bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect and Safety of Ixazomib Plus Dexamethasone (ID) in Patients with Waldenstrom's Macroglobulinemia (WM): Real-World Evidences from a Single Center in China
Authors: Xu Jiadai , Wei Zheng , Li Jing , Zhuang Jingli , Ji Lili , Liu Peng
Published: November 2019

Introdution: As a second-generation proteasome inhibitors (PIs), ixazomib has come to China for 1 years. PIs has been proved efficacy in patients with Waldenstrom macroglobulinemia (WM), a rare form of B-cell lymphoma.

Aims: To evaluated the clinical activity and safety profile of combining ixazomib and dexamethasone (ID) in patients with WM, both in those who previous had not received systematic treatment and in those with refractory or relapsed disease (RRWM).

Patients and Methods: The protocol therapy consisted of oral ixazomib, 4 mg, on days 1, 8, and 15, with oral dexamethasone, 10 mg, on days 1, 8, 15, and 22, every 4 weeks. Progression-free survival (PFS) was set as the primary end point. Response rates, sustained hematologic improvement from baseline, and safety were set as the key secondary end points. Responses were determined according to the 6th International Workshop WM response criteria. Adverse events (AEs) were reported by CTCAE v4.03. The data cut-off is 26 July 2019.

Results: Under the premise of respecting the patient preferences, 9 symptomatic patients with WM have been enrolled, in whom 5 patients were newly diagnosed (NDWM) receiving ID as primary therapy (PT) and the other 4 patients were with RRWM having been received previous systematic therapies. All patients were included in this analysis. The baseline clinical characteristics and major therapy situations are summarized in Table 1. The median reaction time was 4.14 weeks, which was 4.00 weeks in WM patients receiving ID as PT and 7.00 weeks in RR WM patients. After a median follow-up of 6.03 months, in NDWM patients, the overall response rate was 80.0%, and the major response rate (CR+VGPR+PR) was 40.0% (2/5). Patients with RRWM were equally responsive. The overall response rate was 75.0%, and the major response rate (CR+VGPR+PR) was even relatively higher, which was 50.0% (2/4). The median PFS was not reached. The ID regimen was well tolerated with 66.7% patients reporting no drug related AE. Grade 1 adverse events reported in 3 patients included diarrhea (1/9, 11.1%), thrombocytopenia (1/9, 11.1%), digestive disturbance(1/9, 11.1%). Shingles (grade 2) was reported in 1 patient. Serum IgM decreased from 42.66 g/l (median) at baseline to 21.02 g/l (median) in NDWM patients (Fig. 1A), from 43.87 g/l (median) at baseline to 25.59g/l (median) in RRWM patients (Fig. 1B). The median level of hemoglobin in patients with a hemoglobin of less than 115.0 g/l before using ID increased from 71.6 g/dl (median) at baseline to 87.3 g/l in NDWM patients (Fig. 1C), from 73.7 g/dl (median) at baseline to 89.3 g/l in RRWM patients (Fig. 1D). There have been 2 cases suffered disease progression after receiving ID regimen.

Conclusions: ID regimen offers a relatively effective and well tolerated choice for patients with WM.

Disclosures

No relevant conflicts of interest to declare.
Pegylated Interferon in Myelodysplastic/Myeloproliferative Syndrome with Ringsideroblasts and Thrombocytosis
Authors: Benz Rudolf A. , Zimmermann Kathrin , Widmer Corinne C
Published: November 2019

Background:In 2016 WHO recognized the provisional entity refractory anemia with ring sideroblasts and thrombocytosisas a distinct entity and renamed it myelodysplastic/myeloproliferative neoplasm with ring sideroblasts and thrombocytosis (MDS/MPN-RS-T). The disease presents hallmarks of both myelodysplastic and myeloproliferative features. Although the diagnostic criteria are well defined and there are several studies of the molecular landscape of the disease, the treatment regimen of these patients remains inconsistent. Due to lack of clinical trials, treatment options are chosen either on MDS- or MPN-based regimens and individual preferences such as patients characteristics and clinical presentation. Prevention of thromboembolic events is a reasonable goal. However, the use of platelet-lowering drugs is still not well studied and treatment related anemia leading to transfusions with accelerated iron overload limits a general use of cytoreductive treatment. To avoid worsening of the anemia while efficiently reducing thrombocytosis we treated 3 patients with pegylated interferon (pIFN) and provide insight in JAK2-allele burden measurement and additional mutations obtained with next generation sequencing (NGS).

Results:Three patients with diagnosis of MDS/MPN-RS-T were treated with 135µg pIFN during an observation period between 8 and 34 months. All 3 patients had a JAK2V617F mutation, >15% ringsideroblasts and a thrombocyte count above 600G/l. In the additional NGS analysis, two patients showed a mutation in the SF3B1 gene, one with an additional DNMT3A mutation. In the third case, NGS results revealed a mutation in the splicing factor U2AFI, which is a rare finding in patients with MDS/MPN-RS-T. JAK2-allelel burden was obtained at the beginning and showed a decrease of a mean value of 38% as measured by digital droplet PCR. Anemia was present at initial diagnosis in two patients. During observation period, none of the patient became transfusion dependent, in one case even an improvement in hemoglobin levels was achieved. Side effects of the treatment were only minimal and no thromboembolic as well as major bleeding event occurred under additional acetylsalicylic acid medication.

Conclusion: In summary we show that in patients with MDS/MPN-RS-T a therapy with pIFN achieved rapid decrease of thrombocyte values without lowering hemoglobin levels at a maximal dose of 135µg per week. JAK2-allele burden reduction and a low side effect profile further demonstrate the value of this treatment option for these often elderly patients. However, larger studies are needed to confirm our data.

Disclosures

No relevant conflicts of interest to declare.
ZFP91 Regulates Multiple Myeloma Cell Proliferation through IL-1β Mediated IL-6 Secretion in the Tumor Microenvironment
Authors: Jeyaraju Danny V , Kumari Ekta , Amatangelo Michael , Bjorklund Chad C. , Hsu Chih-Chao , Thakurta Anjan
Published: November 2019

Multiple myeloma is a malignancy of terminally differentiated plasma cells that are primarily resident in the bone marrow. Despite novel therapies, myeloma remains a fatal disease. Through interactions with immune cells in the bone marrow microenvironment, myeloma cells have been shown to facilitate their own proliferation and drug resistance. Therefore, targeting the immune support cells in the myeloma bone marrow microenvironment could add further value to existing treatment regimens.

ZFP91, a C2H2-type zinc-finger protein, is a substrate of IMID drugs (An et al, Nat. Commun. 2017). The downstream target of ZFP91 in myeloma has been elusive so far. Our myeloma-specific ZFP91 interactome data and recent publications indicate an immune related function for ZFP91. A recent study also indicated that treatment of healthy volunteers with iberdomide, impaired the ability of monocytes to secrete IL-1β in response to LPS (Schafer PH, et al. Ann Rheum Dis 2018). To further investigate this, we generated CRISPR knockouts of IKZF1, IKZF3 and ZFP91, three major substrates of lenalidomide and iberdomide, in THP-1 monocytic cells. Upon LPS induction, loss of ZFP91 impaired the ability of THP-1 monocytic cells to secrete IL-1β (WT 70 pg/ml of IL-1β vs ZFP91-/- 4.5 pg/ml of IL-1β). Loss of IKZF1/3, the other two substrates of lenalidomide and iberdomide did not impair IL-1β secretion.

IL-1β is a known activator of IL-6, a critical cytokine for myeloma cell proliferation. As a next step, we created a co-culture model to mimic a minimal myeloma tumor microenvironment with ANBL-6 (IL-6 dependent) myeloma cell line and primary bone marrow stromal cells in the presence of pre-conditioned media from LPS stimulated THP-1 monocytes. Preconditioned media from ZFP91-/- monocytes impaired proliferation of myeloma cells in the co-culture model due to lack of IL-1β, the inducer of IL-6 from the stromal cells. Mechanistically, loss of ZFP91 impairs the TLR4 signaling pathway. Thus, targeting ZFP91 impairs proliferation of myeloma cells within the bone marrow microenvironment by acting in trans from monocytes.

Our findings for the first time identify a tumor cell proliferative role for ZFP91 in the myeloma tumor microenvironment. In addition, it establishes ZFP91 as an immune substrate of lenalidomide and iberdomide and could be a key player that mediates the immune functions observed.

Disclosures

Jeyaraju: Celgene Corporation: Employment, Equity Ownership. Kumari:Celgene Corporation: Employment. Amatangelo:Celgene Corporation: Employment, Equity Ownership. Bjorklund:Celgene Corporation: Employment, Equity Ownership. Hsu:Celgene Corporation: Employment. Thakurta:Celgene: Employment, Equity Ownership.
Combination of Chidamide with the CHOEP Regimen in Previously Untreated Patients with Peripheral T-Cell Lymphoma (PTCL): A Prospective, Multicenter, Single-Arm, Phase 1b/2 Trial
Authors: Zhang Wei , Su Liping , Liu Lihong , Gao Yuhuan , Wang Quanshun , Su Hang , Song Yuqin , Zhang Huilai , Shen Jing , Jing Hongmei , Wang Shuye , Cen Xinan , Liu Hui , Liu Aichun , Li Zengjun , Luo Jianmin , He Jianxia , Wang Jingwen , O'Connor Owen A. , Zhou Daobin
Published: November 2019

Background: Chidamide is a novel benzamide class of histone deacetylase (HDAC) inhibitor. In a pivotal phase 2 trial with refractory or relapsed peripheral T-cell lymphoma (PTCL), chidamide produced ORR 28% with CR 14% leading to its approval by the China Food and Drug Administration in 2014 for refractory or relapsed PTCL. We sought to assess the safety, tolerability and efficacy of the novel histone deacetylase inhibitor chidamide in combination with CHOEP (cyclophosphamide, doxorubicin, vincristine, etoposide, and prednisone) (Chi-CHOEP) for previously untreated PTCL.

Patients and methods: This was a prospective, multicenter, single-arm Phase 1b - 2 clinical trial conducted exclusively in China. In the phase 1b study, three dose levels of chidamide were explored. In the phase 2 portion, patients were treated at the RP2D. The primary endpoint of the Phase 1b was determination of the maximum tolerated dose (MTD), dose limiting toxicities (DLT) and RP2D. The primary endpoint of the phase 2 study was determination of the overall response rate (ORR).

Results: A total of 82 patients were enrolled between March 2016 and Dec 2017 at 17 sites across China. The cutoff date for the analysis was July 2018. In phase 1b dose-escalation study, 15 patients were treated and the second dose cohort (20 mg biw) was declared the MTD and thus the RP2D. Subsequently, 67 patients were enrolled and treated at the RP2D in the phase 2 part of the study. Overall, among 82 enrolled patients in the entire phase 1b/2 study, the median number of cycles per person was five (range 1-8) and the relative dose intensity was 75.4%. The most common toxicities in the Phase 1b/2 study included leucopenia, anemia, and neutropenia. No reactivation of Epstein-Barr virus or Hepatitis B virus was observed. For entire phase 1b/2 study of 82 patients, the ORR was 68.3% with a CR rate of 43.9%. In the phase 2 part of the study, the ORR was 73.2% with 47.8% CR. with a median follow-up of 12.7 months (range 0.3-30.8 months), the median PFS for entire phase 1b/2 study was 17.4 months and the 1-year PFS was 52.9%. The median OS had not yet been reached, and the 1-year OS was 74.5%. In the phase 2 study, the median PFS was 17.4 months and the 1-year PFS was 53.6%. The median OS had not yet been reached, and the 1-year OS was 76.3%.

Conclusion: Chi-CHOEP is an effective and feasible novel regimen for untreated PTCL. These data warrant further investigation in a randomized phase 3 study. This trial was registered at www.clinicaltrials.gov as #NCT02987244.

Disclosures

No relevant conflicts of interest to declare.
Successful Treatment of Mucosal Bleeding with Tranexamic Acid in Pediatric Patients with Hemostatic Disorders
Authors: Nakadate Hisaya , Ishiguro Akira , Sakamoto Atsushi
Published: November 2019

Objects

Hemostatic disorders, including thrombocytopenia and/or coagulation disorders, sometimes cause mucosal bleeding and/or bleeding risks. In some cases, severe mucosal bleeds cannot be adequately controlled by standard therapy. Tranexamic acid (TA) is used in preventing and reducing bleeding in such medical settings. But there is little information on useful TA management of mucosal bleeding in hemostatic disorders.

Materials and Methods

4 patients with ITP, 2 hemophilia A patients and one VWD patient were treated with TA (1.5 - 3.0 g/day ) due to recognizable mucosal bleeding. 2 patients were under regular treatment for ITP. The remaining 2 patients with ITP did not require additional therapy. 2 hemophilia A patients were mild case and require on demand therapy of FVIII concentrates. VWD patient did not require any therapy with no mucosal bleeds.

Results

One of the ITP patients were suffering from frequent nasal bleeding and kept her away from school for 1.5 years. She had been going back to school without limitation of physical activity in spite of low PLT count. Remaining 3 patient of ITP who was suffering from frequent nasal bleeds had been managed with TA ( 1.5 g/day ). 2 hemophilia patients who were experienced oral mucosal bleed after tooth extraction were successfully managed with TA ( 1.5 g/day ). VWD patient who were suffering from frequent nasal bleed were ceased after initiation of TA.

Conclusions

Cessation or, at least significant improvement of bleeding was achieved shortly after initiation of TA in all cases. TA was well tolerated and discontinued after cessation of bleeding. We recommend the use of TA in hemostatic disorder patients with bleeding and/or an increased bleeding risk. Ultimately, cessation of bleeding plays a key role in the QOL improvement of such affected patients. However, future studies are required to optimize dose and administration routes (intravenous or oral).

Disclosures

No relevant conflicts of interest to declare.
R4 Rgs Subfamily Proteins Suppress Engraftment and Regulate SDF-1/CXCR4 Signaling in Human CD34+ Hematopoietic Stem/Progenitor Cells
Authors: Chan Kathy , Wong Tsin Sik , Li Karen , Zhang Xiao-Bing , Wang Ronald , Lee King Yiu , Cheng Frankie Wai Tsoi , Li Chi Kong , Leung Kam Tong
Published: November 2019

Members of the Regulators of G-protein Signaling (RGS) are GTPase-accelerating proteins and have been implicated in SDF-1-directed trafficking of mature hematopoietic cells. However, their roles in hematopoietic stem and progenitor cells (HSPC) remain largely unknown. In this study, we investigated the expression, functions and mechanism of R4 RGS subfamily members on migration and engraftment of human HSPC. Our results demonstrated that cord blood (CB), bone marrow (BM) and mobilized peripheral blood (MPB) CD34+ cells expressed specific RGS mRNAs, of which RGS1, RGS2, RGS13 and RGS16 were significantly upregulated by SDF-1 (1.6-1.9 fold, n=5, P<0.05). In the presence of AMD3100, a CXCR4 inhibitor, the stimulating effects of SDF-1 on RGS expression were completely abolished (n=6). SDF-1-directed functions (chemotaxis, trans-matrigel migration and calcium flux) and signaling (phosphorylation of Akt, ERK and Stat3) were significantly inhibited in RGS1, RGS13 and RGS16-overexpressing CD34+ cells (n=4-6, P<0.05) but not in RGS2-overexpressing cells, whereas actin polymerization, adhesion and colony formation were unaffected by these RGS members. In the NOD/SCID mouse xenotransplantation model, overexpression of RGS1, RGS13 or RGS16 in CD34+ cells did not impact their short-term homing but substantially compromised their long-term engraftment efficiency in bone marrow and spleens of recipient mice by 91.4%, 83.7% and 71.2%, respectively (n= 8-9; P<0.05). Genome-wide expression microarray and qPCR validation identified 32 common differentially expressed genes (1 upregulated and 31 downregulated) in RGS1, RGS13 or RGS16-overexpressing CD34+ cells. Network analysis revealed the potential mechanisms of RGS1, RGS13 and RGS16 downstream of SDF1/CXCR4 and Gαi protein, leading to compromised Akt, ERK and Stat3 phosphorylation and negative regulation of stem cell functions (CCNA1, SPP1, LPAR5, IL1RL1, HPSE), complement activation (C3AR1, C5AR2, C5AR1), proteolysis (TIMP3, MMP14) and cell migration (THBS1, F2RL2, PROS1, CCL1). Our results highlight the unprecedented functions of R4 RGS proteins in HSPC migration and engraftment, and provide the foundation of future design of RGS-targeting strategies to enhance the efficiency of clinical HSPC transplantation.

Disclosures

No relevant conflicts of interest to declare.
Cytochromes P450 and Glutathione S-Transferases Polymorphisms: Can They Predict Efficacy of Tyrosine Kinase Inhibitors in Chronic Myeloid Leukemia?
Authors: Mukry Samina N. , Shahni Aneeta , Zaidi Uzma , Shamsi Tahir S.
Published: November 2019

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm characterized by over production of cells due to expression of an abnormal BCR/ABL1 kinase gene. Tyrosine kinase inhibitors (TKI) are the treatment option to achieve deep and prolonged molecular response in CML patients. Resistance or sub-optimal response to TKI such as imatinib has been reported in 5-20% of the patients carrying wild type of Cytochromes P450 (CYP) and glutathione S-transferases (GST). This study was planned to evaluate possible association of presence or absence of polymorphic variation in CYP1A1, GSTT1 and GSTM1 with response to nilotinib in CML patients

A total of 33 CML patients receiving nilotinib treatment (ELN 2013 guidelines) at National Institute of blood Diseases and Bone Marrow Transplantation were enrolled. Informed consent was obtained as per declaration of Helsinki. The presence (+) or absence (-) of CYP 1A1 (A2455G), GSTT1 and GSTM1 was determined by RFLP and multiplex PCR respectively. Treatment response was evaluated by quantification of BCR ABL1 transcripts by real time qPCR. The chi square test was used to compare the genotype distribution between patients and control. A p value less than 0.05 was considered as statistically significant. Odds ratio (OR) with 95% confidence interval (CI) calculated using statistical package SPSS version 22.

The male:female ratio for the study group was 1.75:1 with a mean age of 37±12 years. About 25, 5 and 3 patients were diagnosed with CML in chronic, accelerated and blast crisis phase respectively. There were 25 responders, 5 partial responders and 3 non-responders to nilotinib treatment altogether. The GSTT1- genotype was most frequent in responders (72%) followed by GSTM1+ and CYP 1A1- (p= <0.05). Interestingly, GSTT1-/GSTM1- and heterozygous CYP 1A1+ were less frequent (8% and 20%) but observed only in responders.

This is the first study on nilotinib response and CYP1A1, GSTT1, GSTM1 polymorphisms in CML from Pakistan. Null or GSTT1-, GSTM1+ and CYP 1A1- seemed to be frequent in nilotinib responders only and may have influence on drug response.

Disclosures

No relevant conflicts of interest to declare.
Myeloid Tumor-Related Somatic Mutations in 293 Children and Adults with Acquired Aplastic Anemia in Chinese
Authors: He Hailong , Yin Hua , Liu Xiaoqing , Cheng Lang , He Guangsheng , Li Jianyong
Published: November 2019

ABSTRACT

Objectives and Methods:

It was postulated that the subset of aplastic anemia ( AA ) with somatic mutations typically seen in myeloid malignancies were at risk of clonal evolution[1,2]. But the response rate to immunosuppressive therapy (IST) was different or not between patients with or without mutations, remain unclear[1,2]. Age of patients with AA is a strong predictor of outcome for IST, and the presence of somatic mutations also rise appreciably in frequency with age[3]. Here we checked myeloid tumor-related somatic mutations from 293 children and adult patients who have acquired AA. We excluded Fanconi anemia, mutations of telomere maintenance, and a family history of BM failure (BMF) or cancer. We looked for somatic mutations by identifying previously characterized single-nucleotide variants in 25 genes that are recurrently mutated in myeloid malignancies in the peripheral blood cells by second-generation gene sequencing. And clinical significance of mutations in different ages was also annalyzed.

Results :

Somatic mutations in myeloid tumor-related genes are present in 6.48% of the patients. The the variants occurred in ten genes: ASXL1 (n = 2 ), KRAS (n = 1), PIGA (n = 2 ), TP53 (n = 2 ), BCOR (n = 2 ), TET2 (n = 5 ), SF3B1 (n = 1), DNMT3A (n = 2 ), SH2B3 (n = 1), MPL (n = 1) (Figure 1). The majority of the variants occurred in epigenetic genes: TET2, DNMT3A, ASXL1. Among patients of children and teen agers' group, adult group, the clonal mutations were observed in 2.82%, 9.93% (P= 0.013) (Figure 2). The presence of a somatic mutation was not associated with gender ( male or female, 3.87% vs 9.42%, P=0.054), severity ( non severe AA or severe AA, 7.87% vs 4.35%, P=0.233). Patients with somatic mutations had a simillar response rate, compared with patients without mutations in children and teen agers' group, or adult group. Somatic mutations in AA patients were not associated with high risk of transformation to MDS (P > 0.5 ).

Conclusions:

Age-related clonal hematopoiesis rise along age in patients with AA in chinese, but somatic mutations in AA patients were associated with efectiveness of IST, and risk of clonal evolution.

References

1 Kulasekararaj AG, Jiang J, Smith AE, et al. Somatic mutations identify a subgroup of aplastic anemia patients who progress to myelodysplastic syndrome. Blood. 2014; 124(17):2698-2704).

2 Yoshizato T, Dumitriu B, Hosokawa K et al. Somatic Mutations and Clonal Hematopoiesis in Aplastic Anemia. N Engl J Med. 2015 Jul 2; 373(1):35-47.

3 Jaiswal S, Fontanillas P, Flannick J, et al. Age-Related Clonal Hematopoiesis Associated with Adverse Outcomes. N Engl J Med 2014; 371:2488-98

Disclosures

No relevant conflicts of interest to declare.
Potential Metabolites with Diagnostic Value in Plasma for Angioimmunoblastic T-Cell Lymphoma By LC-MS Based Untargeted Metabonomics Study
Authors: Yang Haiyan , Lei Tao , Li Cong , Yu Haifeng , Chen Zhongjian
Published: November 2019

Background: Angioimmunoblastic T-cell lymphoma (AITL) is an uncommon subtype of mature peripheral T-cell lymphoma (PTCL). and its characteristics is not fully understood. Because it is a rare form of lymphoma, there is difficulty in diagnosis based on current traditional method. Metabolic reprogramming is a major cancer hallmark, which provides cancer cells with vital energy and metabolites. Mass spectrometry-based metabolomics has emerged as an informative technique for profiling metabolic features associated with cancers.

Objective: This study aim to discover metabolites with potential diagnostic value in plasma of AITL patient.

Methods: Plasma was collected from 27 patients newly diagnosed with AITL in Zhejiang Cancer Hospital and 30 age- and gender- matched healthy controls. An aliquot of 50 μL plama was added with 150 μL of chilled acetonitrile, vortexed and centrifuged. the supernatant was dried, reconstituted for metabolomics. Mass spectrometry analysis was performed on a UPLC coupled with a Q Exactive Orbitrap mass spectrometer. "XCMS" and "MetaX" were used to analyze LC-MS data. Significant features were selected as candidate for identification by matching their MS and MS/MS data with online databases HMDB and METLIN.

Results: Eighteen altered metabolites (choline, betaine, L-leucine, creatine, 2-butyl-1H-benzimidazole, hypoxanthine, methionine sulfoximine, L-carnitine, 2-hydroxycinnamic acid, L-tyrosine, D-tryptophan, indoleacrylic acid, lysoPC(P-18:0), 5-aminopentanoic acid, uric acid, sucrose, L-lactic) were indentified (VIP value>1, p<0.05) in AITL patient plasma. The altered metabolites contributed to different metabolic pathways including glycine, serine and threonine metabolism (P=0.003), glycerophospholipid metabolism (P=0.024), Phenylalanine metabolism (P=.030).

Conclusion: This study revealed metabolic reprogram and potential diagnostic metabolites in plasma for AITL, and gave more metabolic information for carcinogenesis of AITL.

Disclosures

No relevant conflicts of interest to declare.
The SQSTM1-NUP214 Fusion Protein Cooperates with Crm1 to Activate Hoxa Genes and Drives Leukemogenesis in Mice
Authors: Aumann Waitman K. , Sze Sei-Gyung K. , Gupta Veerain , Ripple Katelyn , Port Sarah , Kehlenbach Ralph , Wechsler Daniel S. , Lavau Catherine P.
Published: November 2019

Background: The NUP98 and NUP214 nucleoporins (NUPs) are recurrently fused to heterologous proteins in leukemia. The resulting chimeric oncoproteins retain the NUP phenylalanine-glycine (FG) repeat motifs that mediate interaction with the nuclear export receptor Crm1. NUP fusion leukemias are characterized by HOXA gene upregulation; however, their molecular pathogenesis remains poorly understood. To investigate the role of Crm1 in mediating the leukemogenic properties of NUP chimeric proteins, we studied the SQSTM1-NUP214 fusion. Methods: We synthesized a SQSTM1-NUP214 fusion protein which retains only a short C-terminal portion of NUP214 containing FG motifs that mediate interaction with Crm1, and then introduced point mutations targeting these FG motifs (SQSTM1-NUP214FGmut). We compared the activity of these two fusion proteins using co-immunoprecipitation with CRM1, methylcellulose colony assays, murine transplantation, RT-qPCR, and chromatin immunoprecipitation. Results: We found that the ability of the SQSTM1-NUP214FGmut protein to interact with Crm1 was reduced by more than 50% compared with SQSTM1-NUP214. Further, mutation of FG motifs affected transforming potential: while SQSTM1-NUP214 conferred robust colony formation to transduced hematopoietic progenitors in a serial replating assay, the effect of SQSTM1-NUP214FGmut was greatly diminished. Moreover, SQSTM1-NUP214 caused myeloid leukemia in all transplanted mice (6/6), whereas none of the SQSTM1-NUP214FGmut reconstituted mice developed leukemia (0/7). These oncogenic effects coincided with the ability of SQSTM1-NUP214 and SQSTM1-NUP214FGmut to upregulate the expression of Hoxa and Meis1 genes in hematopoietic progenitors. Indeed, chromatin immunoprecipitation assays demonstrated that impairing the interaction of SQSTM1-NUP214 with Crm1 reduced binding of the fusion protein to Hoxa and Meis1 loci. Conclusions: These findings highlight the importance of Crm1 in mediating the leukemogenic properties of SQSTM1-NUP214, and suggest a conserved role of Crm1 in recruiting oncoproteins to their effector genes.

Disclosures

No relevant conflicts of interest to declare.
Creating and Testing a CD207+ Langerhans Cell Histiocytosis-like Cell Line
Authors: Velazquez Jessica , Sengal Amel , Allen Carl E. , Chakraborty Rikhia
Published: November 2019

Langerhans cell histiocytosis (LCH) is an inflammatory myeloid neoplasia characterized by pathological CD207+ dendritic cells (DCs) with persistent mitogen-activated protein kinase (MAPK) activation. Investigations into LCH have historically been challenged by the small percentage of pathologic CD207+ DCs. No cell lines with morphology or function representing LCH lesion CD207+ DCs currently exist. We utilized four strategies to generate cell line(s) mimicking differentiated LCH pathogenic cells. First, CD207+ cells were isolated from the lesion of an LCH patient and cultured in a cytokine cocktail. The cells maintained CD1a and CD207 expression for two weeks, after which there were significant changes in cell morphology and progression to cell death. When a similar approach was implemented to isolate and culture skin CD207+ cells, the cells were viable for only three days, supporting the potential role for somatic activating MAPK mutations in LCH lesion DCs to prolong viability. CD207+ cells were then isolated from HLA-DR+ (lineage negative) cells from the lymphoid stroma of healthy tonsils (tCD207+). The tCD207+ cells were transduced with a lentivirus encoding human telomerase reverse transcriptase (hTERT). These cells, also lacking MAPK activating mutations, died two weeks post-transduction. A fourth approach has been more successful in which tCD207+ cells were cultured in a cytokine cocktail which provided MAPK pathway stimulation. The cells retained CD207 expression and survived in culture for over two weeks. The cells were then immortalized using a lentivirus encoding HOXA9. Immortalized cells maintained CD207 expression. Allele specific MAPK pathway mutations (BRAF and MAP2K1) are being generated by class II CRISPR/Cpf1 genome editing as Cpf1 has been shown to have robust activity to induce specific disruption of only mutant, but not wild-type, BRAF allele. The phenotypic and genomic characteristics of the immortalized cells expressing the different MAPK pathway mutations will be assessed by RHG-banding cytogenetic analysis, fluorescence in situ hybridization, gene expression analysis, and immuno‐cytochemistry and results will be compared to cells isolated from LCH lesions to confirm whether the established cell line may be a viable in vitro mimic of the LCH lesion DC.

Disclosures

No relevant conflicts of interest to declare.
Venous Thromboembolism in Lower Extremity Amputees: A Systematic Review of the Literature
Authors: Alsheef Mohammed Abdullah , Schulman Sam , Donadini Marco , Zaidi Abdul Rehman Z.
Published: November 2019

Patients undergoing lower extremity amputation (LEA) are at risk of developing deep venous thrombosis (DVT) and pulmonary embolism (PE), but no generally accepted prevention guidelines exist. This systematic review aimed at understanding the incidence of VTE with or without thromboprophylaxis in adult patients with major lower extremity amputation (LEA). Primary outcomes were onset of DVT, PE, or mortality. Secondary outcomes were any major adverse events due to treatment. We searched English language full-text papers in multiple databases using keywords, including amputation/adverse effects, amputation/complications, venous thromboembolism, deep vein thrombosis, and pulmonary embolism. Twenty-eight studies providing observations for 4,841 patients were selected. The fatal PE risk was 2.6% without prophylaxis and significantly decreased to a non-zero residual risk of 0.9% with VTE prophylaxis. Above-knee amputees were at greatest risk of VTE and subsequent complications. The risk was not confined to the amputated stump and can involve the contralateral limb. The role of compression ultrasonography screening in asymptomatic patients remains controversial in various populations at risk for VTE. All patients undergoing major LEA should be considered at high risk for the development of VTE, even after discharge from hospital. We recommend prophylactic anticoagulation (if not contraindicated) and clinical surveillance in all patients undergoing LEA and further studies to determine the optimal prophylactic strategy.

Disclosures

No relevant conflicts of interest to declare.
Quality Improvement Project to Evaluate Fever Management and Appropriateness of Lab Draws in Hospitalized Sickle Cell Disease Patients in a Tertiary Care University Teaching Hospital
Authors: Jafri Syed Imran Mustafa , Hook Karen
Published: November 2019

Introduction: Sickle cell disease is the most common inherited blood disorder that affects almost 100,000 people in the U.S. Infection is the most common cause of death in these patients. Early identification and initiation of antibiotics given functional asplenia is the key to appropriate management of hospitalized sickle cell disease patients. Approximately 4-5 billion laboratory studies are ordered every year in hospitals. Unnecessary blood tests lead to frequent blood draws and worsening anemia in this already anemic population as well as contribute towards increasing cost of hospital stay. Total national health expenditures in the U.S. is $3.3 trillion (2016) with percent of national health expenditures for hospital care: 32.4%.

Objective: We reviewed inpatient admissions of sickle cell disease patients to assess for appropriate work up and timely antibiotic initiation in case of fever. Additionally, we looked up the number of blood draws as well as number of days labs were drawn during the inpatient stay.

Methods: This was a retrospective chart review of all patients with sickle cell disease who were admitted to John Dempsey hospital at University of Connecticut with length of stay of at least 3 days between April 2018 to Feb 2019.

Results: 57 inpatient admissions were reviewed. Out of 57 admissions, 14 had fever either at the time of presentation or during the hospital stay. Mean time to first blood culture was 56 minutes with 92% (13/14) drawn within 1 hour of fever. Mean time to first antibiotic was 4.58 hours. Antibiotics were initiated in 7% (1/14) in less than 1 hour and 50% (7/14) within 3 hours. Urine culture was checked 78% and chest X-ray was checked 100% of the time in case of fever. On review of 57 patient admissions, labs were checked every single day of hospital stay in 35% (20/57) and if the day of discharge was excluded in 50% (29/57) of the patients. Overall, the labs were checked 82.33 percent of the days patients were hospitalized. On average, labs were drawn 1.14 times per day on each patient.

Conclusion: Our project addresses safety, efficiency and timeliness with identification of areas of improvement in febrile sickle cell disease patients. It also addresses patient-centeredness raising awareness for decreased needle sticks for our sickle cell disease patients. There is continuing need to educate our care providers on how to manage fever in sickle cell disease for appropriate work up and early initiation of antibiotics. Also, we need to be more considerate when ordering labs on our patients especially sickle cell disease patients with baseline anemia. This will lead to decreased number of needle sticks and iatrogenic anemia as well as better healthcare resource utilization.

Disclosures

No relevant conflicts of interest to declare.
SOX17 Is Essential for Integration of Arterial and HOXA Programs in Hemogenic Endothelium
Authors: Jung Ho Sun , Uenishi Gene I. , Park Mi Ae , Liu Peng , Raymond Matthew , Thomson James , Ong Irene , Slukvin Igor
Published: November 2019

Recent advances in understanding the major bottlenecks in derivation of engraftable HSCs and lymphoid cells from pluripotent stem cells (PSC), have identified deficiencies in NOTCH and HOXA signaling as contributing factors to the observed functional deficits of PSC-derived hematopoietic progenitors. However, little is known about the mechanisms that are essential for establishing these pathways during PSC differentiation. Here, we revealed the critical role of SOX17 in linking HOXA and NOTCH-mediated arterial programs in hemogenic endothelium (HE) and specification of definitive lympho-myeloid hematopoiesis. Using SOX17-knockout (SOX17-/-) and SOX17 DOX-inducible (iSOX17) hESCs, we found that SOX17-deficiency substantially reduces formation of CD144+CD43-CD73-DLL4+CXCR4+/- arterial HE and definitive lympho-myeloid hematopoiesis, while SOX17 upregulation at mesodermal stage of development causes the opposite effect. Molecular profiling of HE generated from iSOX17 hESCs in DOX+ and DOX- conditions using RNAseq, SOX17 ChIPseq and ATACseq, revealed that SOX17 overexpression upregulates 522 genes enriched in NOTCH, TGFb, HEDGEHOG and WNT signaling, including DLL1, DLL4, NOTCH4, LFNG, WNT5a, WNT5b, GLI3, and genes associated with HSC development, CXCR4,KITLG and ALDH1A2. In addition, we noted significant upregulation of HOXA7,HOXA9, HOXA10, HOXB8, HOXC4 and CDX2 homeobox genes in SOX17-induced cultures, with no expression of HOXA genes observed in HE from SOX17-/- cells. ChIPSeq analysis revealed DOX+ specific SOX17 binding at transcriptional start sites (TSS) of 316 significantly upregulated genes, including ALDH1A2, CDX2, DLL1, DLL4, HEY1, HOXA7, HOXB8, HOXC4 and KITLG, suggesting that upregulation of these genes could be explained by their direct activation by SOX17.

Since ALDH1A2 and CDX2 are known to play a role in the activation of HOXA genes, we investigated whether SOX17's effect on HOXA expression could also be mediated by ALDH1A2 and CDX2. We found that adding ALDH1 inhibitor to DOX+ cultures had no effect on arterial HE development and HOXA expression. In contrast, transfection of iSOX17 hPSCs cultures with CDX2 shRNA significantly decreased arterial HE formation and downregulated HOXA7, HOXA9, and HOXA10 expression. Overall, our studies indicate that SOX17 plays a critical role in the activation and integration of arterial and HOXA programs in HE, which is mediated by CDX2. These findings will be important for designing a strategy for direct HSC fate programming from hPSCs.

Disclosures

Uenishi: Casebia Therapeutics: Employment. Slukvin:Cynata Therapeutics: Consultancy, Other: Founder and Stockholder.
Tumor Suppressor Function of Dnmt3b in Hematologic Malignancies Critically Depends on Its Catalytic Activity
Authors: Lopusna Katarina , Nowialis Pawel , Haney Staci L. , Abraham Ajay , Opavska Jana , Opavsky Rene
Published: November 2019

DNA methylation regulates gene transcription and it is involved in various physiological processes in mammals including development and hematopoiesis. It is catalyzed by several DNA methyltransferases, including DNMT1, DNMT3A and DNMT3B that not only mediate methylation but also interact with a number of repressive proteins, thereby contributing to transcriptional repression in methylation-independent manner. Dnmt3b is critical gene in mouse embryogenesis whose loss results in embryonic lethality around E13.5. It also functions as a tumor suppressor in lymphoid and myeloid malignancies in mice. The extent to which Dnmt3b's catalytic activity is involved in these processes is unclear.

To address the role of catalytic activity in embryogenesis and tumorigenesis, we generated mice carrying a catalytically inactive allele of Dnmt3b (Dnmt3bCI) by introducing a double amino acid substitution in enzyme active center (P656V; C657D) in endogenous Dnmt3b locus. By interbreeding Dnmt3b+/CI mice we found that homozygous mutant Dnmt3bCI/CImice survived embryonic development and had a long life span. Such results demonstrated that catalytic activity of Dnmt3b is, surprisingly, dispensable for both pre- and post-natal development. The presence of catalytically inactive Dnmt3b rescued most methylation changes observed in the absence of Dnmt3b in embryogenesis suggesting that Dnmt3b plays a role of accessory protein for methylation mediated by other Dnmts. In contrast, the absence of Dnmt3b's catalytic activity promoted MYC/Ras-induced transformation of mouse embryonic fibroblasts in vitro, and accelerated both MYC-induced T-cell lymphomagenesis and MLL-AF9-induced acute myeloid leukemia. Global gene expression and methylation profiling revealed a number of deregulated events specific for MYC;Dnmt3bCI/CI, resulting in aberrant activation of IL7-R and STAT3 and downregulation of putative tumor suppressor genes including Cdkn2c and Dusp6.

Altogether, our data suggest that Dnmt3b's methyltransferase activity is dispensable for mouse development but critical to prevent hematologic malignancies by controlling events involved in cellular transformation.

Disclosures

No relevant conflicts of interest to declare.
Manufacture and In Vivo Support of Gene-Edited, Regulatory-like, T-Cells (edTreg) Using a Synthetic IL2 Receptor
Authors: Uenishi Gene I. , Mallari Cornell , Gamboa Luis , West Samuel , Jacobs Chester , Dahl Noelle , Boucher Greg , Chin Samantha , Aeran Rangoli , Wodziak Dariusz , Gebremeskel Simon , Vo Karen , Ito Cade E. , Smith Laura , Patel Chandra , Abe Keith , Stankovich Basha , Sommer Karen , Torgerson Troy R. , Rawlings David J. , Scharenberg Andrew M. , Cost Greg J.
Published: November 2019

Regulatory T-cells (Treg) depend on signals from IL2 and their endogenous T-cell receptors to survive, proliferate, and maintain suppressive activity. We have developed a strategy for engineering effector CD4 T-cells into edited, regulatory-like T-cells (edTregs) for treatment of autoimmune disease. edTregs contain a rapamycin-activated synthetic IL2 receptor (designated a chemical-induced signaling complex or CISC) for use in selective cell manufacturing and in vivo trophic support. Our strategy uses gene editing to integrate an MND/ CISC expression cassette into the native FOXP3 locus, resulting in the CISC system cis-linked to expression of the native FOXP3 gene. The resulting high-level and stable expression of FOXP3 converts peripheral T-cells to edTregs with immunosuppressive activity. Cis-linked expression of the CISC system allows for selective cell expansion and in vivo support using rapamycin. Using an optimized protocol, we obtained efficient HDR rates across cells from multiple healthy donors. Edited cells were enriched to >90% purity and expanded 20-50-fold over a 10 day period of culture in rapamycin. Importantly, we demonstrated sustained in vivo suppressive activity of edTregs in a xeno-GvHD mouse model, and successful trophic support of these edTregs by rapamycin (via the CISC) in the absence of effector CD4 T-cells. Along with preliminary data showing successful editing of CD4-positive T-cells from auto-immune disease patients, our data provide pre-clinical proof-of-concept data supporting clinical use of CISC-edTreg in conjunction with rapamycin support for therapy of autoimmune disease.

Disclosures

Uenishi: Casebia Therapeutics: Employment. Mallari:Casebia Therapeutics: Employment. Gamboa:Casebia Therapeutics: Employment. Boucher:Casebia Therapeutics LLc: Employment. Chin:Casebia Therapeutics LLc: Employment. Aeran:Casebia Therapeutics: Employment. Wodziak:Casebia Therapeutics: Employment. Gebremeskel:Casebia Therapeutics: Employment. Vo:Casebia Therapeutics: Employment. Ito:Casebia Therapeutics: Employment. Patel:Casebia Therapeutics: Employment. Abe:Casebia Therapeutics: Employment. Stankovich:Casebia Therapeutics LLc: Employment. Torgerson:Shire: Consultancy; CSL Behring: Consultancy; ADMA Biosciences: Consultancy; UCB: Consultancy. Scharenberg:Generation Bio: Equity Ownership; Casebia Therapeutics LLc: Employment, Equity Ownership; Alpine Biosciences: Consultancy, Equity Ownership. Cost:Casebia Therapeutics: Employment.
Characteristics of Sickle Cell Clinic Visits and Hospital Admissions in a Large Urban Academic Hospital
Authors: Odikadze Levani , McLemore Morgan L. , Stoyanova Neli , El Rassi Fuad A
Published: November 2019

Introduction: Although high rates of emergency department (ED) visits by sickle cell disease patient population is a significant health-care system burden, there are limited studies available that explore outpatient/ED visit patterns. The purpose of this study was to evaluate the pattern of medical care utilization including clinic visits and hospital admission in adults with sickle cell disease in a large urban academic center with a dedicated 24 hour/7 day a week sickle cell emergency room.

Methods: Patients seen in the Acute Care at Grady Memorial Hospital Comprehensive Sickle Cell Center (GMHSCC) over a three years from January 2015 to December 2017 where analyzed. The Acute Care center at GRMHSCC is a unique 24 hour 7 day a week dedicated emergency room for sickle cell patients. Patient demographics, specialized sickle cell clinic visits and hospital admissions were analyzed descriptively. Independent samples t-test was used for comparison of hospitalization and acute care visits between males/females and phenotype groups.

Results: The total of 1,277 patients were included in analysis representing 12,129 acute care visits. 683 (53.5%) females and 593 males (46.5%). Mean age was 32.9 years. Genotypes where similar to known expected distributions, 878 patients had Hb SS (68.8%), 301 had SC (23.6%), 23 had SB0TH (1.8%) and 74 had SB+TH (5.8%). The mean number of acute care visits over 3 years for SS phenotype was 9.71, SC phenotype 9.74, SB+Th phenotype 5.73 and SB0TH phenotype 10.48. Overall admission rate was 16%. There was a significant statistical difference between the mean number of admissions among SS and SC phenotype patients (p=0.009). Rate of hospitalization per clinic visit for SS phenotype was 18.19% , for SC phenotype 10.78% , SB+TH phenotype was 15.58% and SB0TH phenotype 9.55%;

Conclusions and future objectives: We have demonstrated that a dedicated Sickle Cell emergency room is able to achieve a significantly lower admission rate than standard Emergency rooms. Preliminary analysis shows that the distribution of acute care visits is distributed across the genotypes at the known distribution. In addition there is no difference in mean number of sickle cell acute visits between patients with SS and SC phenotype. This is interesting as Hgb SC is considered a less severe form of the disease. There was a statistically significant higher admission rate for SS patients compared to SC patients.

Future analyses will look at additional parameters associated with acute care visits and admission.

Disclosures

No relevant conflicts of interest to declare.
Statins Enhance Killing of Multiple Myeloma Cells By the BCL-2 Inhibitor Venetoclax and the MCL-1 Inhibitor S63845
Authors: Fruman David , Juarez Dennis , Diep Grace , Nguyen Falisha , Gupta Vikas A. , Boise Lawrence H.
Published: November 2019

The BCL-2 inhibitor venetoclax has begun to change the landscape of therapy for diverse blood cancers. Combining venetoclax with standard of care agents has led to impressive response rates, and increased depth and durability of response compared to monotherapy. However, there is a continuing need to develop safe and effective combinations in multiple myeloma (MM), where the BELLINI triple combination trial (venetoclax-dexamethasone-bortezomib) was closed to accrual due to adverse events. We reported that HMG-CoA-reductase inhibitors (statins), a safe and widely prescribed class of drugs, enhance the efficacy of venetoclax in human leukemia and lymphoma cell lines and in a retrospective study of patients with chronic lymphocytic leukemia (JS Lee et al., Sci. Transl. Med.2018, 10(445)). In MM, the subset with t11;14 translocation is BCL-2-dependent and sensitive to venetoclax, whereas other MM subtypes are more dependent on MCL-1. Here we show that statins sensitize MM cell lines and primary cells to killing by either venetoclax or the MCL-1 inhibitor S63845. The compound pitavastatin is particularly potent, active in vitro at a concentration (100 - 300 nM) that is clinically achievable. In OPM2 cells, pitavastatin synergizes with venetoclax and increases apoptosis in triple combination with dexamethasone. Pitavastatin also potentiates venetoclax killing in dexamethasone-resistant MM1R cells. In MCL-1-dependent MM cells, statins increase sensitivity to S63845. Notably, statins do not increase killing of normal human lymphocytes by either venetoclax or S63845. Statin responsiveness in MM cell lines correlates with p53-independent upregulation of PUMA, BAX and BAK, and gene editing experiments suggest that all three contribute to the apoptotic mechanism. Statin-induced apoptosis and PUMA expression are rescued by addition of geranylgeranyl-pyrophosphate, and recapitulated by an inhibitor of geranylgeranyl-transferase I. Together these findings establish a mechanistic framework to explain how statins promote apoptosis by BH3 mimetic drugs like venetoclax and S63845. Further, these results highlight the potential of statins to enhance therapeutic efficacy of BCL-2 and MCL-1 inhibitor regimens in MM, with possibly lesser toxicity than other combination strategies.

Disclosures

Boise: Genentech Inc.: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Honoraria, Research Funding.

OffLabel Disclosure:

Venetoclax is a BCL-2 inhibitor that is approved for some indications. I will discuss the use of venetoclax for multiple myeloma, a disease for which venetoclax is not approved.