bluuebunny/crossref_metadata_2025 huggingface dataset:
Experimental Study on Construction of Human Hiwi Adenovirus Vector and Apoptosis of K562 Cells Induced by Transfected Vector
Authors: Dong Xiaoyan , Jiang Yanfang , Liu Mengmeng , Li Wei , Liu Ziling
Published: October 2019

Abstract 4721

Background:

Leukemia stem cells are responsible for the genesis, progression, drug resistance and relapse of leukemia. Hiwi gene is an important divisive regulatory factor, which plays a role in maintaining the resting stage and down-regulating the cell cycle of stem/progenitor cells. We acquired human overall Hiwi coding region genes and constructed an adenovirus vector carrying human Hiwi with fluorescin. This study will not only establish the foundation of the further study in function and mechanism of Hiwi induce the differentiation and apoptosis of leukemia stem cells, but also provide the theoretical basis for searching new therapeutic target and method for leukemia.

Methods:

Using the overlap extension PCR method to amplify overall Hiwi coding region genes and insert overall Hiwi coding region genes into Flag-IRES –hrGFP vector that carrying green fluorescin with Gateway clone technology to construct pDown-Hiwi-3×flag-IRES-hrGFP. After transform the vector to Stb13, we screen the positive clone with PCR method, extract the plasmid and process recombination reaction between pDown-Hiwi-3×flag-IRES-hrGFP clone vector and pAV.Des1d expression vector to abtain the pAV.Ex1d-Hiwi-3×flag-IRES-hrGFP adenovirus vector recombinant. Screening the positive clone with PCR and extracting the plasmid which were digested by enzyme and then examined by sequencing consequences. At last packaged Ad-Hiwi-3×flag-IRES-hrGFP adenovirus vector recombinant is obtained. Transfect the recombinant adenovirus vector to K562 cells, and then identify the function of the cells.

Results:

Successfully clone human overall Hiwi coding region genes with the technology of overlap extension PCR and construct the adenovirus vector recombinant with enzyme cutting identification and gene sequencing examination.

Conclusions:

Successfully construct Ad-Hiwi-3×flag-IRES-hrGFP adenovirus vector recombinant and establish the foundation for the further study of Hiwi gene in leukemia stem cells. The transfected recombinant adenovirus vector can induce K562 cells to apoptosis. [U1]É¾3ý£z

Disclosures:

No relevant conflicts of interest to declare.
Results of Radiation Therapy for Primary Extranodal Lymphoma of the Head and Neck: A Report of Case Series
Authors: Turaka Aruna , Tianyu Li , Cohen Adam D , Pro Barbara , Millenson Michael , Robu Valentine , Al-Saleem Tahseen I. , Smith Mitchell R
Published: October 2019

Abstract 4761

Purpose:

Radiation therapy is an important treatment modality for the extra-nodal lymphoma (ENL) of the head and neck. Intensity Modulated radiation therapy (IMRT) has been shown for head and neck cancers to be associated with decreased late side effects like Xerostomia compared with conventional RT techniques (Conv.RT). The purpose of the study is to determine the clinico-pathological features, treatment outcomes and late toxicities in patients (pts) with ENL of head and neck treated with different RT techniques.

Methods:

Retrospective review of records from 2007–2010 identified 14 pts with ENL of the head and neck treated with RT at Fox Chase Cancer Center. Eight were treated with IMRT and 6 with conventional RT technique. Thirteen had CD20+ non-Hodgkin lymphoma [NHL; 7 diffuse large B cell (DLBCL), 3 MALT, 3 others], and one Hodgkin's lymphoma (HL; classic). All pts underwent routine staging work-up with negative bone marrow biopsy for all. Pre and post-treatment PET scans were done in 7 pts. Initial chemotherapy (R-CHOP × 4–6 courses for DLBCL, ABVD × 4 for HL), or no chemotherapy (MALT) was followed by involved field radiation therapy (median RT dose: 36 Gy for NHL, 30 Gy HL, 150– 180 cGy per fraction in 20 fractions over 4 weeks).

Results:

The median follow up was 28 months (range: 1– 52). The median age was 60 years (range: 42–95; 7 males and 7 female). Nine had stage IEA disease, 3 had stage II and 2 stage IV. Five had oropharynx involvement, two had paranasal sinus (DLBCL) 2 nasopharynx (1 DLBCL, 1 MALT) and 1 vallecula (HL). The overall response rate after combined modality treatment was 100%. There were no FDG avid lesion noted on post-treatment PET. There were no local or neck node relapses at last follow up. The 2-year actuarial survival rate was 80%. One DLBCL patient had systemic relapse involving stomach and bilateral testes, was treated with second-line chemotherapy and radiation therapy. At the last follow up, 12/14 were alive. RTOG grade 2 acute skin changes were noted in 6 pts (3 by each RT technique), grade 2 mucositis in 5 pts (3 with Conv.RT) and grade 2 xerostomia in 6 pts. None of the pts developed grade 3 xerostomia (both acute and late). Late grade 2 Xerostomia was seen in 5 pts treated with Conv.RT but not with IMRT.

Conclusions:

RT following chemotherapy or used alone in ENL of head and neck was associated with local and distant disease control in the majority of patients, with a toxicity profile that appears favorable for IMRT compared with conventional radiotherapy techniques. Larger studies are required to confirm that IMRT is as effective as conventional involved-field RT.

Disclosures:

Pro: Celgene: Consultancy, Honoraria.
Healthy People 2020 Blood Disorders and Blood Safety: A Vision of Improved Health for Patients with Sickle Cell Disease, Thalassemias and Hemophilia
Authors: Werner Ellen M. , Brown Lorraine , Hassell Kathryn L. , DiMichele Donna
Published: October 2019

Abstract 4771

In December 2010, the US Department of Health and Human Services released Healthy People 2020 (HP2020) Objectives for the nation. For the first time in its thirty-year history, US health objectives include rare blood disorders and blood safety (BDBS). HP2020 is a public health program that incorporates current clinical evidence and best practices in medicine and public health to identify public health priorities. The objectives are not clinical practice guidelines. Rather, they are scientifically-based, quantified goals to improve the health of specific populations, such as children and older adults. Their development and implementation necessitate partnerships among governmental agencies, organizations, private companies, and medical and health associations at the local, state and national levels. Partners collaborate on implementing activities to reach targeted health objectives for specific populations using educational campaigns, media and other communication venues.

NHLBI led the effort with HRSA, CDC, FDA, and the Office of the Secretary to develop, vet and plan implementation of the BDBS objectives. The development process was based on qualitative research methods such as key informant interviews, semi-structured interviews and categorization of data into patterns. For example, categorization of interview results (“data”) identified specific objective topics, such as screening for complications and penicillin prophylaxis in sickle cell patients aged 4 months to 5 years. Other BDBS Objective topics are: vaccinations; patient and family referrals; care in a medical home; disease-modifying therapies; avoidable hospitalizations; high school completion; community health education; awareness of carrier status; joint health; and alloimmunization in chronic transfusion. http://healthypeople.gov/2020/topicsobjectives2020/default.aspx By sponsoring the BDBS objectives, lead governmental agencies commit to collecting, analyzing and reporting data on progress.

We adopted the default target of a 10% improvement for each objective by 2020. If targets for each objective are met, the question will be: What is the impact of reaching these targets on morbidity, mortality and quality of life for patients with sickle cell disease, thalassemias and hemophilia?

Disclosures:

Hassell: NIH: Research Funding.
Gro 3, Osteoprotegerin and Interleukin 32 Function As Hematopoietic Growth Factors
Authors: Moldenhauer Anja , Futschik Matthias , Lu Huili , Helmig Melanie , Götze Patricc , Bal Gürkan , Zenke Martin , Han Wei , Salama Abdulgabar
Published: October 2019

Abstract 4784

The identification of soluble factors involved in stem cell renewal is a major goal in the assessment of the BM niche. We have previously shown that human endothelial cell (EC) supernatants can induce the proliferation of hematopoietic progenitor cells (HPCs), especially after stimulation with IL-1b. To identify new potential growth factors, we compared the expression profile of IL-1b-stimulated ECs over 4, 8 and 16 h with non-stimulated Ecs using oligonucleotide microarrays covering more than 46 000 transcripts. Most significant changes were detected after 4 h. Utilization of Gene Ontology annotation for the stimulated EC transcriptome indicated multiple upregulated genes encoding extracellular proteins with a cell–cell signaling function. Using flow cytometry, delta, colony and cobblestone assays, we assessed the proliferative capacities of 11 gene products, i.e. IL-8, IL-32, FGF-18, osteoprotegerin, Gro 1–3, ENA78, GCP-2, CCL2 and CCL20, which are not known to induce HPC expansion. Notably, IL-32 and to a lesser degree osteoprotegerin and Gro 3 significantly induced the proliferation of HPCs. Furthermore, IL-32 attenuated chemotherapy-related BM cytotoxicities by increasing the number of colony forming cells in mice (7.4±0.87×103 versus 0.7±0.25×103, p=0.003). Our findings confirm that the combination of microarrays and gene annotation helps to identify new hematopoietic growth factors.

Disclosures:

No relevant conflicts of interest to declare.
RhoH Downregulates Rac Through RhoGAP Family Protein
Authors: Katsumi Akira , Kobayashi Miki , Matsushita Tadashi , Kiyoi Hitoshi , Kojima Tetsuhito , Maruyama Mitsuo , Kaibuchi Kozo , Naoe Tomoki
Published: October 2019

Abstract 4792

Rho GTPases have been widely studied because they are the critical regulators of cellular signaling mediated by G-protein-coupled receptors, tyrosine kinase receptors and integrins. Among more than 20 members of the Rho GTPases, Rac1, Cdc42 and RhoA have been extensively characterized. Rac plays essential roles in the control of actin cytoskeleton organization, migration, phagocytosis, metastasis, transformation, gene expression and cell-cycle progression. Moreover, it has been found that Rac plays as a key molecular switch of hematopoietic stem/progenitor cells engraftment and marrow retention. RhoH, a member of the Rho family, is specifically expressed in the hematopoietic cells, including bone marrow progenitor, differentiated myeloid and lymphoid cells. RhoH antagonizes Rac, however, the molecules that inactivate Rac downstream of RhoH have not been identified. Here we perform GTP-RhoH affinity column chromatography by using platelet lysates. Using triple quadrupole liquid chromatography tandem mass spectrometer, 83 different proteins associated with active RhoH were identified. They include Rho GTPase activating proteins (RhoGAP), serine/threonine kinases, phosphatidylinositol kinases, ERM family proteins and others. Among them, we focused on RhoGAP family. RhoGAP binds to RhoH through its GAP domain. Expression of both full length and GAP domain of RhoGAP effectively deactivates Rac, whereas GAP domain deleted RhoGAP did not. These results suggest that RhoH negatively regulates Rac through RhoGAP.

Disclosures:

Naoe: Kyowa-Hakko Kirin.: Research Funding; Dainipponn-Sumitomo Pharma.: Research Funding; Chugai Pharma.: Research Funding; Novartis Pharma.: Honoraria, Speakers Bureau; Zenyaku-Kogyo: Research Funding; Otsuka Pharma.: Research Funding.
Cord Blood Hematopoietic Progenitor Cell in Co-Culture with Bone Marrow Mesenchymal Stromal Cells: A Synergic Effect
Authors: Almici Camillo , Neva Arabella , Verardi Rosanna , Braga Simona , Di Palma Andrea , Bianchetti Andrea , Russo Domenico , Marini Mirella
Published: October 2019

Abstract 4811

The number of hematopoietic stem and progenitor cells (HPCs) in cord blood units are limited and this can result in delayed engraftment. In vitro expansion of HPCs provides a perspective to overcome these limitations. Different combinations of cytokines as well as mesenchymal stromal cells (MSC) have been shown to separately support HPCs ex vivo expansion, but the combining effects are under evaluation. Data derived from ex vivo co-culture systems using MSC as a feeder layer suggest that cellular contacts could have a significant impact on expansion. We have evaluated the expansion rate of thawed cord blood samples (n=6) in a medium containing SCF (100 ng/ml) and G-CSF (100 ng/ml) plated over a pre-established bone marrow derived MSC layer in comparison to the absence of either MSC layer or cytokines. After 7 days cultures were demi-depopulated. At 14 days of culture adherent and non-adherent cells were harvested, counted and evaluated for antigens expression and clonogenic capacity. Immunophenotypic analysis was performed using CD34-PE, CD38-FITC, CD45-PE-Cy7, CD133-APC. Clonogenic assay was performed in semisolid methylcellulose culture medium (MethoCult, Stem Cell Technologies), CFU frequencies and total CFU numbers per cord blood sample were determined. After 14 days of culture, in the presence of MSC layer, an 11.2-fold increase (range 4.4–18.4) in total number of cells was observed, in comparison to a 4.8-fold increase (range1.1-10.35) in the absence of MSC layer. The presence of MSC layer generated a 4.3-fold increase (range 1.5–7.2) in the number of CD34 positive cells, compared to a 3.3-fold increase (range 0.9–5.7) in the absence of MSC; when considering the more immature CD34+/CD38− subpopulation the corresponding increase were 26.9-fold vs 2.85-fold, respectively. Moreover, the percentage of the CD34+/CD38− subpopulation was higher in the adherent compared to the non-adherent fraction (76% vs 15%). The selection effect given by the MSC layer was confirmed by the presence of hematopoiesis foci growing onto the MSC layer. Our data show that cord blood HPCs can be expanded in vitro, moreover the co-culture on a MSC layer shows a synergistic effect on TCN, CD34+ cells and on more primitive CD34+/CD38− cells. Therefore, a clinical protocol of cord blood HPCs and MSC co-culture could represent a promising approach for improving engraftment kinetics in cord blood transplant recipients.

Disclosures:

No relevant conflicts of interest to declare.
Ex Vivo Expansion of CD34+ Cells From Cord Blood, Bone Marrow, and Mobilized Peripheral Blood Using NANEX Nanofiber Scaffold
Authors: Fischer Stephen L , Fonseca Jacqueline M , Zhao Yukang , Kelley Linda L. , Sakthivel Ramasamy
Published: October 2019

Abstract 4816

Hematopoietic stem cell (HSC) transplantation has become the standard of care for patients with hematologic cancers, anemia, and a variety of other malignant and non-malignant disorders, with greater than 50,000 such procedures being performed globally each year, according to the Worldwide Network for Blood and Marrow Transplantation. Although mobilized peripheral blood (MPB) has become a preferred source of HSCs for transplants, bone marrow (BM) and umbilical cord blood (UCB) are also frequently utilized. Regardless of source, several groups have reported that grafts containing lower total nucleated cell (TNC) and CD34+ cell doses contribute to delayed engraftment and higher graft failure rate. Therefore, methods to increase the total cell number while maintaining the progenitor phenotype, especially the CD34+ progenitor cells, from individual grafts would have a significant clinical impact.

Ex vivo expansion of HSCs prior to transplantation is one approach that offers tremendous promise for increasing cell doses and improving clinical outcomes. In many ex vivo culture systems, HSCs are cultured as a suspension cells and cultured in the presence of various media additives that act to enhance cell proliferation while reducing differentiation. An often-overlooked factor influencing fate decisions is the interaction of HSCs with a substrate. In the natural bone marrow microenvironment, HSCs maintain close contact with a complex network of stromal cells and extracellular matrix, likely indicating that cell-cell and cell-matrix interactions play an important role in maintaining their stem cell phenotype. With the goal of mimicking the bone marrow stem cell niche, Arteriocyte, Inc. has developed a 3-D NANEX nanofiber based cell culture substrate. The functionalized NANEX substrate is designed to provide topographical and substrate-immobilized biochemical cues that act in synergy with media additives to enhance HSC proliferation while maintain the progenitors stem cell phenotype.

Here, we present our recent work with the NANEX platform towards comparing and achieving a high yield ex vivo expansion of CD34+ cells from MPB, BM, and UCB. Additionally, through the use of flow cytometry and CFU assays, we quantify and characterize NANEX-expanded cells from each source. Furthermore, we compared NANEX to a variety of commercially available products and demonstrate that NANEX significantly improves expansion and reduces phenotype loss during ex vivo culture. Our data indicates that NANEX technology provides a robust ex vivo expansion of HSCs and, with further GMP and clinical development, offers great potential for clinical applications.

Disclosures:

No relevant conflicts of interest to declare.
Gender Difference in Periodic Limb Movements in Adults with Sickle Cell Disease
Authors: Roizenblatt Suely , Roizenblatt Marina , Filho Frederico Pollack , Matsuda Sandra S , Mecabo Grazielle , Arruda Martha M A S , Tufik Sergio , Figueiredo Maria Stella
Published: October 2019

Abstract 4843

Periodic limb movements during sleep (PLMS) are commonly described in children with Sickle Cell Disease (SCD), however the relevance of such events in sleep disturbance or complaint of daytime fatigue remain questionable. It has been known that the dopaminergic neurotransmission has a modulator role in pain perception. Since PLMS have been suggested to be a potential biological marker of dopaminergic mechanisms, this study aimed to assess PLMS in adults with SCD and its correlations with clinical and sleep parameters.

Methods:

Seventy adults with SCD (50% females, matched for age and body mass index), underwent Brief Pain Inventory, overnight polysomnogram and laboratorial tests for hemoglobin, reticulocytes, ferritin, transferin saturation, haptoglobin, fetal hemoglobin, lactate dehydrogenase (LDH), and bilirrubins.

Results:

The mean PLMS index was higher in females (16.5±10.7/h vs. 8.7±8.2/h, p< 0.05), with 88.6% of the females having increased PLMS index (≥ 15/h) in comparison to males (22.6), p<0.01. Total arousal index and PLMS arousal index were increased in females with PLMS ≥ 15/h compared to those with PLMS < 5/h (p<0.05, both), but not in males. Females also exhibited correlation of increased PLMS index and clinical parameters such as pain score (rS =0.71), indirect bilirrubin (rS =0.42) and LDH (rS =0.38), p<0.01 in all. An interaction effect of the non-use of hydroxyurea treatment in PLMS abnormality was also observed (p<0.05).

Conclusions:

Elevated PLMS were more common in females with SCD than in males and correlated with pain symptoms and hematological parameters of hemolysis. Hydroxyurea treatment showed a protective effect against the PLMS index increase.

Supported by AFIP, CEPID-FAPESP 98/14303-3, and CAPES/SUS.

Disclosures:

No relevant conflicts of interest to declare.
A Study of Chronic Hydroxyurea Administration in Sickle Cell Patients
Authors: Koutsouka Freideriki , Makis Alexandros , Lagos Konstantinos , Pliakou Xanthi , Bourantas Dimitrios , Kapsali Eleni , Briasoulis Evangelos
Published: October 2019

Abstract 4857

Background.

The hemoglobin F (HbF) promoting agent hydroxyurea (HU) has been established as an effective therapy for adults with sickle cell disease (SCD) who develop frequent pain episodes, acute chest syndrome, severe vaso-occlusive events, or severe symptomatic anemia. However, concerns have been raised for long-term safety issues of HU in relation to its cytotoxic effects, which are attributed to ribonucleotide reductase inhibition. We analyzed the clinical effects of chronic use of HU in adult SCD patients treated in a referral Academic center.

Methods.

Medical files of 29 adult patients (10 male, median age 42 years, range 29–68 years) followed at our Thalassemia Unit for the last 17 years were retrieved, and detailed data were recorded and analyzed. Four patients had homozygous SCD and 25, sickle cell/beta thalassemia.

Results.

The median follow up time was 10 years (range 3–17 years), the total follow-up time was 289 patient-years and the total HU exposure period was 308 patient-years. The mean dose of HU administered was 29.3 mg/kg/day. The main clinical outcomes of HU therapy are shown in Table 1. In terms of efficacy, HU induced marked increase of HbF and substantial reduction of painful crises and transfusions needed, all outcomes designated statistically significant. HU therapy was also associated with significant increase of hemoglobin (Hb) levels, as well as with reduction of serum lactate dehydrogenase levels (LDH) and white blood cell count (WBC). In terms of toxicity, one poor HU compliant female patient died of pulmonary embolism. Disease complications observed during the long follow up time were: pulmonary hypertension in 2 patients, leg ulcers in one patient and renal impairment in one patient. Seven patients discontinued HU therapy (mean treatment duration 8.4 years) because of scheduled pregnancy (3 patients), severe neutropenia (2 patients) and non-compliance (2 patients). No patient developed cancer.

Conclusions.

This retrospective study, which analyzed data of most prolonged administration of HU, provides data supporting the efficacy and safety of chronic administration of HU in SCD adults.

Disclosures:

No relevant conflicts of interest to declare.
Paraneoplastic Pemphigus (PNP) As a Presenting Finding in a Patient with Hodgkin's Lymphoma
Authors: Marjon Philip , Kim Byung , Lazic Tamara , Cernik Christina , Rathore Ritesh
Published: October 2019

Abstract 4864

Introduction:

We report a case of PNP in a patient who was subsequently diagnosed with classical Hodgkins Lymphoma mixed cellularity type. PNP is an autoimmune mediated blistering disorder that presents with skin lesions demonstrating characteristics of erythema multiforme and pemphigus vulgaris.

Case:

A 76 year-old Hispanic male presented to the Emergency department with intermittent low fevers of 4 months duration associated with progressive weakness and fatigue; he now had mouth ulcers of 1-week duration along with anorexia. Physical exam was significant for a temperature of 102.4F, bilateral conjunctivitis with mild white discharge. Labs included WBC 5,000/mm3, hemoglobin 9.5 gm/dL, platelets 99,000/mm3; differential showed lymphocytes 22%, monocytes 11%, eosinophils 3%, and myelocytes 9%. Oral examination revealed dry mucosa with bleeding perioral ulcers. An ulcer biopsy was obtained and demonstrated necrotic keratinocytes and necrotic neutrophilic inflammation consistent with PNP. Chest CT showed a multinodular goiter, enlarged mediastinal and hilar nodes with calcified granulomas bilaterally as well as a new noncalcified nodule in the right lung. PET scan revealed FDG avid lymph nodes in the left supraclavicular, hilar and left axilla areas as well as multiple regions in the spleen and retroperitoneal nodes. A left axillary node biopsy showed classical Hodgkins lymphoma of mixed cellularity type. The patient was started on prednisone 75 mg per day in divided doses and his oral ulcerations and clinical status improved after 2 days of therapy. Subsequently the patient was started on gemcitabine, vinorelbine and liposomal doxorubicin (GVD) chemotherapy for his malignancy.

Discussion:

Although PNP has been described as being associated with hematologic malignancies such as Non-Hodgkins Lymphoma, Chronic Lymphocytic Leukemia and Castleman's disease, it has rarely been demonstrated in association with classical Hodgkins disease. This case report highlights the need for a higher index of suspicion of underlying malignancy including classical Hodgkins disease when confronted with a patient presenting PNP.

Disclosures:

No relevant conflicts of interest to declare.
Hodgkin's Lymphoma (HL) Presenting As An Abnormal Mammogram: A Case Report
Authors: Le Kim , Lorenzo-Medina Mercedes , Friedman Paul , Dane Langsjoen , Noble Luis , Richards Matthew , Arango Carmen , Gupta Anuradha , Campos-Gines Angel F , Landeros Mark M , Herrada Juan
Published: October 2019

Abstract 4865

Background:

Although not a common presentation, Hodgkin's lymphoma (HL) can first manifest as an enlarged axillary lymph node. We report a case of a patient diagnosed with nodular sclerosing HL, presenting as an abnormal axillary lymph node incidentally noted during routine mammogram.

Case report:

A 79 year-old asymptomatic female was referred to our center after being discovered a 3 cm left axillary lymph node detected during a routine screening mammogram. Except from the enlarged left axillary lymphadenopathy, the remaining physical exam was within normal limits. An ultrasound-guided fine needle biopsy revealed histologic features consistent with Hodgkin's lymphoma, nodular sclerosing type. Immunostains confirmed the presence of Reed Sternberg cells that were positive for CD 30, focally positive for CD 15, and negative for CD 20 and EMA. Bilateral bone marrow showed no evidence of HL involvement. A positron-emission tomography detected abnormal uptake in multiple enlarged lymph nodes above and below the diaphragm, and in the thoracic spine, at T9, T11 and T12 vertebral body levels. The patient was successfully treated with doxorubicin-based combination chemotherapy regimen.

Conclusion:

There are multiple clinical presentations for Hodgkin's lymphoma. Routine mammograms can aid in the detection of cancer types other than breast cancer.

Disclosures:

No relevant conflicts of interest to declare.
Metabolism of Human Leukemia Cells in NOD/Scid Mice: Effectiveness of 2-Deoxy-D-Glucose
Authors: Tsunekawa Norikazu , Miwa Hiroshi , Mizuno Shohei , Gotoh Mineaki , Takahashi Miyuki , Goto Mayuko , Suganuma Kazuto , Hanamura Ichiro , Shikami Masato , Nitta Masakazu
Published: October 2019

Abstract 4881

We have recently shown that NB4,an APL cell line, is dependent on glycolysis, while THP-1, a monocytic cell line, is relatively dependent on oxidative phosphorylation in mitochondria, where oxidation of fatty acid is important. (Leukemia and Lymphoma 51:2112,2010)

Here, we examined the energy metabolism of these leukemia cell lines in immunodeficient mice. 1.0×107 cells of each cell line were inoculated subcutaneously in NOD/scid mice with the treatment of anti-asialo GM1 antibody. Mice were divided into two groups: normal diet (carbohydrate;65.3%, fat;6.3%) or carbohydrate-restricted (high fat) diet (carbohydrate;19.6%, fat;62.2%). At day 42, tumor volume (TV) of NB4 in three normal diet-fed mice became 694±358mm3, which was greater than that in three high fat diet-fed mice:173±216mm3 (P= 0.043). On the other hand, tumor volume of THP-1 in three normal diet-fed mice became 1130 ± 600mm3, which was smaller than that in three high fat diet-fed mice: 3300 ± 1053mm3 (p = 0.050).

Then, since 14 days after inoculation of leukemia cells in NOD/scid mice fed with normal diet, glycolysis inhibitor 2-deoxy-D-glucose (2-DG) (35mg/mouse) was administered intraperitoneally once a week. Tumor size at day 41 of both leukemia cells were suppressed by 2-DG treatment. (THP-1: 2-DG (-): TV = 3099 ± 193mm3, 2-DG (+): TV = 513 ± 550mm3) (THP-1: p = 0.040). (NB4: 2-DG (-): TV = 495 ± 417mm3, 2-DG (+): TV = 87 ± 89mm3) (NB4: p = 0.313).

Pathological examination of tumors have shown that 2-DG treated tumor had larger areas of dead cells in both cell lines.

Our study demonstrated that NB4 is dependent largely on glucose and THP-1 on fat. In addition, 2-DG is effective in growth suppression of leukemia cells in vivo. These findings will help the future therapy for leukemia targeting energy metabolism.

Disclosures:

No relevant conflicts of interest to declare.
Analysis of the Clinical Features and the Prognosis of Acute Lymphoblastic Leukemia with Complex Chromosomal Aberrations
Authors: Ye Xiujin , Zhu Lixia , Li Li , Zhang Jie , He Jingsong , Zhu Jingjing , Huang He , Cai Zhen
Published: October 2019

Abstract 4914

Background:

Acute lympoblastic leukemia (ALL) with complex chromosomal aberrations (CCAs) which patients harboring three or more acquired chromosomal aberrations always have a poor outcome. Philadelphia chromosome–positive (Ph+) ALL is the largest genetically defined subtype in adult ALL with the most unfavorable prognosis. It is necessary to investigate the clinical and laboratory features of ALL patients with CCAs, analyze the distribution of Ph+ in these patients and assess the prognosis.

Methods:

In a retrospective follow-up study from 2005.1∼2011.6, 38 ALL patients with CCAs were investigated. All cases were diagnosed with morphological,immunological,cytogenetic classification and molecular biology. The karyotypes were interpreted according to the International System for Human Cytogenetic Nomenclature (ISCN). Clinical characteristics, laboratory features, treatment response and prognosis of ALL patients with CCAs were analyzed.

Results:

The study included 17 Ph+ ALL patients with an age range of 12 to 81 years (mean, 34.5 years) and a male to female ratio of 1:1. Ph+ ALL patients with CCAs were older than Ph- cases (P=0.096). The median survival time was 9.5 months (range, 1–44 months). The complete remission (CR) rate after two cycles of systemic chemotherapy was 53.6% (15/28) and the relapse rate was 53.3% (8/15). Ph+ ALL patients with CCAs showed a lower CR rate than Ph- cases. Eleven patients died during the follow-up period, six of them died within 2 months from the initial ALL diagnosis, and nine patients were failure to achieve CR. Three patients underwent allogeneic bone marrow transplantation (BMT): two of them presented Ph+ relapsed after 2 and 10 months, respectively. Using Kaplan-Meier survival analysis, we found the survival outcome of Ph+ ALL patients with CCAs is inferior to Ph- cases.

Conclusion:

Treatment outcomes of ALL patients with CCAs receiving chemotherapy or BMT are very poor. Ph+ as an unfavorable parameter influences on the CR rate and survival outcome. It is necessary to accumulate more clinical data to find innovative treatments that can improve the prognosis of this refractory leukemia.

Disclosures:

No relevant conflicts of interest to declare.
A Multicenter, Randomized, Open-Label Study to Evaluate Safety and Efficacy of Posaconazole Vs Fluconazole in the Prophylaxis of Invasive Fungal Infection in a Chinese Population
Authors: Yang Shen , Huang Xiaojun , Wang Jianxiang , Jin Jie , Hu Jianda , Yu Kang , Wu Depei , Wang Shujie , Yu Li , Chen Xiequn , Liu Ting , Liang Yingmin , Fangping Chen , Li Yan , Shen Zhixiang
Published: October 2019

Abstract 4916

Background

Invasive fungal infection (IFI) is a common and fatal complication in neutropenic patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS).

Methods

In this multicenter, randomized study, we enrolled patients with persistent neutropenia resulting from chemotherapy for the treatment of AML and MDS. Prophylaxis with either posaconazole or fluconazole was given to the patients to compare the efficacy and safety of the 2 drugs. The treatment was given with each cycle of chemotherapy until the patient recovered from neutropenia and had complete remission, an IFI occurred, or until the patient had received a maximum of 12 weeks of treatment. The primary end point was the incidence of possible, proven, or probable diagnosis of IFI during the treatment phase in *the 2 groups; clinical failure rate, all-cause mortality, and time to first systemic antifungal treatment were secondary end points.

Results

A total of 252 patients were included in this study, and 7 patients were excluded due to withdrawal of informed consent or deviation of eligible criteria. Finally, 124 patients from the posaconazole group and 121 patients from the fluconazole group were included; and 234 patients (117 in each group) entered into statistical analysis, respectively. The incidence of proven, probable, and possible IFI was 9.4% (11/117) and 22.2% (26/117) in the posaconazole and fluconazole groups, respectively (P = 0.0114). The clinical failure rate was lower in the posaconazole group (31.6% [95% CI: 23.3–40.9], 37/117) than in the fluconazole group (41.88% [95% CI: 32.8–51.4], 49/117); however, statistical significance was not reached (P = 0.168). The patients receiving posaconazole had a later onset of first systematic antifungal therapy than those receiving fluconazole (P = 0.0139). The most common clinical adverse reactions were liver function abnormalities, with 11 cases (9.4%) in the posaconazole group and 6 cases (5.1%) in the fluconazole group (P = 0.221).

Conclusions

Posaconazole demonstrates efficacy as prophylaxis against IFI in high-risk neutropenic patients with AML and MDS undergoing chemotherapy and is well tolerated during long-term use. (ClinicalTrials.gov number, NCT00811928)

Disclosures:

No relevant conflicts of interest to declare.
Using Auto Dendritic Cells Accompanied with CIK Plus Chemotherapy As Consolidation Treatment for 59 Patients with Acute Leukemia
Authors: Zheng Zhuanzhen , Qiao Zhenhua , Chen Wenliang , Gong Rong , Wang Yalin , Xu Lianrong , Ma Yanping , Hou Lihong , Zhang Li , Lu Yujin , Jiang Bo , Li Guoxia , Dong Chunxia
Published: October 2019

Abstract 4923

Objective

To explore the safety and clinic therapeutic effects of using tumor activated dendritic cells (DC) accompanied cytokine induced killer (CIK) in order to feinforce opposing anti-tumor function and to eliminate minimal residual disease.

Methods

59 patients entered into DCIK plus chemotherapy consolidation teams as achieved the 1st complete remission and after 1 or 2 cycle themotherapy. Collect 56 case's bone marrow monocyte cells at diagnosising to prepare tumor antigen, Collect bone marrow monocyte cells at complete remission to culture DC and CIK, using tumor antigen to activate DCs. After 7 days culturing separately then co-culture together for 1 day, collect these cells as DCs activated CIK, after scrubbing, infusing back to patients. Then these patients accept 10–15 day's immuno-regulation treatment, such as interleukine-2(IL-2) 500000u/d for one week or interferon -α(IFN-α)3,000,000u/d three times one week or thymopentin 1mg/d,2 times every week for two weeks. Those cases (one case AML-M3a,one case ALL-B,one case AML-M4a)only have routine culturing and infusion of DCs and CIK and the DCs are not activated by tumor antigen because of having no conservation leukemic cells.

Results

59 cases accept DCIK immuno-therapy for 6 to 16 times respectively, in the 24 to 78 months observation stage, 42 cases OS(overall survival)and 33 cases DFS(disease free survival). No one developed therapeutic-dependent auto-allergic disease, such as fever, swollen, diarrhea, rash et al. Those three cases who accepted routine culturing and infusion of CIK and DCs that had not activated by tumor antigen didn't occure any treatment-dependency side-effects and keep hematologic complete remission, minimal residual disease test outcome persist lower under 103.

Conclusion

Tumor antigen activated DCs combined with CIK can reinforce anti-tumor immune function and have no obviously side-effects,accompanying with chemotherapy,those patients maybe benefit of this kind consolidation treatment and avoid routine chemo-therapeutic side reaction, temporary curative effect are satisfaction and worth popularized.

Disclosures:

No relevant conflicts of interest to declare.
NR4A1 Mediated Apoptosis of Aggressive Lymphoma Cells Suppresses Tumor Growth in a Xenograft Model.
Authors: Deutsch Alexander JA , Rinner Beate , Pichler Martin , Reitter Sonja , Beham-Schmid Christine , Neumeister Peter
Published: October 2019

Abstract 2408

NR4A1 (Nur77) and NR4A3 (Nor-1) are two members of the orphan nuclear receptors (NRs). Their function as critical tumour suppressor genes (TSG) is demonstrated by the rapid development of acute myeloid leukemia (AML) of NR4A1 and NR4A3 double knock out mouse and by their reduced expression in leukemic blasts from human AML patients. The aim of our study is to comprehensively study NR4A1 and NR4A3 expression B-cell malignancies and to define and functionally characterize the nuclear orphan receptors NR4A1 as TSGs in B-cell malignancies. We found a more than 50% reduction of both, NR4A1 and NR4A3, in B-CLL (71%) and Follicular Lymphoma (70%), and in diffuse large B cell lymphoma (DLBCL) (74%) compared to normal controls. In DLBCL low NR4A1 expression was significantly associated with non-germinal center B-cell subtype and with poor overall survival (p=0.042, HR=2.2, CI=1.01–4.9). To investigate the function of NR4A1 in lymphomas, we over-expressed NR4A1 in a lymphoma cell line (Sc-1) by using an inducible lentiviral expression system and performed apoptotic assays by determing cleaved caspase 3, the sub-G1 peak and Annexin V positivity. Induction of NR4A1 expression led to apoptosis in a significantly higher proportion of induced Sc-1 cells compared to their uninduced controls in all assays analysed. Additionally, treatment of an immortalized B cell line (UH3) and three lymphoma cell lines (Karpas422, SC-1 and Ly8) with Cytosporone B (Csn-B), a NR4A1 ligand known to induce NR4A1, caused NR4A1 mediated apoptosis. To test the tumor suppressor function of NR4A1 in vivo, the stably transduced Sc-1 lymphoma cell lines were further investigated in a NOD/SCID/IL-2rγnull (NSG) mouse model. Induction of NR4A1 in Sc-1 suppressed tumor growth in the NSG mice, in contrast to vector controls and uninduced Sc-1 cells, where massive tumor formation was observed. Our data suggest that NR4A1 has pro-apoptotic functions in vitro and that Csn-B induces a NR4A1 mediated apoptosis in lymphoma cells. Our xengraft experiments define NR4A1 as novel tumor suppressor in vivo. Hence, regulation of NR4A1 is a promising new therapeutical target for future anti-lymphomatherapy.

Disclosures:

No relevant conflicts of interest to declare.
Prospective Comparison of Sperm Parameters in SCD Adult Patients Before and after a 6 Months Treatment with hydroxyurea (HU)
Authors: Bachir Dora , Lionnet Francois , Dautremer Justine Gellen , Stankovic Katia , Habibi Anoosha , Bartolucci Pablo , Chalas Céline , Berthaut Isabelle , Eustache Florence , Ravel Célia , Kuntsman Jean Marie , Girot Robert , Simon Tabassome , Mandelbaum Jacqueline , Galacteros Frederic
Published: October 2019

Abstract 242

Introduction

Hydroxyurea (HU) has proved its efficacy in reducing vaso-occlusive events in patients with SCD and increasing life expectancy. However, effects on fertility in adult males represent a major issue for acceptance and adherence, as HU has been reported to impair spermatogenesis through direct cytotoxic effects and hypogonadism. There are only limited data in the literature in this field and mainly retrospective.

Patients and methods

49 adult patients aged 20–52 years with homozygous SCD eligible for first line HU treatment were enrolled after informed consent between June 2010 and April 2012 in this prospective study: HYDREP.The main objective was to compare at Day 0 and 180 of HU treatment the semen parameters according to the WHO (1999) criteria(volume, sperm concentration, viability, forward motility and morphology ). Vaso-occlusive events and other complications, number of transfusions during the 6 months preceding and following HU initiation were recorded. Hematological, biochemical, hormonal parameters, HU dosage were recorded at Day 0, Day 90 and 180. HU was begun at 15mg/Kg dose (Platt *) and modulated following haematological tolerance and renal function.

Results and discussion

Preliminary results concern the 24/49 patients, who complete the 6 months protocol.

A statistically significant impairment in all sperm parameters was observed, after a 6 months HU therapy, with great variations between individuals, which are to be explained by complementary tests. These preliminary deleterious findings must be interpreted with caution as in our experience 1) spermatogenesis may recover after drug withdrawal 2) Repeated vaso-occlusive events are also potentially deleterious on fertility (Berthaut **)and 3)this study must be completed with a survey on effective fertility seen in men treated with HU. Thus, regarding to the great benefit offered by HU on prognosis allowing a real “life project” in young symptomatic adults with SCD, it is worth providing each patient of the most accurate information on fertility as an essential prerequisite for their adhesion to HU treatment.

Conclusion

Sperm cryopreservation is advised before beginning a treatment with HU. Prospective follow up studies of HU male cohort regarding fertility are warranted. Health education has a major role to play to increase HU compliance especially in view of these results.

Disclosures:

No relevant conflicts of interest to declare.
Efficacy Analysis of the Novel Dual PI3K-MTORC1/2 Inhibitors NVP-BGT226 and NVP-BEZ235 in Acute Leukemia.
Authors: Schittenhelm Marcus M , Doehner Hartmut , Doehner Konstanze , Kanz Lothar , Heinrich Michael C. , Kampa-Schittenhelm Kerstin M
Published: October 2019

Abstract 2474

Introduction:

Dysregulation of the PI3Kinase/AKT pathway is involved in the pathogenesis of many human malignancies. Constitutive phosphorylation of AKT is frequently found in acute leukemia but the underlying molecular mechanisms remain unclear and mutations in the PI3K/AKT pathway are uncommon in leukemia. In some cases, constitutive AKT activation can be linked to gain-of function tyrosine kinase mutations upstream of the PI3K/AKT pathway. While inhibitors of the PI3K/AKT pathway appear attractive for tumor therapy, so far response rates to PI3K inhibitor treatment of various neoplasms are moderate. Furthermore, MTORC1 inhibitors, targeting downstream of AKT, have the disadvantage of activating AKT via feed-back mechanisms. We here comparatively studied two novel dual PI3K-MTORC1/2 inhibitors, NVP-BEZ235 and NVP-BGT226, with regard to their ability to inhibit proliferation and to induce apoptosis in different leukemia cell models as well as in primary leukemia samples.

Methods:

Expression of phospho-AKT protein levels was determined in 74 leukemia patient blood and bone marrow samples by flow cytometry. Protein and functional viability assays evaluating the effects of NVP-BEZ235 and NVP-BGT226 were performed in several leukemia cell lines, mutant-tyrosine kinase cell models and with primary leukemia blasts and bone marrow cells. Antineoplastic activity was assessed in proliferation and apoptosis experiments. Immunoblots were performed to confirm consecutive suppression of AKT signaling.

Results:

AKT is generally expressed in acute leukemia at significantly higher levels as compared to healthy donor samples (p < 0.05, students t-test). Dual targeting of the PI3K-AKT-MTOR pathway profoundly inhibited AKT signaling, leading to antiproliferative effects in vitro and ex vivo. Moreover, both agents potently induced apoptosis in a subset of leukemia samples with BGT226 being the more effective agent and displaying IC50s at the low nanomolar level. Cell cycle analyses revealed that NVP-BGT226 overrides the G1-arrest observed with NVP-BEZ235-treated cells. Importantly, normal mononuclear cells revealed lower phospho-AKT expression levels and relative insensitivity towards dual PI3K-MTORC1/2 inhibition.

Conclusion:

Our data provide a strong rationale for clinical evaluation of the tested dual PI3K-MTORC1/2 inhibitors in acute leukemias.

Disclosures:

No relevant conflicts of interest to declare.
Native and Fc Enhanced Therapeutic Human Monoclonal Antibodies Targeting the Intracellular WT1 Oncogene Product in Leukemia.
Authors: Veomett Nicholas , Dao Tao , Pankov Dmitry , Yan Su , O'Reilly Richard J. , Liu Cheng , Scheinberg David A.
Published: October 2019

Abstract 2599

The Wilms' tumor oncogene protein (WT1) is an intracellular, oncogenic transcription factor that is over-expressed in leukemias and a wide range of cancers. WT1 may be expressed in leukemia stem cells. RMFPNAPYL, “RMF”, a WT1-derived CD8 T cell epitope presented by HLA-A0201, is a validated target for T cell-based immunotherapy, used in multiple clinical trials of vaccines and cellular therapies. Therefore, we hypothesized that a “TCR-like antibody” specific for the WT1 peptide/HLA-A0201 complex might be an effective therapeutic agent. Using phage display technology, we generated 2 lead, high avidity (Kd < 0.2nM), fully human monoclonal antibodies (mAb) specific for the WT1 RMF peptide/HLA-A0201 complex. One version, ESK1, is a native human IgG1. A second version, ESKM, with enhanced antibody dependent human effector cell cytotoxicity (ADCC) function due to altered Fc glycolsylation was also prepared. ESK mAb bind to leukemia lines and other cancer cell lines, as well as primary leukemia cells that are both WT1+ and HLA-A0201+. In vitro, both ESK mAb mediated ADCC against CML cells, at concentrations below 3 ug/ml, but ESKM was about 8–10 fold more potent and could kill targets with far fewer peptide/MHC complexes expressed on the cell surface. At therapeutic doses of ESKM, there was no difference in biodistribution between the 2 mAb in C57BL6 mice or in mice that were transgenic for HLA-A0201. Low doses of ESK (25–100ug twice) effectively treated an established disseminated, ALL or human bcr/abl + lymphoid leukemias in a NSG mouse model (T, B and NK deficient) and prolonged survival. In mice, ESKM was slightly more effective than ESK1. An F(Ab')2 version of the antibody had no anti-tumor effect, indicating that an Fc-mediated mechanism plays the major role in therapy. When combined with a single infusion of human CD34-, CD3-, human NK and monocyte effectors in the NSG mice, therapeutic effects of the mAb were more pronounced and more durable. There was no therapeutic effect of either mAb on WT1 low/A0201 negative disseminated Daudi ALL in mice. There was no observed toxicity in HLA-A0201 transgenic mice at the therapeutic mAb dose and schedule. ESK mAb are potential therapeutic agents for ALL, CML, other leukemias and cancers over-expressing the WT1 oncoprotein. Its expression in early leukemia cells may allow for elimination of the progenitors. The data also provide proof of concept for developing therapeutic mAb targeting important intracellular oncogenic proteins.

Disclosures:

Yan: Eureka: Employment, Equity Ownership. Liu:Eureka: Employment, Equity Ownership.