bluuebunny/crossref_metadata_2025 huggingface dataset:
Propylene Glycol-Free Melphalan Induces Higher Remission Rates in Multiple Myeloma Patients Undergoing Autologous Transplantation
Authors: Aljitawi Omar S. , Ludlow Anna , Ganguly Siddhartha , Abhyankar Sunil , Lin Tara , Pipkin J.D , McGuirk Joseph
Published: October 2019

Abstract 4551

Background:

In a recent phase IIa open-label, randomized, cross-over design study, we evaluated the pharmacokinetics of Propylene Glycol-Free Melphalan (PG-free Mel) HCL and Alkeran in multiple myeloma patients undergoing transplantation. In this study, we have shown through statistical tests of the geometric means of AUC that PG-free Mel was bioequivalent to Alkeran, and provided marginally higher blood drug levels.

Hypothesis:

Because this new formulation is associated with marginally higher blood drug levels and because melphalan is associated with a linear dose-response curve, we speculated that patients treated on study would have a better multiple myeloma response compared to matched controls.

Goal:

To assess day +100 multiple myeloma responses from the study patients (n=24) receiving PG-free Mel conditioning in combination with Alkeran. Responses were assessed based on International Uniform Response Criteria for Multiple Myeloma. Comparison was made to a matched cohort of patients who received conventional melphalan.

Methods:

We compared the study patient responses to matched controls (n=24) treated exclusively with Alkeran or generic melphalan conditioning within the same time frame in a case-control study design. The controls were matched to study population based on disease stage, age, and pre-transplant response.

Results:

The study population and the control cohort were well matched in terms of age, gender, disease stage, and pre-transplant response. However, the study population had more patients with high-risk cytogenetics (10 vs 3). Overall response (CR, VGPR, and PR) was higher in the study population (22 vs 19). While no patient on the study progressed within 100 days of transplant, 3 patients in the matched cohort progressed within 100 days. The median duration of follow up of surviving patients was 650 days in the study cohort and 409 days in the matched control cohort. Twenty three study patients and 22 patients in the matched control cohort were reported alive. On the other hand, 7 study patients and 8 patients in the matched cohort progressed during follow up.

Conclusion:

Compared to matched controls, PG-free Mel, as part of the melphalan conditioning regimen, resulted in higher remission rates and less disease progression at day 100 post autologous transplant for multiple myeloma.

Disclosures:

Off Label Use: Propylene glycol free melphalan for high-dose therapy and autologous transplant for multiple myeloma.
Pilot Study: Favorable Outcomes with Addition of Topotecan to Busulfan/Melphalan As High-Dose Myeloablative Therapy Followed by Autologous Hematopoietic Cell Transplantation
Authors: Rosenthal Joseph , Pawlowska Anna , Cheng Jerry , Kechichian Steven , Hitt Debbie , Suarez Mary , Tsai Ni-Chun , Palmer Joycelynne
Published: October 2019

Abstract 4553

High dose (HD) therapy followed by autologous hematopoietic stem cell transplantation (AHCT) has been the mainstay of treatment in patients diagnosed with advanced neuroblastoma (NBL). Busulfan and Melphalan (Bu/Mel) was demonstrated to be superior to other HD regimens and is widely considered as standard treatment. (Ladenstein, Bone Marrow Transplant. 2008 Jun;41 Suppl 2:S118-27), Topotecan (Topo) with or without cyclophosphamide has demonstrated efficacy in treatment of advanced NBL (Kushner, Cancer 116: 3054, 2010). We hypothesize that adding Topo to Bu/Mel as HD therapy followed by AHCT is well tolerated and will result in favorable outcomes.

Patients and Methods:

Patients with NBL, stage III or IV were eligible and consented to participate in the study. Seven patients were enrolled, the median age was 2.65 yrs (range 2.3 – 4.x yrs), and there were 4 males (57%). All patients were initially treated on COG protocols and in all cases a complete response was documented prior to proceeding with AHCT. Autologous hematopoietic cells were collected, following mobilization with G-CSF, after 2 (n=6) or 3 (n=1) cycles of chemotherapy. All patients were given post transplant radiation therapy to the primary tumor bed and cis-retinoic acid, 3 patients were also given monoclonal antibodies. Data on engraftment, toxicities, event free survival (EFS) and overall survival (OS) were analyzed.

Results:

The median cell dose was 6.62 × 106 CD34+/kg (5.57×106−7.33 × 107). Engraftment occurred promptly in all patients. Myeloid and platelet recovery occurred at a median of 12 (8–17 days) and 22 (17–41 days), respectively. The treatment was well tolerated with no grade 4 or higher toxicities. Grade 3 toxicities included; mucositis (n=7, 100%), electrolyte imbalance (n=3), diarrhea (n=3), autoimmune hemolysis (n=1) and epistaxis (n=1). Three patients suffered disease recurrence, 156, 821 and 899 days post AHCT. One patient is dead and two continue salvage therapy 55 and 52 months, post transplant, respectively. The 4-year OS and EFS were 87% to 57%, respectively.

Conclusion:

This novel regimen of Topo/Bu/Mel appears to be well tolerated with low TRM and promising EFS and OS. Further and larger studies are required to establish its role as HD therapy prior to AHCT in patients with advanced NBL.

Disclosures:

No relevant conflicts of interest to declare.
Hematopoietic Stem Cell Transplantation Combined with Imatinib to Treat Philadelphia Chromosome-Positive Acute Lymphocytic Leukemia: A Comparative Research
Authors: Chen Jia , Chen Feng , Depei Wu
Published: October 2019

Abstract 4555

Objective

To compare the effect of the hematopoietic stem cell transplantation with and without imatinib to treat adult patients sufferred from Philadelphia chromosome-positive acute lymphocytic leukemia by evaluating the survival post-transplantation and the quality of life.

Method

35 acute lymphocytic leukemia patients with Philadelphia chromosome-positive have taken hematopoietic stem cell transplantation between 2003 and 2011, in which 23 cases who were treated combined with imatinib were conducted in imatinib group, and the rest cases who haven't utilized imatinib were conducted in the control group. The incidence of relapse, incidence of graft-versus-host disease (GVHD), overall survival (OS) and disease-free survival (DFS) of the groups were compared so as to identify the advantage of combining treatment.

Results

The age, gender, cytogenetic classification, doner type, preparative regimen and counts of stem cells were comparative between the two groups. The proportion of patients who were in the first remission (CR1) in the imatinib group was higher than that in control group, however, the single factor analysis showed that it didn't affect the survival significantly. The incidences of relapse were 17.4% in the imatinib group and 16.7% in the control group (P = 0.9569), and the incidences of acute GVHD of Grade II to Grade IV were 35.3% and 41.7% (P = 0.4506), respectively. The 2-year-OS of two groups showed statistical difference of 62.6% versus 41.7% (P = 0.028), and 2-year-DFS were 53.7% and 33.3% (P = 0.054), respectively. Patients who survived more than 2 years post-transplantation would have a favorable prognosis.

Conclusion

Patients of imatinib group had a better survival, but the incidences of relapses and severe GVHD between the two groups had no significant difference. Thus, patients of Philadelphia chromosome-positive acute lymphocytic leukemia may benefit from the combination of hematopoietic stem cell transplantation and imatinib.

Disclosures:

No relevant conflicts of interest to declare.
Bortezomib-Based Induction Therapy Followed by High-Dose Chemotherapy and Autologous Stem Cell transplantation in Newly-Diagnosed Myeloma: Initial Report From a Chinese Multiple-Center Study
Authors: Hu Jiong , Shen Zhi-Xiang
Published: October 2019

Abstract 4557

Bortezomib-based induction therapy followed by high-dose chemotherapy and autologous stem cell transplantation (ASCT) is considered as standard treatment for patients with newly-diagnosed myeloma. Though front-line treatment of Bortezomib containing regimen is widely used in China, first-line treatment of high-dose chemotherapy with ASCT is not widely accepted for Chinese patients. There is also no prospective study in Chinese population to evaluate the efficacy, safety and feasibility of such treatment strategy. This is a prospective study in young Chinese population (18–65 year old) with newly-diagnosed myeloma (NCT00984828). All patients will receive 4 cycles of Bortezomib and Dexamethasone as frontline therapy. After that, patients will receive either a single ASCT conditioning with melphalan 200mg/m2 (mel 200) or ASCT with mel 200 + bortezomib conditioning and another 3 cycles of Bortezomib monotherapy as consolidation after ASCT. From Jan 2010, a total of 37 patients (median age 54, 31∼64) were enrolled in this study. There were 2 patients died of treatment toxicity 1 and 3 months after entering study. In the remaining 35 patients, 17 already finished ASCT and other 18 patients declined the ASCT. In 23 evaluable patients, 11 achieved CR (n=10) and VGPR (n=1), 9 with PR, 2 with SD and 1 PD. At the last follow-up at May 2012, a total of 6 patients experienced disease progression and no patients died of disease or treatment associated toxicity. As a whole group, with a median follow-up of 10 months (1∼33 months), the estimated 2-year OS and PFS were 94.3±4% and 66.2±12.3% respectively. Though further analysis was impossible with limited number of patient enrolled in the study with short follow-up, overall, the treatment was safe particularly no significant treatment associated toxicity or treatment mortality documented during and after ASCT procedure. The overall 43% CR rate was comparable to most previous reports. The early analysis of the study demonstrated that Bortezomib-Dexa followed by ASCT can be safely performed in Chinese population and further enrolled and follow-up may provide evidence of long-term efficacy of such strategy in Chinese population.

Disclosures:

No relevant conflicts of interest to declare.
CD34+ Marker As Prognostic Factor in Chronic Lymphoid Leukemia
Authors: De Salvo Cardullo Luigi , De Salvo Dalila , Salas Rodolfo J , Hassanhi Manzur , Paz Luis S , Weir Jesus E. , Salas Dalida , Fuenmayor Alba
Published: October 2019

Abstract 4591

From 2008 to date we marked all Chronic Lymphocytic Leukemia (CLL) with monoclonal CD34. The authors consider presence of said marker on the CLL cell membrane is a prognostic added factor to positivity for CD38 and ZAP70. CLL frequency is lower in countries with mixed races, compared with Europe and North America. In Venezuela is 5.4% (Luigi De Salvo personal communication) where I have never diagnosed a CLL case in a pure Venezuelan Indian. Similar reports are documented from Japan and China, provided racial genetics. Since 2008 to date we studied 28 patients for CLL: male 18 and female 10 with CD34-labeled. All patients presented lymphadenopathy, splenomegaly, and hypogammaglobulinemia. Flow cytometry revealed: HLA-Dr (33–100%), CD19 (44–100%), CD20 (28–95%), CD23 (63–100%), CD19 + CD5 (44–100%), CD23 + CD5 (28–100%) and CD19 + CD23 (28–100%) ZAP-70 (31–97%) and CD38 (12–75%). Of 28 patients studied, 21 individual CD34+ presented a variability of 10–99%. All had diffuse bone marrow infiltration, anemia and a white cell count range of 20.000–325,000/dl, with more than 80% lymphocytes of mature appearance. All patients presented discrete thrombocytopenia 70,000–120,000 plt/ml and responded well to standard treatment with Fludara + Rituximab. We believe presence of CD34 antigen on the cell membrane is an added risk factor for patients with CLL, producing an increased angiogenesis, and infiltration of organs and bone marrow by the leukemia cell. Lack of complete remission or negativity of Minimal Residual Disease may be CD34-linked.

Disclosures:

No relevant conflicts of interest to declare.
Silencing c-Myc Using Myc Inhibitor JQ1 and EZH2 Inhibitor DZNep Blocks Converging Survival Signals in Aggressive B-Cell Lymphomas
Authors: Zhao Xiaohong , Lwin Tint , Zhang Xinwei , Huang Andy , Wang Jian , Marquez Victor E. , Chen-Kiang Selina , Bradner James E , Dalton William S. , Sotomayor Eduardo M. , Tao Jianguo
Published: October 2019

Abstract 4618

c-Myc (hereafter Myc) overexpression has been recognized in aggressive B-cell lymphomas and linked to adverse prognosis. Myc activation results in widespread repression of miRNA expression and lymphoma aggressive progression. Our recent study identified a Myc-miRNAs-EZH2 feed-forward loop linking over-expression of Myc, EZH2, and miRNA repression. Here, using a novel small-molecule bromodomain inhibitor, JQ1, to silence Myc and the EZH2 inhibitor DZNep, we demonstrated that combined inhibition of Myc and EZH2 cooperatively disrupted Myc activation, resulting in restoration of miR-26a expression, which subsequently suppressed lymphoma growth and clonogenicity in aggressive lymphoma cells. Furthermore, Myc and EZH2 cooperatively regulated miR-26a expression in aggressive lymphoma cells. MiR-26a functioned as a tumor suppressor miRNA and mediated the combinatorial effect of JQ1 and DZNep. These findings represent a promising, novel approach for silencing Myc with JQ1 for combinatorial therapy of aggressive B-cell lymphomas.

Disclosures:

No relevant conflicts of interest to declare.
Tyrosine Kinase FLT3 and C-KIT Receptor Expression in Patients with Myelodysplastic Syndromes – A Preliminary Study
Authors: Cortesão Emília , Goncalves Ana Cristina , Sousa Isabel , Moucho Carlos , Rito Luís Carlos , Magalhaes Emília , Carda José Pedro , Geraldes Catarina , Sousa Patrícia , e Silva Nuno Costa , Espadana Ana , Pereira Amélia , Teixeira Adriana , Ribeiro Graça , Costa José Nascimento , Ribeiro Ana Bela Sarmento
Published: October 2019

Abstract 4620

Myelodysplastic syndrome (MDS) is a clonal hematopoietic stem cell disorder characterized by dyshematopoiesis and high susceptibility to acute myeloid leukemia.

Deregulated epigenetic mechanisms are likely involved in the pathogenesis of MDS. Gene silencing through aberrant CpG island methylation is the most extensively analyzed epigenetic event in human tumorigenesis and has huge diagnostic and prognostic potential. Aberrant methylation of gene promoter region is responsible for inappropriate gene silencing, and it has been associated to initiation and progression of cancer. However, in the MDS disease process, more and more gene dysfunction has been related with the pathogenesis. FLT3 and c-KIT are important members of the receptor tyrosine kinase family that are overexpress or dysexpress in many malignant hematologic diseases. However, little is known about the distribution and the role of these proteins in MDS.

The study is to investigate the role of receptor tyrosine kinase FLT3 and c-KIT expression in patients with myelodysplastic syndromes (MDS) and their clinical implication.

We have at moment examined c-kit protein (CD117) expression by flow cytometry, in CD34 bone marrow cells collected at diagnosis of 12 patients with de novo MDS and 5 non-neoplastic patients (controls). FLT3 mutations, in particular Internal Tandem Duplications (ITD) and the D835 mutation were analysed by PCR-RFLP. The median age was 72 years (22–89), gender M/F=5/7, WHO subtypes: RCMD (n=6), RA (n=3), RARS (n=1), AREB-2 (n=1), CMML (n=1) and IPSS: low (n=6), intermediate-1 (n=5) and intermediate-2 (n=1). None of the patients evolved to acute leukemia, with a median follow up of 24 months (7–74).

Our preliminary results show an increase in c-KIT expression in CD34 positive cells in MDS patients as compared with controls. However, the percentage of c-KIT protein expressing cells was also higher than in the controls in particular in CD34 negative cells. There was a correlation of the c-kIT protein expression with the CD34 antigen of the cells. Expression is correlated with the WHO MDS classification and with IPSS, being highest in RAEB-2 and INT2 MDS prognostic group. These results suggest that the elevated c-KIT expression could maintain the affected clone in MDS. Besides that we didn't find any FLT3 mutations in our population However further data and refinement of data analysis are needed to confirm our results and to predict clinical outcomes. The preliminary results suggest that c-KIT expression could be helpful to the pathogenesis and prognosis prediction of MDS patients.

Disclosures:

No relevant conflicts of interest to declare.
Towards Cell Therapy for FPD/AML with Patient-Specific IPS Cells
Authors: Connelly Jon P , Horwitz Marshall S , Liu Pu Paul
Published: October 2019

Abstract 4677

Induced pluripotent stem cells (iPSCs) have the potential to differentiate into any cell type. This makes them a valuable source of cells for treating genetic diseases. If the disease causing mutation can be corrected in patient-specific iPSCs and the cells differentiated to therapeutically relevant cell types and transplanted back, the symptoms of the disease may be alleviated or possibly cured. Hematopoietic diseases are an attractive target due to the ability of hematopoietic stem cells (HSCs) to reconstitute the entire hematopoietic system. However, directing the differentiation of iPSCs towards a HSC fate has proven difficult. Our lab aims to (1) Derive iPSCs from patients with a hematopoietic disease and correct the mutant gene through gene targeting. This will provide iPSCs that could be used in treating the patients. (2) Develop methods that direct the differentiation of iPSCs towards a HSC fate. This will be accomplished through novel iPSC reporter lines that we will use in small molecule and RNAi screens to identify factors that direct iPS cell differentiation towards a HSC fate.

Familial Platelet Disorder with propensity to acute myeloid leukemia (FPD/AML) is an autosomal dominant disorder caused by a mutation in the gene RUNX1 resulting in thrombocytopenia, platelet defects, and a predisposition to AML. Our first goal is to derive iPSCs from FPD/AML patients and correct the genetic defect using zinc finger nuclease mediated gene targeting. This will provide a potential source of cells for therapy. Towards this goal, we have obtained fibroblasts from four members of a FPD/AML family and derived iPSCs from two of the individuals. Currently we are correcting the RUNX1 mutation in these iPSC lines using zinc finger nuclease mediated gene targeting. We have developed a pair of zinc finger nucleases that bind and cleave RUNX1, resulting in gene targeting frequencies of 80–90% in 293 cells and iPSCs respectively.

Our second goal is to develop RUNX1 iPSC reporter lines for use in screening small molecule and RNAi libraries to find methods of directing iPSC differentiation to the HSC fate. RUNX1 is a key player in the development of adult hematopoiesis and is expressed at the earliest stages of hematopoiesis. We have targeted fluorescent proteins and a luciferase cassette into the RUNX1 locus of wild type iPSC lines using the RUNX1 zinc finger nucleases at high efficiency. We will use these RUNX1 reporter iPSC lines in screens to identify inducers of RUNX1 expression soon. We believe such inducers have the potential to direct iPSCs towards a HSC fate.

Disclosures:

No relevant conflicts of interest to declare.
Implementation of an End-of-Life Care Pathway in Cancer Patients
Authors: Karthaus Meinolf , Riesle-Keil J , Weber Ursula , Wolf Ingeburg
Published: October 2019

Abstract 4689

Background:

Supportive care for dying hemato-oncological patients in the hospital remains an unmet need. The Liverpool Care Pathway (LCP) has been developed in Europe for patients in the final 24–72 h of life to help physicians and nurses in the end-of-life care (ELC). We evaluated appropriately timed cessation of treatment in a Hemato-Oncology Department after integration of a modified LCP in a tertiary Munich cancer center. The End-of-life Care Pathway was modified to suit local conditions. The aim of the trial was to include over 40% of dying patients in the ELC. The effectiveness of symptom control which included termination of not necessary drugs, sufficient pain relief, control of agitation, bronchial fluid secretion, dyspnoea, nausea and emesis was compared with a control group (CG). The ELC was in accordance to local ethics. Nurses and physicians were trained. The ELC was periodically reviewed by a local multidisciplinary steering group for improvements.

Patients and Methods:

From 01/10 until 06/11 a total of 228 cancer deaths were observed. Criteria for entry onto the ELC were that the multidisciplinary team agreed the patient was dying, and was at least two of the following: bedbound; semi-comatose; only able to take sips of fluid; no longer able to take tablets. 96 pts (41 male/55 female) went on the ELC (42%), while 132 dying cancer pts were not enrolled due to a variety of reasons (e.g. lack of informed consent, rapid deterioration). The mean age was 72,6 ys (range 33 to 91 ys). The median duration of ELC use was 41,3 hours (range 0.5 to 189 h). Six patients improved after they had entered the ELC and left the ELC consecutively. Out of these four patients reentered the ELC and died, one of these six patients died without being reentered on the ELC. Symptom control (ELC/CG in %) was achieved in pain in 92/50%; agitation 89/50%, nausea 96/40%, dyspnoea 92/55% while unnecessary medication was terminated in 95/90%. All patients had current medication assessed and non-essential drugs were discontinued. Inappropriate interventions with antibiotics (85/65%), blood tests (95/75%), iv fluids (90/70%) were terminated more often when pts entered the ELC.

Conclusion:

Integration of an ELC in a hematology unit provides a better symptom control for dying patients. Inappropriate interventions could be reduced. Appropriate template design and supervision are the keys to success for end-of-life care in cancer patients.

Disclosures:

No relevant conflicts of interest to declare.
Serial Imaging of Luciferase Positive Bone Marrow Stromal Cell Migration to Form Radiation Pulmonary Fibrosis
Authors: Goff Julie P. , Dixon Tracy M. , Epperly Michael W. , Sprachman Melissa , Wipf Peter , Zhang Xichen , Greenberger Joel S.
Published: October 2019

Abstract 4734

Thoracic irradiation of C57BL/6 mice leads to an acute reaction phase in the lungs characterized by increased cytokine production and inflammation days 1–14 post irradiation. This is followed by a latent period where inflammation, histologic appearance and cytokine response returns to control levels. The late reaction phase occurs 100+ days post irradiation and is characterized by organizing alveolitis/fibrosis and involves migration of marrow origin macrophages and proliferating mesenchymal stem cells (fibroblasts), a subpopulation which migrates from marrow to the lungs. To quantitate migration in real time, thoracic irradiated mice were either made chimeric for luciferase positive (luc+) whole marrow or were injected with cells from a positive luc+ bone marrow stromal cell line and serially imaged at day 7, 60 or 120 using an IVIS.. 200 Optical Imaging System. As a control for migration to the lung, another group of mice received 20Gy to the right hind leg and 1.5 ×106 luc+ bone marrow stromal cells i.p. Imaging of chimeric mice revealed luc+ cell lung migration only after day 120. C57BL/6NTac female mice that received 20Gy thoracic irradiation followed by an i.p. injection 1.5 × 106 luc+ positive bone marrow stromal cells revealed no migration of luc+ cells to the lungs at day 7 or day 60. Furthermore there was no migration to 20Gy irradiated leg at any timepoint. In marked contrast, at the time of the late reaction phase, at day 100, fibrosis was revealed as an increase in luc+ cell migration in lungs. The lung fibrosis model in C57BL/6 mice combined with live imaging allows sequential measurement of the effect of agents which may alter migration of bone marrow cells that contribute to radiation pulmonary fibrosis.

Disclosures:

No relevant conflicts of interest to declare.
Hematopoietic Stem Cells Have an Intrinsic Expansion Limit
Authors: Rojas-Sutterlin Shanti , Haman André , Hoang Trang
Published: October 2019

Abstract 4749

Hematopoietic stem cell (HSC) transplantation is the first successful cellular therapy and remains the only treatment providing long-term cure in acute myeloblastic leukemia. At the apex of the hematopoietic system, quiescent HSCs are spared by chemotherapeutic treatments that target proliferating cells and therefore can regenerate the entire blood system of a patient after drug exposure. Nevertheless, the consequence of repeated chemotherapy regimen on HSC function remains to be clarified. We previously showed that Scl/Tal1 gene dosage regulates HSC quiescence and functions when transplanted at limiting dilutions (Lacombe et al., 2010). In the present study, we investigate how massive expansion in vivo influences stem cell functions. To address this question, we optimized a protocol based on 5-fluorouracil (5-FU), an antimetabolite that has been used to treat colon, rectum, and head and neck cancers. In addition, we used Scl+/− mice to address the role of Scl in controlling HSCs expansion post-5-FU. We show that within 7 days following 5-FU treatment, HSCs exit quiescence and enter the cell cycle. To deplete cycling HSCs, we injected a second dose of 5-FU and showed that the stem cell pool was disseminated. Nonetheless, the remaining HSCs proliferated extensively to re-establish the HSC pool, which was twice larger than that of untreated mice. At this point, most HSCs have exited the cell cycle and were back to quiescence. Despite a near normal stem cell pool size and a quiescent status, HSCs from these 5-FU treated mice could not compete against untreated cells to regenerate the host in transplantation assays. Furthermore, we show that this extensive proliferation in vivo severely impaired the clonal expansion of individual HSC as measured by the mean activity of stem cell (MAS). Our results demonstrate that HSCs lose their competitive potential after two 5-FU treatments, suggesting that HSCs have an intrinsic expansion limit beyond which their regenerative potential is impaired. In addition, Scl is haplodeficient for cell cycle entry and cell division but Scl gene dosage does not affect this expansion limit. Therefore, our data dissociate the control of HSC expansion under extensive proliferative stress from cell cycle control during steady state. We surmise that chemotherapy regimen based on repeated administration of 5-FU or other antimetabolites are likely to severely impair long-term stem cell functions.

Disclosures:

No relevant conflicts of interest to declare.
Primary CNS Lymphoma in a HIV Negative Population: A Review of Clinicopathologic Characteristics, Therapy, and Outcomes of 59 Patients
Authors: Jaglal Michael V. , Shah Bijal D. , Cultrera Jennifer L. , Sotomayor Eduardo M. , Tomblyn Michael B , Bello Celeste M. , Sokol Lubomir
Published: October 2019

Abstract 4858

Background

Primary central nervous system lymphoma (PCNSL) is a rare aggressive variant of diffuse large B cell lymphoma (DLBCL) with a poor prognosis. Optimal therapeutic strategies have not been defined yet in primary CNS lymphoma. High dose Methotrexate (HD MTX) is an effective chemotherapeutic agent with superior outcomes compared to historical studies using whole brain radiation therapy (WBRT). The combination of HD MTX with WBRT showed improved response rates compared to chemotherapy (CT) alone, but was associated with greater risk of neurotoxicity in patients >60 years old.

Purpose

To review clinicopathologic characteristics, therapy and outcomes of 59 patients with primary CNS DLBCL without HIV.

Methods

This was a single center retrospective review of pts with confirmed diagnosis of primary CNS DLBCL from 1999 to 2012. Data was extracted from the Moffitt Cancer Center (MCC) electronic records. Baseline demographics, clinical, pathological and treatment data were collected and analyzed. Patients were stratified according to their treatment regimens including HD MTX (3g/m2) alone or in combinations and WBRT alone or in combination with CT.

Descriptive statistical analyses were utilized. Chi square analysis and t- test were performed to compare categorical and continuous variables. Kaplan-Meier method was used to estimate OS and log rank test was used to compare the groups. All data was analyzed using SPSS version 19.0 statistical software.

Results

59 patients who underwent CT and/or WBRT for PCNSL between 1999 and 2012 were identified. The age range at diagnosis was 17–85 years with median age of 64. 35 of 59 patients (59%) were ≥ 60 years old. Male to female ratio was 1.27:1 (33:26). The median ECOG PS was 1. A majority of patients presented with motor deficits, 29 out of 59. The most common location of lymphoma was in the cerebral hemispheres. The median survival of the entire cohort was 37 months. 18 of 59 pts (25%) survived ≥ 60 months. In the cohort of pts that survived ≥ 60 months, a majority 16 of 18 (89%) received HD MTX. Patients treated with initial WBRT and chemotherapy revealed inferior overall survival (OS) compare to patients treated with induction chemotherapy alone (OS 37 months vs. 66 months) (p=0.011). Patients over the age of 60 had worse outcomes compared to patients who were less than the age of 60 (OS 33 months versus 70 months) (p=0.023).

Conclusions

HD MTX was the most frequently utilized CT regimen in the cohort of patients surviving > 60 months. Administering WBRT combined with chemotherapy was associated with worse outcomes in this retrospective analysis. Patients with primary CNS lymphoma who are older than 60 have worse outcomes in this retrospective analysis compared to patients younger than 60.

Disclosures:

Sokol: Celgene: Honoraria, Speakers Bureau.
An RNAi-Therapeutic Targeting Tmprss6, in Conjunction With Oral Chelator Therapy, Ameliorates Anemia and Additively Diminishes Secondary Iron Overload In a Mouse Model Of β-Thalassemia Intermedia
Authors: Schmidt Paul J , Racie Tim , Butler Jim S , Fitzgerald Kevin , Fleming Mark D
Published: October 2019

β-Thalassemias are a group of inherited blood disorders caused by loss of β-globin synthesis and are characterized by anemia, extramedullary hematopoiesis and ineffective erythropoiesis leading to secondary iron overload. Increased iron absorption is due to inappropriately low levels of the liver hormone, hepcidin (HAMP). The membrane serine protease Matriptase-2 (TMPRSS6) attenuates BMP-mediated HAMP induction by cleaving the BMP co-receptor, hemojuvelin (HJV). Previously, we demonstrated that an RNAi-therapeutic targeting Tmprss6 elevates hepcidin expression and reduces disease severity in the Hbbth3/+ mouse model of β-Thalassemia intermedia (Blood. 2013; 14;121(7):1200-8). To further interrogate the efficacy of this therapeutic approach, Hbbth3/+ animals were treated with a siRNA directed against Tmprss6 on a replete 50ppm iron diet, a low iron diet (3-5ppm iron) or a 50ppm iron diet containing deferiprone.

Systemic administration of an siRNA directed against Tmprss6 in the three diet conditions leads to significant inhibition of Tmprss6 mRNA in the livers of Hbbth3/+ mice with concomitant elevation in hepcidin expression. In correspondence with earlier studies, we demonstrate here that Tmprss6 silencing in animals under each of the three diet regimens leads to a significant improvement in the anemia of Hbbth3/+ mice as evidenced by increased total hemoglobin. Furthermore, hallmarks of ineffective erythropoiesis, including splenomegaly and reticulocytosis, were decreased in all Tmprss6 silenced Hbbth3/+ animals. If untreated, excessive iron loading in humans with β-Thalassemia leads to tissue iron deposition and eventual organ damage and failure. Importantly, here we demonstrate that the total body iron burden of Hbbth3/+ mice, as assessed by non-heme liver iron, is decreased by almost 30% in animals chelated with oral deferiprone and treated with Tmprss6 siRNA. A similar diminution of iron deposition is not evident in animals on a low iron diet or in mice fed deferiprone alone. Taken together, this data suggest that siRNA suppression of Tmprss6, in conjunction with chelation therapy, may provide an improved modality for treatment of the anemia and secondary iron loading seen in hemoglobinopathies such as β-Thalassemia.

Disclosures:

Racie: Alnylam Pharmaceutical, Inc: Employment. Butler:Alnylam Pharmaceutical, Inc: Employment. Fitzgerald:Alnylam: Employment. Fleming:Alnylam Pharmaceutical, Inc: Research Funding.
Novel NGS-Based Platforms For Molecular Diagnosis Of Severe Congenital Neutropenia
Authors: Racek Tomas , Puchalka Jacek , Kohistani Naschla , Klein Christoph
Published: October 2019

Congenital neutropenia (CN) is a heterogeneous disorder. More than 30 distinct genetic defects have been discovered in patients with genetic diseases associated with decreased numbers of peripheral neutrophil granulocytes. Currently, most molecular diagnostic laboratories use Sanger-based sequencing techniques to define disease-causing mutations in patients with CN. In approximately 50% of patients no known genetic disorder can be found.

To identify novel genes that can be causative for unexplained CN cases we embarked on next-generation whole-exome sequencing using SOLiD 5500™ and Ion Proton™ sequencers. Up to date we sequenced whole exomes of 49 families, in which children were diagnosed with CN. The fragment libraries were constructed using the SureSelect™ V4+UTRs System (Agilent) allowing us to target whole coding sequence and the majority of UTRs of human genome (approx. 71 Mb). The vast majority of the families were analysed in the “Trio” approach and suitable homozygous or compound heterozygous rare variations (frequency below 1%) in protein coding regions or in splice sites were chosen for further validations.

In seven cases mutations previously described as causative for neutropenia were identified including G6PC3, HAX1, and ELANE. Four other rare variants are currently being analysed for their potential to cause CN. In 35 patients, no plausible candidate could be identified so far.

When we assessed variants within the genes related to CN, our data revealed unequal coverage pattern over these genes. Around 10% of the exons were insufficiently covered (coverage of less than 10) to allow for reliable variant and genotype call. These facts limit the power of whole exome sequencing as a diagnostic tool, as mutations at the non-covered positions cannot be ruled out, and demonstrate the need of an alternative comprehensive approach.

We are currently assessing sensitivity and specificity of a robust, rapid, and cost-effective approach that comprehensively analyses the sequence of 34 CN-relevant genes. Our approach is based on enrichment of specific exon regions by amplification using custom made AmpliSeq™ (Life Technologies) panel. For 25 genes we are able to sequence coding region as well as both UTR sequences, for 9 genes sequencing is limited to coding regions. This approach will provide a reliable, quick, and inexpensive diagnostic strategy for CN patients which will be offered free-of-charge to patients worldwide, independent of ethnic, national, or financial considerations.

Disclosures:

No relevant conflicts of interest to declare.
AGM-Derived Endothelial Cells and Notch Ligands Provide Embryonic Hematopoietic Stem Cell-Supportive Niches In Vitro
Authors: Hadland Brandon K , Varnum-Finney Barbara , Moon Randall T , Butler Jason M , Rafii Shahin , Bernstein Irwin D.
Published: October 2019

Greater knowledge of embryonic niche signals regulating the establishment, maintenance, and expansion of hematopoietic stem cells (HSC) during development will be essential in deriving therapeutically useful HSC from pluripotent stem cells (PSC). To this end, we have used the murine embryo model to dissect components of embryonic hematopoietic microenvironments which are sufficient to support nascent HSC and their precursors in vitro. We demonstrate that Akt pathway-activated endothelial cells (ECs) derived from the AGM (aorta-gonad-mesonephros) region, a critical site of HSC emergence during development, can substantially increase short and long-term multilineage engraftment potential from isolated embryonic day 11 (E11) VE-Cadherin+/CD45+ AGM-derived hematopoietic cells by co-culture in vitro. Furthermore, preliminary experiments show that co-culture with AGM-ECs also promotes high level, multilineage engraftment capacity from VE-cadherin+/c-kit+ precursors isolated from younger embryos (E9-E10). These results suggest that endothelial cells from an embryonic HSC-producing niche provide signals sufficient to promote maturation of HSC from embryonic precursors and subsequently support early HSC expansion in vitro. Further dissection of required signals for embryonic HSC expansion identified a unique combination of Notch activation by immobilized Notch ligands, cytokines, and small molecule inhibition of the TGF-β pathway, which is sufficient to inhibit differentiation and enhance self-renewal of embryonic, definitive-stage hematopoietic precursors in vitro. Notably, these conditions significantly increased short and long-term, multilineage repopulating HSC from E11 VE-Cadherin+/CD45+, but not E9-10 VE-Cadherin+/c-kit+ AGM-derived hematopoietic cells, indicating AGM-ECs provide additional, yet to be identified, signals for HSC maturation from developmental precursors. These findings have important implications for dissecting critical niche signals for HSC formation and expansion that will be essential for addressing the elusive goal of deriving HSC from pluripotent precursors.

Disclosures:

Rafii: Angiocrine Bioscience: Founder Other.
Long Non-Coding RNAs As Components Of The MYC Network In B Cell Lymphoma
Authors: Kluiver Joost , Winkle Melanie , Tayari Mina , Terpstra Martijn , Kortman Gertrud , de Jong Debora , Sietzema Jantine , Kroesen Bart-Jan , Kok Klaas , van den Berg Anke
Published: October 2019

MYC is an important oncogenic transcription factor in B-cell lymphoma and high MYC expression is associated with aggressive behavior and poor clinical outcome. A large number of genes are regulated by MYC, several of which are shown to contribute to the MYC induced phenotype. Long non-coding (lnc)RNAs have recently emerged as a novel class of regulatory RNAs acting at the epigenetic, transcriptional or posttranscriptional level. Aberrant expression of several lncRNAs has already been implicated in various aspects of tumorigenesis. It is currently unknown to what extend MYC can regulate lncRNA expression and whether these lncRNAs contribute to the pathogenesis of B-cell lymphoma.

Using an inducible MYC B cell lymphoma model and a custom microarray we investigated the expression >10,000 lncRNA loci and identified 1,820 lncRNA probes that show a MYC regulated expression pattern. Of these, 355 responded already after 4h, indicating direct MYC regulation. To identify transcripts relevant to lymphoma pathogenesis, we determined if these 355 lncRNAs were differentially expressed between primary lymphoma cases with high and low MYC expression and in addition also between MYC-high lymphoma cell lines and normal germinal center B cells. This revealed an overlap of 176 lncRNAs that were MYC regulated, aberrantly expressed in B cell lymphomas and differentially expressed between MYC-high and MYC-low lymphomas. Differential expression patterns were validated by qRT-PCR. As a first indication for lncRNA function, we isolated RNA from nuclear and cytoplasmic fractions of B cell lymphoma cell lines and determined enrichment fold in comparison to RNA isolated from the total cell lysates. Approximately 40% of all lncRNA transcripts showed specific subcellular localization, 80% nuclear and 20% cytoplasmic enriched. 31 of the 176 candidate lncRNAs were enriched in a specific cellular fraction. Furthermore, we analyzed which lncRNAs are enriched in Argonaute 2 containing complexes as an indication for lncRNA-miRNA interaction. For ∼5% of all expressed lncRNAs we found evidence for miRNA-lncRNA interactions, including 8 of the 176 differentially expressed MYC-induced lncRNAs.

This study identified 176 MYC responsive lncRNAs that are deregulated in B cell lymphoma. To establish a definitive role in B cell lymphoma pathogenesis a further characterization is warranted.

Disclosures:

No relevant conflicts of interest to declare.
Dietary Determinants Of The White Blood Cell Count
Authors: Balkaransingh Pauline D , Wheeler David , Ning Yi , Helou Marieka A. , Massey Gita
Published: October 2019

Introduction

White blood cells are key components of the immune system. They defend the body against a host of diseases and infections. Various studies have explored the possible influence of dietary nutrients on the white blood cell (WBC) count. If it can be demonstrated that nutrients affect the WBC count, they may offer an inexpensive way of modulating the immune system and in turn, the body's ability to fight against disease and infection. The sample sizes of previous studies, however, have been relatively small. The results have therefore been variable and conflicting. The purpose of this study is to continue to explore the dietary determinants of the WBC count, using a database that allows for a larger sample size, the 2005-2006 National Health and Nutrition Health Survey (NHANES).

Methods

The main outcome variable, WBC count, was recoded to the normal range for ages ≥ 18 years, of 4-11(±2). This was done in order to account for extreme values; particularly high and low values secondary to disease or infection. The population means and 95% confidence intervals were obtained using the statistical weights for the key variables of interest (Table 1). Multiple linear regression and backward elimination were used to predict the final model (Table 2). Variables with p < 0.1 were kept in the model. SAS 9.3 was used for all statistical analyses.

Results

Associations for age, gender, race and Body Mass Index (BMI) and WBC count, were consistent with previous studies and were all statistically significant (p< 0.0001). It has been shown that more intense physical activity usually leads to an increase in the WBC count. In this study, however, more vigorous forms of activity were associated with a lower WBC count (p < 0.0001). Increases in dietary copper and iron were associated with a subsequent decrease in the WBC count (Table 2, p < 0.05). An increase in vitamin K was also associated with a decrease in WBC count, however, it was not statistically significant (Table 2, p > 0.05). Increases in vitamin B1 were associated with an increase in WBC count (p < 0.05).

Conclusion

The results of this study suggest that further prospective studies are needed to investigate the role of these nutrients as determinants of the WBC count.

Disclosures:

No relevant conflicts of interest to declare.
Survival In Advanced Diffuse Large B-Cell Lymphoma In Pre- and Post-Rituximab Eras In The United States: A Population Based Study
Authors: Bista Amir , Shafii Bahman , Shah Binay K.
Published: October 2019

Background

Rituximab was approved by FDA as first line agent for treatment of advanced diffuse large B-cell lymphoma (DLBCL) on February 2006. We conducted this population based study to determine if the results from clinical trials have translated into survival benefit in general population.

Methods

We selected patients with advanced diffuse large B cell lymphoma (Distant stage based on LRD Summary Staging 1998+ in SEER*Stat) from Surveillance, Epidemiology, and End Results (SEER) 18 database (1973-2010), and calculated relative survival rates for patients diagnosed from 2002-2005(pre-rituximab) and March 2006 to 2009 (post-rituximab). We used Z-test in the SEER*Stat to compare the relative survival rates in cohorts categorized by race (white, black and other), gender, and age groups (<60 and 60+). The “Cansurv” software, and specifically, Cox proportional hazard function were used to investigate the influence of age (<60 ; 60+ years), sex (male; female), race (white; black; others) and marital status (married; single; separated/divorced/widowed) on the relative survival.

Results

A total of 15627 patients with advanced stage DLBCL as the only primary cancer were identified. Patients were predominantly white of non-Hispanic, non-Spanish/non-Latino origin, males, belonging to the older age category and married. The median age at the diagnosis was 65 years. One-year relative survival in ‘Whites’ and ‘Others’ improved significantly in post-rituximab era compared to pre-rituximab era (64.80 ± 0.6% vs 61.3 ± 0.6%; p= 0.0002 and 64.5 ± 1.9% vs 54.9 ± 2.2%; p= 0.0011, respectively). Also, three year relative survival improved significantly in ‘Whites’ and ‘Others’ in post-rituximab era compared to pre-rituximab era (53.7 ± 0.7% vs 50.3 ± 0.7%; p= 0.0001 and 52.0 ± 2.3% vs 40.8 ± 2.3%; p= 0.0002, respectively). However, no significant improvement were observed in one-year and three-year relative survival in blacks, during post rituximab era comapared to pre-rituximab era. Interestingly, in pre-rituximab era, relative survival in blacks was comparable to that of whites, but it was significantly better than that of others (p<.05).

Although there was significant improvement in one- and three-year relative survival in young females, old males and old females, no significant improvement (p>.05) in survival was observed in the young males. Factors such as young age, female sex, non-Hispanic origin, white race, married status and post-rituximab era were associated with significantly better survival (p<.05).

Conclusion

The relative survival rates among young males and black patients with advanced diffuse large B cell lymphoma has not improved during post-rituximab era.

Disclosures:

No relevant conflicts of interest to declare.
Transdifferentiation Of Malignant B-Cells Into Macrophages In a Murine Model Of Burkitt´s Lymphoma
Authors: Bruns Heiko , Mueller Sebastian , Pasemann Shirin , Mackensen Andreas , Gerbitz Armin
Published: October 2019

Background

The presence of abundant macrophages is characteristic morphological hallmark for the diagnosis of Burkitt´s lymphoma. They are considered to be the removers of cellular debris which occurs due to the rapid proliferation of such lymphomas. However, although macrophages represent the main non-malignant population within such lymphomas and therefore can be considered a substantial part of the stroma, little is known about how these cells are being recruited and how they influence malignant growth. Since malignant B–cells show high plasticity and have capacity to differentiate into various phenotypes we analysed the origin of macrophages in a murine model of Burkitt´s lymphoma. In this transgenic model a human c-myc gene is overexpressed in a B-cell specific fashion.

Results

When long term cultured lymphoma cells from transgenic animals were transferred into wild type animals expressing the congenic marker CD45.1 lymphomas grew out within 14 days post transfer. Lymphomas were minced into single cell suspension and macrophages were sorted by flow cytometry based on the expression of CD11b and F4/80. Approximately 6% of the cells expressed F4/80 and CD11b as macrophage markers and displayed an M2 phenotype. Surprisingly approximately 50% of these macrophages also expressed CD45.2 and therefore were not of host origin but derived from the transferred lymphoma cells. These cells were positive for the c-myc transgene, but displayed reduced expression of c-myc as shown by RT PCR in comparsion to the lymphoma cell line in culture. Furthermore, lymphoma derived macrophages displayed reduced PAX5 expression and were negative for CD19 surface expression. When lymphoma cells were retrovirally transduced to express bcl2 to block apoptosis, and were treated with chemotherapy in vivo, we observed a massive increase in macrophage numbers in remaining tissues suggesting transdifferenziation as an alternative exit strategy in case of cellular stress.

Conclusion

These results demonstrate that BL cells are able to differentiate into a macrophage phenotype to form their own supporting stroma. In addition, differentiation after DNA damage such as chemotherapy is increased suggesting a new exit strategy when classic apoptotic pathways are blocked.

Disclosures:

Mackensen: Eli Lilly: Consultancy.