a practical, safe, and well tolerated treatment for leptomeningeal malignant disease. Further research is needed to determine the long-term effects and side-effects.
Differences in Fetal Hemoglobin Induction in Sickle Cell Disease and beta-Thalassemia.
Authors: Fathallah Hassana , Taher Ali , Bazarbachi Ali , Atweh George F.
Published: October 2019

A number of therapeutic agents including hydroxyurea, butyrate and decitabine have shown considerable promise in the treatment of sickle cell disease (SCD). However, the same agents have shown less clinical activity in β-thalassemia. As a first step towards understanding the molecular basis of the different clinical responses to these agents, we have studied the mechanisms of induction of fetal hemoglobin (HbF) by butyrate in BFU-E derived cells from 5 patients with SCD and 9 patients with β-thalassemia intermedia. Exposure to butyrate resulted in a dose-dependent augmentation of γ-globin mRNA levels in erythroid cells from patients with SCD. In contrast, induction of γ-globin expression in erythroid cells from patients with β-thalassemia intermedia was only seen at a high concentration of butyrate. The increase in γ-globin mRNA levels in patients with SCD and β-thalassemia intermedia was associated with opening of the DNA structure as manifested by decreased DNA methylation at the γ-globin promoters. Interestingly, butyrate exposure had markedly different effects on the expression of the β- and α-globin genes in the two categories of patients. Butyrate decreased the level of β-globin mRNA in 4 out of 5 patients with SCD (P = 0.04), while in β-thalassemia the levels of β-globin mRNA did not change in 7 patients and decreased in 2 patients after butyrate exposure (P = 0.12). Thus in patients with SCD, the effects of the induction of the γ-globin gene on the γ/(β+γ) mRNA ratios were further enhanced by the butyrate-mediated decreased expression of the β-globin gene. As a result, γ/(β+γ) mRNA ratios increased in all patients with SCD, with a mean increase of 31% (P = 0.002). In contrast, butyrate increased γ/(β+γ) mRNA ratios only in 4 out of 9 patients with β-thalassemia, with a more modest mean increase of 12% (P = 0.004). Interestingly, the decreased β-globin expression in patients with SCD was associated with closing of the DNA configuration as manifested by hypermethylation of DNA at the promoter of the β-globin gene while methylation of the same promoter did not change following butyrate exposure in patients with β-thalassemia intermedia. More surprisingly, the expression of the α-globin genes increased following butyrate exposure in 4 out of 9 patients with β-thalassemia, while the levels of α-globin mRNA decreased in 4 out of 5 patients with SCD. As a result, the favorable effects of the butyrate-induced increase in γ-globin gene expression on the α: non-α mRNA imbalance in patients with β-thalassemia intermedia were partly neutralized by the corresponding increase in α-globin gene expression. These differences may explain, at least in part, the more favorable effects of inducers of HbF in SCD than in β-thalassemia. Further studies are necessary to fully understand the molecular bases of the different responses to agents that induce HbF in patients with these disorders.
Quantitative Assessment of Minimal Residual Disease Predicts Outcome of Patients of Acute Myeloid Leukemia with Nucleophosmin (NPM) Mutation.
Authors: Chou Wen-Chien , Tang Jih-luh , Wu Shang-Ju , Yao Ming , Tien Hwei-Fang
Published: October 2019

Purpose: In patients of acute myeloid leukemia (AML) with mutations in Nucleophosmin (NPM), minimal residual disease (MRD) can be quantified by real-time polymerase chain reaction (qPCR). However, the validity of MRD detection by qPCR on a long-term basis and its prognostic significance remain undetermined.

Patients and Methods: We quantified NPM mutants by qPCR in genomic DNA of 202 bone marrow samples serially collected during a median follow-up of 21.9 months (range 2.5 to 204) from 40 AML patients bearing this mutation.

Results: With a relatively homogeneous number of mutants at diagnosis, induction chemotherapy resulted in a median of 2.78 logs of reduction (p<0.001). The mutant numbers determined by qPCR matched well with clinical courses: clinical relapse was accompanied by rise of mutants (p<0.001) and mutant numbers at morphological complete remission from patients without any subsequent relapse were significantly lower than others (p<0.001). Detection of mutant signals predicted relapse if no further chemotherapy was administered. Failure to achieve at least 2 logs of mutant reduction after consolidation led to shorter overall survival (OS) (11.3 months vs. not reached, p=0.01) and relapse-free survival (RFS) (3.8 months vs. 21.2 months, p=0.001). Patients with any rise of mutants during serial follow-up relapsed more frequently than those with persistently low or undetectable signals (p<0.001). Along serial follow-up, patients obtaining any mutant reduction to < 0.1% of internal control (INC) after treatment had longer OS (a median of 57.9 months vs. 20.2 months, p=0.002) and RFS (11.3 months vs. 4.0 months, p<0.001). On the other hand, detection of mutants above 1.5% of INC in any post-induction sample predicted a poorer outcome (a median of 22.1 months vs. not reached, p=0.002 for OS and 9.7 months vs. not reached, p<0.001 for RFS).

Conclusion: Quantification of MRD by qPCR on genomic DNA in AML bearing NPM mutation predicts outcomes of this group of patients.
A New Oral Aniticoagulant, Dabigatran Etexilate, Is Effective and Safe in Preventing Venous Thromboembolism after Total Knee Replacement Surgery (The RE-MODEL Trial).
Authors: Eriksson Bengt I. , Dahl Ola E. , van Dijk Cornelis N. , Frostick Simon P. , Kurth Andreas A. , Rosencher Nadia , Schnee Janet , Hettiarachchi Rohan , Christiansen Anita V. , Hantel Stefan , Büller Harry R.
Published: October 2019

BACKGROUND: Oral DVT prophylaxis not requiring monitoring is an advantage in orthopaedic patients. Dabigatran etexilate is an oral direct thrombin inhibitor undergoing evaluation for the prevention of venous thromboembolic events (VTE) following orthopaedic surgery.

METHODS: In a phase III, multicenter, non-inferiority, double-blind study, patients undergoing total knee replacement were randomized to 3 treatments. The patients received 8±2 days of oral dabigatran etexilate, 150 or 220 mg once daily starting with a half dose (i.e.75 or 110 mg) 1–4 hours after surgery, or subcutaneous enoxaparin 40 mg once daily starting 12 hours prior to surgery. The primary efficacy outcome was the composite of total VTE and all causes of mortality during the treatment period. All efficacy and safety outcome events were adjudicated by blinded independent committees.

RESULTS: Efficacy could be evaluated for 1541 (75%) treated and operated patients. Total VTE and death occurred in 40.5%, 36.4% and 37.7% of patients assigned to dabigatran etexilate 150 or 220mg once daily or enoxaparin, respectively. Proximal DVT and/or PE occurred in 3.8%, 2.6% and 3.5% of patients receiving dabigatran 150 or 220mg or enoxaparin, respectively. Three deaths occurred during the treatment period, one in each of the treatment groups. Safety was evaluated for all 2076 patients receiving study treatment. The rate of major bleeding was 1.3%, 1.5% and 1.3% of patients receiving dabigatran 150 or 220mg or enoxaparin. Elevated LFTs (ALT >3xULN) occurred in 3.7%, 2.8% and 4.0% of the patients treated with 150 and 220 mg dabigatran or enoxaparin during the study. A late temporary rise in LFTs was observed in 6 patients (0.5%) who had received dabigatran.

CONCLUSIONS: Non-inferiority for the primary efficacy endpoint was met for both doses of dabigatran etexilate compared to enoxaparin. There was no difference in bleeding rates between the treatment groups. Oral administration of dabigatran etexilate once daily, given early in the postoperative period, was effective and safe for the prevention of total VTE in patients undergoing total knee replacement surgery.
ZAP-70 Enhances IgM Signaling in Chronic Lymphocytic Leukemia Cells Independent of Its Tyrosine Kinase Activity.
Authors: Chen Liguang , Huynh Lang , Weiss Arthur , Kipps Thomas J.
Published: October 2019

We found that expression of the zeta-associated protein of 70 kD (ZAP-70) by chronic lymphocytic leukemia (CLL) B cells enhanced IgM-receptor signaling, even though such CLL cells also expressed a highly-related and more active tyrosine kinase, p72Syk. To investigate whether the kinase activity of ZAP-70 is necessary for this effect, we transfected ZAP-70-negative primary leukemia cells with expression vectors encoding either wild-type ZAP-70 or ZAP-70-KI, a mutant ZAP-70 that has an inactivating point mutation in the ATP-binding site (Lys369-Ala) and that lacks tyrosine kinase activity. We achieved high-level expression of ZAP-70 or ZAP-70-KI in transfected CLL cell samples and compared these cells with each other and with the same CLL cell samples that had been mock transfected with a control vector and that remained negative for expression of ZAP-70 (n = 7). To examine the B-cell-receptor (BCR) signaling potential of these cells we assessed the extent of tyrosine phosphorylation of p72Syk, B-cell linker protein (BLNK), and phospholipase C gamma (PLC-γ), and measured intracellular calcium flux ([Ca2+]I) before and 5–10 minutes after surface IgM ligation with F(ab)2 anti- μantibody. Treatment of control mock-transfected ZAP-70-negative CLL cells with anti-μ resulted in negligible-to-minimal increases in phosphorylation of these cytosolic proteins, with mean increases in phosphorylation of p72Syk, BLNK, and PLC-γ of only 46% ± 47% (S.D), 173% ± 112%, and 73% ± 76%, respectively. CLL cells engineered to express non-mutated ZAP-70 experienced significantly higher levels of protein tyrosine phosphorylation following treatment with anti-μ, with mean increases in phosphorylation of p72Syk, BLNK, and PLC-γ of 186% ± 102%, 490% ± 323%, and 722% ± 836%, respectively, as had been noted in earlier studies. Surprisingly, CLL cells made to express the tyrosine-kinase-defective mutant ZAP-70KI also experienced significantly higher levels of protein tyrosine phosphorylation following treatment with anti-μ than did the mock-transfected CLL cells, with mean increases in phosphorylation of p72Syk, BLNK, and PLC-γ of 172% ± 94%, 431% ± 261%, and 759% ± 637%, respectively. These values were similar to those noted for anti-μ treated CLL cells that expressed the wild-type ZAP-70 protein. These results were reflected also in the ([Ca2+]I) induced by anti-μ in each of the three groups of CLL cells. Whereas anti-μ treatment of mock-transfected CLL cells resulted in negligible-to-minimal increases in ([Ca2+]I) of 0.25 units ± 0.19, anti-μ treatment of ZAP-70-transfected or ZAP-70KI-transfected CLL cells resulted in increases in ([Ca2+]I) of 1.05 units ± 0.53 and 0.95 units ± 0.46, respectively. These values each were significantly higher than that noted for mock-transfected CLL cells. We conclude that the tyrosine-kinase activity of ZAP-70 is not required for ZAP-70 to enhance BCR signaling in CLL cells.
An EVI1 Point Mutant Unable To Bind GATA1 Rescues EVI1−Induced Defects in Erythroid Differentiation.
Authors: Laricchia-Robbio Leopoldo , Rinaldi Ciro R. , Fazzina Raffaella , Nucifora Giuseppina
Published: October 2019

EVI1 is a nuclear oncoprotein deregulated by recurring chromosomal abnormalities in MDS. The expression of this gene in MDS patients represents a very poor prognostic marker and is associated with erythroid and megakaryocytic defects. We have previously shown that the forced expression of EVI1 in mice results in a fatal disease with features characteristic of MDS including dyserythropoiesis, dysmegakaryopoiesis, and anemia. More recently we have shown that EVI1 directly interacts with GATA1 and disrupts GATA1−binding to DNA leading to deregulation of GATA1−dependent genes.

Here we describe the effects of an EVI1 mutant unable to bind GATA1, on the regulation of GATA1 target genes and on the erythroid differentiation of murine bone marrow progenitors. The structure of two zinc finger motifs of this mutant, EVI1(1+6Mut), were destroyed by His to Ala and Cys to Ala changes. Semi−quantitative RT−PCR showed that most of the analysed genes were down−regulated in 32Dcl3 cells expressing EVI1 but not EVI1(1+6Mut). Bone marrow lineage negative cells infected with EVI1, EVI1(1+6Mut), or the empty vector as control, selected in G418, and plated in presence of Epo were utilized to determine the progenitors’ potential to differentiate in response to this cytokine. The EVI1−expressing cells were virtually unable to generate erythroid colonies after Epo stimulation and only scarce small colonies were observed. In contrast, the EVI1(1+6Mut)−cells produced about 65% of the colonies formed by the control cells. The cells were recovered and their morphology was analysed after Wright−Giemsa staining. All EVI1−positive erythroid cells showed an impaired differentiation that was arrested at the basophilic−erythroblast stage. These cells were bi− and tetra−nucleated with chromatin bridges, budding nuclei and nuclear−cytoplasmatic maturative asynchronizations. All of these features are known as dysplastic erythroid aspects in MDS patients. These dysplastic characteristics were less prominent in EVI1(1+6Mut)−positive cells and were observed only in a minority of cells rather than in the entire population. As expected, the control cells had the appearance of normal erythroblasts and none of the aberrant features were observed. These results parallel the morphology observation of peripheral blood smears obtained from EVI1−mice and EVI1(1+6Mut)−mice in that erythropoiesis defects such as aniso−poikilocytosis and orthochromatic erythroblasts were less striking and observed only in a limited number of erythroid cells in EVI1(1+6Mut)−mice. Based on these results, we propose that EVI1 blocks erythroid differentiation by direct interaction with GATA1 leading to impairment of GATA1 regulation. This differentiation block is significantly reduced in vitro and in vivo with the EVI1(1+6Mut) mutant, which re−establishes to a high degree the normal functions of GATA1 and diminishes the erythroid dysplastic aspects observed in EVI1 cells.
Tribbles Homolog 2 (Trib2) Inactivates C/EBPalpha and Causes Acute Myelogenous Leukemia.
Authors: Keeshan Karen , He Yiping , Wouters Bas J. , Shestova Olga , Xu Lanwei , Sai Hong , Rodriguez Carlos G. , Maillard Ivan , Valk Peter , Carroll Martin , Aster Jon C. , Delwel Rudd , Pear Warren S.
Published: October 2019

Trib2 is a member of the Trib family of serine/threonine kinase-like proteins (Trib1, Trib2, Trib3), whose function in hematopoiesis is not documented. To investigate the effects of Trib2 in hematopoietic progenitors, mice were reconstituted with hematopoietic stem cells retrovirally expressing Trib2. Trib2-reconstituted mice uniformly developed fatal transplantable acute myelogenous leukemia (AML), with a median survival of 179 days. Retroviral Trib2 expression in hematopoietic stem cells perturbed myeloid development, enhanced progenitor proliferation, and directly inhibited the function of C/EBPα, a critical transcription factor that is frequently dysregulated in AML. Furthermore, an analysis of microarray data generated from 285 AML patient samples identified elevated Trib2 expression in a distinct subset of patients in a cluster with a high frequency of C/EBPα mutations. In mechanistic studies, we found that Trib2 associated with and led to the proteasomal-dependent degradation of C/EBPα. To determine the structural requirements for Trib2 to inhibit C/EBPα, and to induce AML, we have initiated a structure/function analysis of Trib2. Together, our data identify Trib2 as a novel oncogene that induces AML through a mechanism involving inactivation of C/EBPα. The identification of Trib2 as potent leukemogen points to new pathogenic mechanisms and possible therapeutic opportunities in this aggressive cancer, which is not currently curable in the majority of patients.
Xanthohumol: A New Potential Proteasome Inhibitor That Activates the Pro-Apoptotic Arm of the Unfolded Protein Response in B-CLL Cells.
Authors: Lust Sofie , VanHoecke Barbara , Van Gele Mireille , Boelens Jerina , Philippé Jan , Vincken Petra , Bracke Marc , Poelman Martine , Offner Fritz
Published: October 2019

B-Chronic Lymphocytic Leukemia (B-CLL) is incurable by current methods because of increasing resistance to chemotherapy. Therefore, new options are needed for the treatment of CLL. The proteasome-inhibitor bortezomib was described to promote apoptosis in CLL cells and to induce endoplasmic reticulum (ER) stress and unfolded protein response (UPR) in refractory multiple myeloma. The UPR is mainly a self-protective mechanism activated when protein folding is disrupted and misfolded proteins are accumulating in the ER. However, sustained ER stress eventually leads to cell death. Three major ER sensors are involved during UPR, namely ATF6, PERK and IRE1. In inactivated state, they are bound to the ER-chaperone BiP/GRP78. Upon ER stress, BiP is released from the luminal domain of the ER stress transducers resulting in their activation and downstream signaling. Pro-survival signals are delivered by IRE1 (splicing of XBP1) and ATF6 (cleavage), while pro-apoptotic signals are generated by PERK (upregulation of CHOP). Previously, we demonstrated that the xanthohumol (X) is able to kill B-CLL cells in a dose- and time-dependent way as evidenced by PARP cleavage and Annexin V staining (Lust et al., 2005). In this study, we first demonstrated that X induces apoptosis of B-CLL cells in part via activation of ER stress and the UPR and identified the associated molecular markers. Treatment of freshly isolated B-CLL cells with X, stimulated the expression BiP and Hsp70 (suggestive for ER stress), the phosphorylation of eIF2a (suggestive for PERK activation), and the splicing of XBP1 mRNA (indicative for IRE activation). In contrast, ATF6 activation seemed not to be implicated since no cleaved ATF6 could be demonstrated. Induction of UPR was associated with a pro-apoptotic outcome evidenced by upregulation of CHOP, generation of ROS, downregulation of the anti-apoptotic proteins Mcl-1, Bcl-xL, Bcl-2, cleavage of PARP, and processing of caspase-3. Next, we showed that X inhibited the 20S proteasome activity in reticulocyte lysates but also functionally in B-CLL as demonstrated from the accumulation of polyubiquitin-conjugates under X treatment. The activation of UPR and influence on the anti-apoptotic proteins can mostly be explained by this proteasome inhibitory activity. In conclusion, we identified proteasome inhibitory capacities of xanthohumol in B-CLL in vitro. Proteasome-inhibition was accompanied by ER stress, UPR and apoptosis. Our results further suggest that tackling organelles like the proteasome and the ER is a valuable strategy in treatment of B-CLL.
APcK 110, a novel Inhibitor of Kit, Extends Survival of NOD-SCID Mice in An AML Xenograft Model.
Authors: Faderl Stefan , Bueso-Ramos Carlos , Liu Zhiming , Pal Ashutosh , Bornmann William , Harris David , Hazan-Halevy Inbal , Kantarjian Hagop M. , Estrov Zeev
Published: October 2019

Abstract 1045

Poster Board I-67

Kit is a type III receptor protein tyrosine kinase and membrane receptor for stem cell factor (SCF). Tight regulation of the activity of kit is a crucial component of normal hematopoiesis. Mutations of the KIT gene have been identified in blasts of patients with AML predominantly those with core-binding factor leukemias. As the presence of mutations of KIT has been associated with higher likelihood of relapse and shorter survival, targeting Kit may be of therapeutic benefit. Therapeutic activity may extend to cases with Kit overexpression. We have recently described the activity of APcK110, a novel inhibitor of Kit, in vitro where we have shown potent proapoptotic and antiproliferative effects in AML cell lines and primary AML samples irrespective of mutation status but with measurable expression of Kit (Faderl S et al. Cancer Res 2009; 69: 3910-3917). Here we extend our investigations to the activity of APcK110 in vivo in a xenograft mouse model. After exposure of 8-weeks-old female NOD-SCID mice (NOD.CB17-PRKDS SCID\J) to 30cGy sub-lethal WBXRT, 1 × 105 OCI/AML3 cells (human AML cell line) were injected I.V. Ten days later, the mice were injected I.P. every other day with the same volume of either APcK110 or PBS. The mice were sacrificed according to experimental protocol or when they became moribund, unable to obtain food or water, or if they lost > 20% of their body weight. Necropsy was performed on all animals. Marrow, blood and organs were preserved in formalin for histological analysis. Kaplan-Meier estimates were applied to calculate survival of the mice. We could show that 1) all mice injected with OCI/AML3 cells developed a clinical and histological picture consistent with AML. Wright stains of blood and marrow confirmed presence of a large, heterogeneous blast population, which was negative for myeloperoxidase, but stained positive for butyrate and anti-human CD45. Histological analysis of lung and liver tissue revealed dense infiltration with AML blasts as confirmed by anti-human CD45-positive sheets of cells in these tissues; 2) survival of APcK110-treated mice was significantly improved compared with mice which were injected with PBS (p=.02); 3) Marrow histology showed residual AML following injection of mice with APcK110. In conclusion, APcK110 is a novel kit kinase inhibitor with anti-AML activity in vitro and in vivo. Further evaluation in toxicology and clinical studies should be pursued.

Disclosures:

Pal: Patent Office: patent application pending. Bornmann:Patent office: Patent application pending. Estrov:Patent Office: Patent application pending.
The Cyclin Dependent Kinase 2, 7 and 9 Inhibitor SNS-032 Has Single Agent Activity in Acute Myeloid Leukaemia Cells and Is Highly Synergistic with Cytarabine.
Authors: Walsby Elisabeth J , Pepper Chris , Knapper Steven , Burnett Alan K
Published: October 2019

Abstract 1059

Poster Board I-81

Cyclin dependent kinases (CDKs) are a family of serine/threonine kinases that require activation by binding to cyclin partners. These complexes of CDK-cyclin play an essential role in the regulation of cell cycle progression (e.g. CDK2) and some have also been implicated in the regulation of mRNA transcription (e.g. CDK9). CDK7 is involved in both these processes. Cyclins, CDK complexes and other cell cycle regulators have been shown to be mechanistically involved in the development of human tumours making them tractable drug targets. SNS-032 (BMS-387032) is a 2-aminothiazole derivative that has been shown to function as a selective inhibitor of CDK2, 7 and 9. Blocking the action of CDK2 and 7 is thought to block cell cycle progression in tumour cells. Additionally blocking CDK7 and 9 may also reduce transcription of survival factors in the tumour cells by abrogating their ability to promote transcription. Primary mononuclear cells isolated from acute myeloid leukaemia patients at diagnosis (n=87) were treated with SNS-032 for 48h resulting in a mean LD50 of 0.14μM ± 0.2 as measured by MTS assay. By comparison, cytarabine (AraC) was more than 35 times less potent in the same cohort (mean LD50 = 4.88μM ± 5.02). In combination with AraC (ratio SNS-032 1:1.9 cytarabine) showed a mean combination index (CI) of 0.13 in the 25 primary samples tested indicating strong synergism between SNS-032 and AraC. SNS-032 induces apoptosis as evidenced by dose-dependent increases in AnnexinV/propidium iodide (PI) staining and caspase-3 activation. SNS-032 affects activity of CDK 2 and 9 at the expression level as quantitative PCR showed that levels of CDK 2 and 9 mRNA were decreased at six hours of treatment to SNS-032 (CDK2 to 20% untreated levels (0.26μM), CDK9 to 36% untreated levels) but CDK7 mRNA levels were not significantly altered. However, CDK7 phosphorylation was reduced at 6 hours suggesting the SNS-032 modulates CDK7 at the post-translational level by suppressing activation. Treatment of the cell lines with the combination of SNS-032 and AraC resulted in reduced mRNA levels of CDK2 and 9. In contrast, AraC alone did not induce these reductions in mRNA expression. Additionally, the pro-survival proteins XIAP and Bcl-2 and Mcl-1 mRNA levels were decreased in SNS-032 treated cells, increased in AraC treated cells and reduced in cells treated with the combination. In conclusion, SNS-032 is effective in AML cells in vitro as a single agent and has also demonstrated a remarkable degree of synergy with AraC. One putative mode of action of this synergy is the SNS-032-induced decrease in pro-survival proteins. This may sensitise AML cells to the actions of cytotoxic agents LIKE AraC leading to the levels of synergy seen in this study.

Disclosures:

No relevant conflicts of interest to declare.
High Dose Cytarabine, Clofarabine and Gemtuzumab Ozogamicin (CLAC-MYL) in Relapsed or Refractory AML Patients.
Authors: Scappini Barbara , Gianfaldoni Giacomo , Salvatore Palmieri , Susini Maria Chiara , Izzo Teresa , Mannelli Francesco , Longo Giovanni , Fanci Rosa , Ferrara Felicetto , Bosi Alberto
Published: October 2019

Abstract 1060

Poster Board I-82

Clofarabine has been shown to be effective in AML patients, either as single agent or, mainly, in association with intermediate dose cytarabine. On the basis of these reports, we conducted a preliminary study combining clofarabine, intermediate dose cytarabine and gemtuzumab ozogamicin (Mylotarg) in AML patients who relapsed or failed to respond to at least two induction therapies.

We treated 20 patients affected by relapsed/refractory AML with a regimen including clofarabine at 22,5 mg/m2 daily on days 1-5, followed after three hours by cytarabine at 1 gr/m2 daily on days 1-5, with the addition of gemtuzumab ozogamicin 6 mg/m2 on day 6 (CLAC-Myl). Six patients received a further consolidation cycle with clofarabine at 22,5 mg/m2 and cytarabine at 1 gr/m2 day 1-4.

Among the twenty patients, six were in first relapse, six in second relapse, eight with resistant disease. The mean age was 52 years (range 33-68 years), the white blood count at the accrual was 31.500 mcc (range 2140-153.000). 10/20 (50%) patients achieved a complete remission, 1/20 a partial response, 7/20 had resistant disease, 2/20 died of complications during the aplastic phase (a case of multiorgan failure an a septic shock caused by Pseudomonas Aeruginosa).

The most frequent non hematologic adverse events were vomiting, diarrhea, transient liver toxicity (2/20 grade 3-4), febrile neutropenia (7/20), infections microbiologically documented (2/20 Pseudomonas Aeruginosa sepsis).

Comparing with other salvage strategies, in this small cohort of patients we did not observe a significant delay in bone marrow recovery (median time to ANC recovery 31.5 days), except in a patient (female, 34 years old, relapsed after ABMT) that experienced an unexpected, irreversible aplasia after the consolidation course, complicated by an unusual HHV6 reactivation.

Among the ten responding patients, three underwent allogeneic bone marrow transplantation, one patient still in CR after 7 months died for complications of an acute myocardial infaction occurred during the consolidation course, one relapsed after 6 months, and five not eligible for transplant procedures are still in complete remission with a median follow up of 6 months.

These very preliminary results suggest that the CLAC-Myl regimen is effective in this particularly poor prognosis category of patients, with safety data consistent with previously reported salvage therapies. Further studies are warranted.

Disclosures:

Off Label Use: Clofarabine in relapsed/refractory AML.
Lenalidomide and Dexamethasone Promote Erythropoiesis in Both Normal and RPS14 and RPS19 shRNA Infected Human CD34+ Cells: Implications for Therapy of Myelodysplastic Syndrome and Diamond-Blackfan Anemia.
Authors: Narla Anupama , Dutt Shilpee , McAuley J. Randall , Ebert Benjamin L.
Published: October 2019

Abstract 1092

Poster Board I-114

Lenalidomide, a derivative of thalidomide, induces erythroid responses in patients with myelodysplastic syndrome while dexamethasone induces erythroid responses in patients with Diamond Blackfan anemia. In vitro, both lenalidomide and dexamethasone have been shown to stimulate erythropoiesis. In the present study, we explored the activity of lenalidomide and dexamethasone in stimulating erythroid differentiation of primary hematopoietic progenitor cells and CD34+ cells expressing RPS19 or RPS14 shRNAs. Both agents increase the absolute number of erythroid cells produced from normal human CD34+ bone marrow cells by 1.3 to 1.6-fold. Importantly, both lenalidomide and dexamethasone increased the diminished erythroid proliferative capacity of CD34+ cells expressing either RPS19 or RPS14 shRNAs. However, the drugs have distinct effects on the production of erythroid progenitor colonies, Blast Forming Units – Erythroid (BFU-E) and Colony Forming Units – Erythroid (CFU-E). Dexamethasone caused a dose-dependent and statistically significant increase in the number of BFU-E colonies by 2-fold, while it had little effect on CFU-E. In marked contrast, lenalidomide caused a dose-dependent and statistically significant increase in the number of CFU-E colonies by 2.5-fold but had no effect on BFU-E colonies. In addition, the combination of lenalidomide and dexamethasone had an additive effect on the production of erythroid cells and on erythroid colony formation. Our findings imply that lenalidomide and dexamethasone regulate different stages of erythropoiesis and lend support to the concept that combination therapy may be effective in minimizing the potential toxicity of these two drugs in the clinical management of patients. These findings have important implications for the treatment of patients with Myelodysplastic Syndrome as well as with Diamond Blackfan Anemia.

Disclosures

Off Label Use: Lenalidomide is approved for 5q- MDS. We are investigating its possible use in Diamond Blackfan Anemia.
Deletion of IKZF1 in Pediatric Precursor-B ALL Is a Strong Prognostic Marker for Relapse.
Authors: Kuiper Roland P. , Waanders Esmee , Van der Velden Vincent H.J. , van Reijmersdal Simon V , Scheijen Blanca , Hehir-Kwa Jayne , Keijzers-Vloet Suzanne T.M. , Sonneveld Edwin , van Dongen Jacques JM , van Leeuwen Frank N. , van Kessel Ad Geurts , Hoogerbrugge Peter M.
Published: October 2019

Abstract 1104

Poster Board I-126

Relapse is the most common cause of treatment failure in pediatric acute lymphoblastic leukemia (ALL), and is difficult to predict from information at diagnosis in the majority of cases. To explore the prognostic impact of recurrent copy number abnormalities on relapse in children diagnosed with precursor-B cell ALL, we performed genome-wide copy number profiling of 34 paired diagnosis-relapse samples. Lesions detected at diagnosis were often absent at relapse, including recurrent targets in precursor-B ALL like PAX5 (not preserved in 2 out of 7 cases with deletions at diagnosis), CDKN2A (not preserved in 1 out of 15 cases), and EBF (not preserved in 2 out of 5 cases), which illustrates that these lesions are often secondary events that are not present in the therapy-resistant progenitor that causes relapse. In contrast, deletions and nonsense mutations in IKZF1, which encodes the lymphoid differentiation factor IKAROS, were highly frequent (38%) and always preserved at time of relapse. Locus-specific copy number screening of IKZF1 in an additional cohort of diagnosis samples from children enrolled in the Dutch treatment protocol DCOG-ALL9 with (n=40) or without (n=51) relapse revealed that IKZF1 deletions were significantly enriched in relapse-prone cases (22.5% vs 3.9%; P=0.007). An independent and unbiased validation cohort of 150 DCOG-ALL9 cases was used to confirm these findings, which established that 28.6% of the cases with IKZF1 deletion at diagnosis developed a relapse. Together, we conclude that deletions of IKZF1 in DCOG-ALL9 treated pediatric precursor-B ALL patients provide a strong prognostic marker for relapse.

Disclosures

No relevant conflicts of interest to declare.
Proteomic Screening Applied to the Diagnosis of Chronic Graft-Versus-Host-Disease.
Authors: Mischak-Weissinger Eva M. , Stadler Michael , Holler Ernst , Schleuning Michael , Greinix Hildegard , Kolb Hans-Jochem , Dickinson Anne M , Krons Annika , Diedrich Helmut , Kontsendorn Julia , Hahn Norbert , Metzger Jochen , Ganser Arnold
Published: October 2019

Abstract 1164

Poster Board I-186

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative treatment for many hematologic malignancies or hematopoietic dysfunction syndromes, but the application is still limited due to major complications, such as severe graft versus host disease (GvHD) and infectious complications. Diagnosis chronic GvHD is based on clinical features and biopsies, a non invasive, unbiased laboratory test does not exist.

We used the urine collected from 20 patients (10 with limited cGvHD, 10 with extensive cGvHD) to establish a proteomic pattern that allowed the diagnosis of cGvHD development and tested the resulting set of polypeptide markers (27 differentially excreted peptides) on more than 200 patients prospectively and blinded for the correct classification of cGvHD samples. The majority of the patients included were transplanted for hematological malignancies (n=209), 6 for hematopoietic failure syndromes. Conditioning regimens included dose reduced conditioning regimens (FLAMSA and ClaraC for the majority of the patients of MHH), as well as standard conditioning regimens (TBI+Cy or Busulfan+Cy) for about 35% of the patients, with GvHD-prophylaxis including cyclosporine A and mycophenolate (MMF) or metothrexate (MTX) as appropriate. Eighty percent of the patients received ATG (antithymocyte globulin) prior to HSCT.

A peptide pattern of 27 peptides, differentiating chronic from acute GvHD was developed. Controls were patients at least 100 days post HSCT, with no GvHD in the history, no infections and without relapse at the time of sampling. Prospective and blinded evaluation of the patients revealed the correct classification of patients developing cGvHD with a sensitivity of 85% and specificity of 95%.Further evaluation of the cGvHD patterns specific for particular organ manifestations of cGvHD are currently ongoing. Interestingly, the cGvHD pattern seems to be predictive for GvHD developing post DLI, while the aGvHD-specific proteomic pattern only predicts GvHD of the intestine, which may be more similar to “late acute GvHD”.

Disclosures

Krons: mosaiques-diagnostics GmbH: Employment. Metzger:mosaiques-diagnostics GmbH: Employment.
Salvage Autologous or Allogeneic Stem Cell Transplantation After the Failure of First Autograft in Patients with Multiple Myeloma.
Authors: Bashir Qaiser , Thall Peter , Hosing Chitra , Mendoza Floralyn L , Han Eric , Wang Michael , Shah Jatin , Alousi Amin , Anderlini Paolo , Kebriaei Partow , Khouri Issa F. , Popat Uday , de Lima Marcos , Champlin Richard , Giralt Sergio A , Qazilbash Muzaffar H
Published: October 2019

Abstract 1186

Poster Board I-208

Background:

Almost all patients with multiple myeloma (MM) who receive autologous hematopoietic stem cell transplantation (auto HCT) eventually relapse. The treatment options for these patients include novel agents or second (salvage) auto or allogeneic (allo) HCT. Use of reduced intensity conditioning (RIC) has significantly reduced the transplant-related mortality (TRM) with allo HCT. We evaluated the outcomes of patients who received salvage auto or allo HCT for relapsed MM.

Methods:

Sixty-two patients (24 females and 38 males), with a median age of 55 years (range: 37-73) received a salvage auto HCT between January 1992 and December 2008, whereas 44 patients (19 females and 25 males), with a median age of 51 years (range 32-65) received salvage allo HCT (12 unrelated and 32 related donor) between October 1988 and December 2006. Among 12 patients with unrelated allo HCT, ten were matched at 10/10 HLA loci, while two patients were mismatched at 1 or 2 loci, respectively. Among 32 patients with related donor allo HCT, 29 matched at 10/10 loci, while 3 patients had one or two antigen mismatches. In the allo HCT group eight patients received myeloablative regimens (MA), while thirty six patients received RIC regimens. MA regimens were fludarabine + melphalan 180 mg/m2 in 4 patients, busulfan + cyclophosphamide in 2 patients, busulfan + melphalan in one patient and TBI-based in another patient. RIC regimens were fludarabine + melphalan ≤140 mg/m2 in 34 patients and cyclophosphamide + fludarabine in 2 patients.

Results:

Median follow-up for both auto and allo HCT patients was 24 months. Median prior treatment regimens in auto and allo HCT patients were 4 (2-16) and 5 (2-10), respectively. Overall response rates in evaluable patients in auto HCT and allo HCT were 63% and 75%, respectively. Cumulative incidence of grade II-IV acute GVHD was 27% and limited or extensive chronic GVHD was 43% in allo HCT group. One-hundred day TRM in auto HCT and allo HCT groups was 3% and 9%, respectively. Most common causes of nonrelapse mortality were infections (12%) in auto, and acute or chronic GVHD (24%) in allo HCT group. Median progression free survival (PFS) and overall survival (OS) for auto HCT were 15.5 and 43.3 months, and for allo HCT were 6.9 and 14 months, respectively. Patients receiving MA regimens had significantly shorter PFS and OS than patients receiving RIC regimens.

Conclusions:

Both second auto and allo HCT are feasible for salvage therapy in patients with advanced MM, who had relapsed after an auto HCT. Disease progression remains the major cause of treatment failure. RIC regimens have improved the outcome of allo HCT.

Disclosures:

No relevant conflicts of interest to declare.
“An Intention-to-Treat Analysis of Rome Transplant Network Policy for Alternative Donor Search in Patients Lacking a HLA Matched Related donor”.
Authors: Picardi Alessandra , de Fabritiis Paolo , Dentamaro Teresa , De Rossi Giulio , Caniglia Maurizio , Pinto Rita , Petti Maria Concetta , Mengarelli Andrea , Annino Luciana , Chierichini Anna , Monarca Bruno , Montefusco Enrico , Avvisati Giuseppe , Tirindelli Maria Cristina , Mangione Ilaria , Cudillo Laura , Cerretti Raffaella , De Angelis Gottardo , Di Veroli Ambra , Arcese William
Published: October 2019

Abstract 1195

Poster Board I-217

The policy of the Rome Transplant Network (RTN), a metropolitan network of transplant Centers, for patients candidates to an allogeneic hematopoietic stem cell transplant (HSCT) and lacking an HLA identical sibling is the contemporary search for one the HSC alternative sources such as Matched Unrelated Donor (MUD),Cord Blood Unit (CBU) or Haploidentical Related Donor (HRD). The main aim of the RTN policy is the identification of a suitable donor in order to perform transplant in adequate timing. The selection criteria for MUD consist of a 8/8 HLA loci matching tested at low resolution for class I HLA and at high resolution (HR) for class II; one difference in C Ag is considered acceptable in case of both I and II class HR identity. CBU's selection criteria are instead based on cell doses (TNC≥2.5×107/kg and CD34+≥1× 105/kg) and on a HLA-compatibility ≥to 4/6 HLA Ag. From April 2006, the haplodentical option was also simultaneously considered, so all closer family members have been tested for the HLA. Here, we report the results of the intention to treat (ITT) analysis on the potential therapeutic impact of our transplant policy. Data were obtained from RTN database. From April 2006 to date, 196 pts have been candidated to receive an allogeneic HSCT for hematological disease. Sixty-six out of 196 (34%) underwent HSCT from HLA identical sibling, while a search process for an alternative donor was activated for 130 pts. Of 130 pts, 9 (7%) lost the eligibility to transplant early during the search process and 19 (15%) died of early disease progression in most cases: a suitable MUD or CBU had been identified for 13 of 19 within 3 months from the start of the search and only 6 pts (5%) died without an alternative donor had been found. To date, 73/102 evaluable pts (72%) lacking an HLA identical sibling have been transplanted (n=66: 23 MUD; 24 CBU; 19 HRD) or are willing to proceed towards the transplant (n=7: 3 MUD;2 CBU; 2 HRD). In summary, for all 196 candidates to an allogeneic transplant the eligibility was confirmed for 187 (95%), a suitable donor could be identified for 181 (92%) of all pts or 97% of the eligible ones and an allogeneic transplant could be performed for 168 (86%) of all candidates or 93% of those eligible. From this ITT analysis, we can conclude that, by adopting the RTN policy of widespread donor search and multiple transplant options, the allogeneic transplant can be offered as potential therapeutic procedure to a large majority of pts.

Disclosures:

No relevant conflicts of interest to declare.
ON 01910.Na Suppresses Cyclin D1 Accumulation in trisomy 8 Myelodysplastic Syndromes Patients While Decreasing Bone Marrow CD34+ Blast Counts and Aneuploid Clone Size.
Authors: Sloand Elaine M , Olnes Matthew J. , Galili Naomi , Shenoy Aarthi , Pfannes Loretta , Wilhelm Francois , Weinstein Barbara , Scheinberg Phillip , Groopman Jerome E. , Raza Azra
Published: October 2019

Abstract 120

Patients with high risk MDS can be successfully treated with 5-azacytidine, or with lenalidomide but non-responding patients have few treatment options. Chemotherapy produces significant morbidity and very short remissions and most patients are too old for bone marrow transplantation. We previously demonstrated up-regulation of c-myc, survivin, and cyclin D1 in CD34+ cells in patients with trisomy 8 (and selected patients with monosomy 7). siRNA-mediated knockdown of survivin or c-myc decreased trisomy 8 cell growth in vitro (Sloand et al, Blood 2007, 110: 822). We postulated that increased cyclin D1 causes upregulation of survivin, resulting in resistance of these cells to apoptosis. The styryl sulfone, ON 01910.Na, decreases cyclin D1 accumulation in cultured bone marrow from patients with high risk trisomy 8 MDS and in some monosomy 7 patients (who also show upregulation of cyclin D1), while selectively decreasing blasts and aneuploidy with this cytogenetic abnormality (ASH Abstracts Nov 2008; 112: 1651). Here we examine the clinical response to ON1910 in an ongoing phase I/II clinical trial in which 13 evaluable patients with intermediate-1(int-1) to high risk MDS and treatment-refractory trisomy 8 AML were enrolled. Patients were treated with escalating doses of ON 01910.Na at 800 mg/m2 × 2 days every 3/4 weeks, 800 mg/m2 × 3 days every 2 weeks, 800 mg/m2 × 5 days every 2 weeks, and 1500 mg/m2 × 2 days every 3/4 weeks at two institutions. No significant toxicity could be ascribed to the drug. Patients with trisomy 8 and monosomy 7 demonstrated significant declines in aneuploidy measured by florescence in situ hybridization (FISH) (mean aneuploidy; 50% before and 24% after 1 cycle of treatment; p=0.02 :Fig below). Rather than becoming cytopenic, many patients showed substantial improvements of blood counts and one patient (01-02; graphic shown below) became red cell transfusion-independent and maintains his remission 14 months after stopping therapy. Cyclin D1 measurement by flow cytometry showed decreases of this protein in both CD34 and CD33 cells during infusion of ON 1910 infusion (example shown in Fig below). Results from individual evaluable patients are shown in table 1. These results indicate that modulation of cell cycle control by cyclin D1 may represent a novel targeted approach for trisomy 8 and monosomy 7 MDS.

Disclosures:

Sloand: Onconova: Research Funding. Olnes:Onconova: Research Funding. Galili:Onconova: Research Funding. Wilhelm:Onconova: Employment. Groopman:Onconova: Membership on an entity's Board of Directors or advisory committees. Raza:Onconova: Research Funding.