bluuebunny/crossref_metadata_2025 huggingface dataset:
Spontaneous Mutations of Bcor and Jak1/2 genes Lead to an Aggressive Leukemia of B-1 Progenitor B Cells
Authors: Gough Sheryl M , Goldberg Liat , Pineda Marbin , Walker Robert L , Zhu Yuelin J , Bilke Sven , Meltzer Paul S , Aplan Peter D
Published: October 2019

NUP98 gene fusions, generated by non-random chromosomal translocations, are associated with a wide spectrum of high risk hematologic malignancies and have been shown to alter normal hematopoietic stem and progenitor cell (HSPC) gene expression programs. A recurrent t(11;17)(p15;p13) translocation in patients with AML leads to the production of a NUP98–PHF23 (NP23) fusion gene. The consequent NP23 fusion protein retains the PHD domain, known to bind H3K4me3, and is thought to have aberrant chromatin regulation properties. We have generated a transgenic mouse model of the NUP98-PHF23 gene fusion which develops a range of hematologic malignancies, most commonly pre-T LBL and AML. However, approximately 10% of NP23 mice develop an aggressive B-1 progenitor acute lymphoblastic leukemia (pro B-1 ALL).

B-1 and B-2 lymphocytes have distinct developmental pathways and are thought to represent arms of the innate and adaptive immune systems, respectively. Mature B-2 lymphocytes predominate in the peripheral circulation, and are characterized by expression of B220; whereas B-1 lymphocytes are more prevalent in the pleural and peritoneal cavities, and do not express B220. Murine B cell malignancies typically stain positive for B220, and represent transformed B-2 cells. In the present study, NP23 progenitor ALLs displayed an immunophenotype (Lin-B220- CD19+ AA4.1+) that was identical to that of the recently described B-1 progenitor cell. All B-1 progenitor ALLs exhibited clonal rearrangements of the IgH gene locus. Specifically, these rearrangements involve favored usage of 3’ VH regions, similar to observations with fetal B-1 progenitor cells, further supporting the notion that these are leukemias of B-1 progenitors. Using whole exome sequencing, we found acquired mutations in the BCL6 interacting corepressor (Bcor) gene in 5 out of 7 B-1 progenitor leukemias. The mutations were all frame shift or nonsense mutations, and were located within a 9 bp “hot spot” in Bcor exon 8. In addition, 4 of 7 cases had somatic mutations of Janus kinase 1 (Jak1) or 2 (Jak2), and 7/7 cases showed hyperphosphorylation of Stat3 or Stat5, consistent with the contention that the Jak1/2 mutations are activating mutations, and leading to a hypothesis that the NP23 pro B-1 ALLs which do not harbor Jak1/2 mutations may have acquired an unidentified mutation in the Jak-Stat pathway. Of note, Jak1/2 mutations have previously been identified in a subset of high-risk pediatric B-cell precursor ALL patients. The striking correlation between Bcor and Jak1/2 mutations, occurring specifically in a subset of NP23 leukemias, implies that these three mutations (NP23, Bcor, and Jak1/2) collaborate and provide the oncogenic setting for B-1 progenitor transformation.

Disclosures

No relevant conflicts of interest to declare.
GNA13, a Novel Component of the GPR84/Beta-Catenin Signaling Axis in AML Stem Cells, Regulates the Maintenance of MLLAF9 Leukemogenesis
Authors: Dietrich Philipp A , Norris Murray D , Wang Jenny Yingzi
Published: October 2019

Beta-catenin is required for the establishment of leukemic stem cells (LSC) in mixed lineage leukemia (MLL)-rearranged acute myeloid leukemia (AML). Targeted inhibition of beta-catenin signaling has been hampered by the lack of pathway components amenable to pharmacological inhibition. Our recent study has identified a new beta-catenin regulator, GPR84, a member of the G protein-coupled receptor family that represents a highly tractable class of drug targets. Since no GPR84-specific anti-cancer compound has been developed to date, we aim to further delineate the GPR84/beta-catenin axis in order to inhibit other components of this signaling pathway pharmacologically.

Preliminary data from our lab has implicated GNA13 as a potential downstream target gene of GPR84. In this study we sought to further examine the involvement of GNA13 in the GPR84/beta-catenin signaling axis. Functional studies showed that shRNA-mediated ablation of GNA13 significantly decreased the capacity of MLLAF9-LSC to form colonies in vitro (P=0.0184). We further demonstrated that GNA13 inhibition in LSC induced a significant G1 cell cycle arrest in vitro (P=0.0192). Subsequent in vivo studies showed that GNA13 downregulation in MLLAF9-LSC significantly delayed the onset of AML (P=0.0016) and significantly decreased LSC proliferation (P=0.0066). Hence GNA13 inhibition mimics the previously described GPR84-deficient phenotype in MLLAF9-LSC (Blood 2013;122:3781). In contrast, the GPR84-deficient phenotype could be rescued in MLLAF9-LSC by GNA13 overexpression in vitro (P=0.0130) and in vivo (P=0.0012). Further analysis showed that there was a significant increase in the mitotic index of GPR84-deficient LSC transduced with GNA13 cDNA in vitro, evidenced by an increase in G2/M-phase accumulation (P=0.0030) and p-histone H3 (P=0.0040). In addition, our data demonstrated a significant increase in BrdU incorporation of GPR84-deficient LSC overexpressing GNA13 in vivo (P<0.0001).

In conclusion, our studies have identified GNA13, a novel component of the GPR84/beta-catenin axis. Since this pathway is pivotal for MLLAF9 AML maintenance by sustaining LSC self-renewal, targeting the GPR84/GNA13/beta-catenin signaling axis may provide a novel and promising strategy for improving the therapy and outcome of AML patients.

Disclosures

No relevant conflicts of interest to declare.
Influence of Dihydrofolate Reducatse (DHFR) Gene Polymorphisms on Outcome of Methotrexate Maintenance Therapy in Patients with Acute Lymphoblastic Leukemia
Authors: Biswajit Dubashi , Kodidela Sunitha , Pradhan Suresh Chandra , Basu Debdatta , Prasad Ravi , Gurusamy Umamaheswaran
Published: October 2019

Objectives: 1)To determine the influence of DHFR gene polymorphisms on outcome of methotrexate-based maintenance therapy in patients with acute lymphoblastic leukemia (ALL) 2)To compare the relapse free survival between DHFR genotype 3)To establish the normal allele and genotype frequencies of the DHFR polymorphisms in the South Indian population Methods: A total of thirty one patients with ALL were included for the study. A total of 85 healthy volunteers from the South India were recruited to establish the allelic frequencies of the A-317G & C-680A variants of DHFR gene. DNA was extracted by phenol chloroform method and genotyping was done by allelic discrimination method. Patients were followed up for two years for the outcome. Results: Homozygous mutant genotype carriers (GG) were at two-fold increased risk of relapse when compared to the other genotypes (AA & AG) [Relative risk= 2.6; 95% CI, 1.260 to 5.365, P<0.05]. Relapse-free survival rate was lower in GG genotype carriers when compared to wild and hetero genotypes [Log-rank test, p<0.05]. The genotype frequencies of A-317G variant in healthy volunteers were- AA=51.76%, AG=37.64%, GG=10.58% and C-608A variant genotype frequency were-CC=0%, AC=20.98%, AA=79.01%. Conclusion: GG variant of DHFR A-317G is associated with increased relapse and lower relapse-free survival in ALL patients.

Disclosures

No relevant conflicts of interest to declare.
Ectopic Expression of Lin28 Biases Myeloid Differentiation to Promote an Immature Mast Cell State and Is Implicated in Aggressive Mastocytosis
Authors: Wang Leo D. , Nguyen Phi , Rowe Robert G , Nageswara Rao Tata , Daley George Q. , Wagers Amy J.
Published: October 2019

Dysregulated mast cell development leads to systemic mastocytosis, a clinically variable but often devastating family of hematologic disorders. Lin28 is a heterochronic gene and pluripotency factor implicated in many types of malignancy, and prior studies suggest that Lin28 expression can restore a fetal hematopoietic program in adult mice. However, the role of Lin28 in hematologic malignancy remains controversial. In our study, we induced expression of Lin28 in adult mice using a doxycycline-responsive transgenic system. Lin28 induction caused marked mast cell accumulation in target organs such as the skin and peritoneal cavity. In vitro assays revealed a skewing of myeloid commitment in Lin28-expressing hematopoietic progenitors, with increased levels of Lin28 in common myeloid progenitors and basophil-mast cell progenitors altering gene expression patterns to favor cell fate choices that enhance mast cell specification. In addition, Lin28-induced mast cells appeared phenotypically and functionally immature, and in vitro assays suggested a slowing of mast cell terminal differentiation in the context of Lin28 upregulation. Finally, interrogation of human mast cell leukemia samples revealed upregulation of LIN28 in abnormal mast cells from patients with aggressive systemic mastocytosis (ASM). This work identifies Lin28 as a novel regulator of innate immune function and a new protein of interest in mast cell disease.

Disclosures

No relevant conflicts of interest to declare.
Limited Efficacy of BMS-911543 in a Murine Model of JAK2V617F Myeloproliferative Neoplasm
Authors: Pomicter Anthony D. , Eiring Anna M. , Senina Anna V. , Marvin James E , Prchal Josef T. , O'Hare Thomas , Deininger Michael W.
Published: October 2019

Activation of JAK2, frequently as a result of the JAK2V617F mutation, is a characteristic feature of the classical myeloproliferative neoplasms (MPN) polycythemia vera, essential thrombocythemia and myelofibrosis and thought to be responsible for the constitutional symptoms associated with these diseases. BMS-911543 is a JAK2 selective inhibitor that induces apoptosis in JAK2-dependent cell lines and inhibits the growth of CD34+ progenitor cells from patients with V617F+ MPN. To explore the clinical potential of this inhibitor, we tested BMS-911543 in a murine retroviral transduction – transplantation model of JAK2V617F MPN. Treatment was initiated at two dose levels (3 mg/kg and 10 mg/kg) when the hematocrit exceeded 70%. Following the first week, white blood cell counts were reduced to normal in the high dose group and were maintained well below the vehicle-treated mice throughout the study. However, BMS-911543 had no effect on red cell parameters. After 42 days of treatment, the proportion of JAK2V617F - positive cells in hematopoietic tissues was identical to controls or slightly increased compared to controls. Plasma concentrations of IL-6, IL-15, and TNFα were elevated in MPN mice and reduced in the high dose treatment group, while other cytokines were unchanged. Collectively, these results show that BMS-911543 has limited activity in this murine model of JAK2V617F – driven MPN and suggest that targeting JAK2 alone may be insufficient to achieve effective disease control.

Disclosures

Deininger: BMS, Novartis, Celgene, Genzyme, Gilead: Research Funding; BMA, ARIAD, Novartis, Incyte, Pfizer: Advisory Board, Advisory Board Other; BMS, ARIAD, Novartis, Incyte, Pfizer: Consultancy.
Comparison of DNA Methyltransferase Inhibitors in Patients with Myelodysplastic Syndrome
Authors: Avsar Esin , Salim Ozan , Toptas Tayfur , Deveci Burak , Kurtoglu Erdal , Tombak Anil , Tiftik Eyup Naci , Undar Levent
Published: October 2019

The aim of this retrospective study was to compare the response rates (RR), overall survival (OS), time–to–acute myeloid leukemia–transformation (TTAMLT), hematologic toxicity and transfusion requirement of azacitidine and decitabine in patients with Myelodysplastic Syndrome Refractory Anemia with Excess Blasts (MDS RAEB) types 1 and 2. A total of 51 patients (35 male, 16 female, median age (years): 67) diagnosed with MDS RAEB-1 and RAEB-2 treated with azacitidine (n=31) or decitabine (n=20) in three Turkish institutes were evaluated . Response to treatment was classified according to International Working Group response criterias. Competing risk analysis was used to compare TTAMLT. There was no significant difference between the two groups in terms of age, gender, performance score, diagnosis, etiology, treatment cycles, bone marrow cellularity and blast percentage. Most patients received at least 4 cycles of azacitidine or decitabine. Patients who received azacitidine or decitabine had comparable response rates (58.1% vs 66.7%, respectively, p= 0.55). There was no difference in terms of red blood cell and platelet transfusion requirement and febrile neutropenia episodes (p= 0.46, p= 0.27, p= 0.19, respectively). Median follow–up was 31 months for azacitidine and 20 months for decitabine. Median OS was 18.4 months for azacitidine and 13.4 months for decitabine (p= 0.16). Only predictor of OS was at least 4-cycle treatment with any DNA methyltransferase inhibitors. 1–year risk of AML transformation was comparable across the groups (26% vs 33%, p= 0.55). In conclusion, both azacitidine and decitabine have similar efficacy and toxicity profiles in the treatment of MDS RAEB-1 and RAEB-2. Until a head–to–head comparison in prospective, randomized studies are conducted, the current source of available data will derive from meta–analyses that consist of the indirect comparison of treatment arms, or retrospective analyses with inevitable biases.

Disclosures

No relevant conflicts of interest to declare.
Cryptic Unbalanced Chromosomal Changes in a Case of Chronic Lymphocytic Leukemia / Small Lymphocytic Lymphoma (CLL/SLL) Identified By Single Nucleotide Polymorphism (SNP) Microarray, but Not By Conventional Karyotyping
Authors: Kurniali Peter C. , Al-Janadi Anas , Chai Hongyan , Wei Sainan
Published: October 2019

Genomic abnormalities have been widely used to diagnose and provide prognostic significance in many hematologic malignancies (HM). The use of conventional G-banded chromosome and fluorescence in situ hybridization (FISH) analyses sometimes fail to detect genomic abnormalities due to the need for actively dividing cells and lower resolution of chromosome analysis (G-banded), as well as the inability to detect copy number of neutral loss of heterozygocity (G-banded and FISH). The addition of single nucleotide polymorphism (SNP) microarray has been shown to increase the detection rate of genomic abnormalities in HM.

We report a 62-year-old Caucasian woman with a diagnosis of chronic lymphocytic leukemia /small lymphocytic lymphoma (CLL/SLL). Cytogenetic studies done at the time when she required initial treatment in 2011 showed a balanced translocation between chromosomes 6 and 13 {46,XX,t(6;13)(p10;q10)[10]/46,XX[10]}. She was treated with bendamustine plus rituximab and achieved complete response. In 2013, she developed worsening of anemia and marked lymphocytosis. Blood and bone marrow examination showed polymphocytic transformation. G-banded cytogenetic studies again showed a balanced translocation between chromosomes 6 and 13 (same as in 2011). However, FISH analysis detected a deletion of 13q. G-banded cytogenetic study was re-examined and showed balanced translocation: 46,XX,t(6;13)(p10;q10)[10]/46,XX[10]. Re-analysis of bone marrow cytogenetic from 2011 also suggested possible deletion of 13q, but the resolution was poor. Cytogenetic microarray validation was performed and revealed not only deletions of 13q14.2q14.3, but also 6p21.1p21.1. Karyotypic changes of chromosome 6 have been associated with prolymphocytic transformation in CLL/SLL.

Deletion of 13q14.3 usually carries good prognosis in CLL/SLL. However, additional abnormalities and deletions of different sizes of 13q reportedly cooperate to change its favorable value. An accurate cytogenetic analysis may be useful in aiding in diagnosis, prognosis, and possibly therapy. Our findings suggest that microarray analysis is a suitable companion test to G-banded chromosome and FISH analyses, especially in CLL/SLL.

Disclosures

No relevant conflicts of interest to declare.
Differences in the Original Diagnostic Markers Including the "CRAB" Criteria in Multiple Myeloma Patients from Different Ethnicities: A Single Center Experience
Authors: Mattour Ahmad Hatem , Vollstaedt Joshua , Kuriakose Philip
Published: October 2019

LEARNING OBJECTIVES:

Determine if there are any differences in the original diagnostic markers, including the “CRAB” criteria, in Multiple Myeloma patients from different ethnicities.

BACKGROUND:

About 24,050 new cases of Multiple Myeloma are expected to be diagnosed in 2014. In a metropolitan institution, such as Henry Ford Hospital, newly diagnosed patients are drawn from multiple ethnic backgrounds. While several epidemiological studies have demonstrated a positive correlation between race and values of several diagnostic markers (such as hemoglobin, creatinine and albumin), most of the research focused on molecular and genetic, as opposed to clinical, differences in patients from multiple ethnicities.

Our study, therefore attempted to offer a more customized diagnostic approach to patients with newly diagnosed Multiple myeloma, based on their ethnicity, and to determine if there was a correlation between the patients' races and common diagnostic elements, including the CRAB criteria, at time of diagnosis.

METHODS:

We conducted a retrospective study. Data from 300 patients over 5 years (2007-2012) with newly diagnosed with Multiple myeloma was collected. Only 197 patients fulfilled the diagnostic criteria and were included in the final analysis. The following data was extracted: Age at diagnosis, gender, diagnostic elements according to the CRAB criteria (serum Calcium, serum Creatinine, Anemia, Bone lytic lesions) at date of diagnosis, serum beta-2-microglobulin level, M-protein serum concentration, and whether Urine monoclonal protein excretion was present. The patients were divided into two groups by race: African American and Caucasian. An overall score was created by summing the incidence of each diagnostic marker.

RESULTS:

Categorical data was compared between the two groups using chi-square tests, and CRAB score was compared using a Wilcoxon rank-sum test due to its ordinal nature. Our study demonstrated that there were no statistically significant differences between the two groups in any of the components collected, or in the final total score

CONCLUSIONS: Our study demonstrated that there was no statistically significant difference in the original diagnostic markers, including the CRAB criteria, in Multiple Myeloma Patients from Different Ethnicities presenting to our Institution.The effect of baseline cytogenetic characteristics on the diagnostic markers between multiple myeloma patients from different ethnicities is still not fully understood, and might be a factor that needs to be studied further.

Disclosures

No relevant conflicts of interest to declare.
Exomic microRNA Profiling of Bone Marrow Aspirate Plasma and Comparison with mRNA Profiles Used in the Clinical Management of Multiple Myeloma
Authors: Van Laar Ryan K , Flinchum Rachel , Nathan Brown , Ramsey Joseph , Shaughnessy John , Epstein Joshua , Heuck Christoph , Barlogie Bart
Published: October 2019

Exosome-mediated processes are increasingly being implicated in the pathobiology of many forms of solid tumors and hematologic malignancies. In the case of blood cancers, an oncogenic role for exosomes has been demonstrated for multiple myeloma (MM). An increased understanding of the abundance and disease association of extra-cellular microRNAs in particular, may lead to opportinities to improve early detection and management of MM and precursor conditions.

We performed microRNA profiling of cell-free bone marrow aspirate plasma on a series of 100 patients with diagnoses of MGUS, active multiple myeloma or relapsed multiple myeloma. The microRNA profiles were compared between disease status groups and also individually with each patient’s routine MyPRS results (ie. GEP70 high/low risk and molecular subtype gene signatures), generated from their malignant CD138+ plasma cells1. MicroRNAs with patterns of expression associated with these clinically validated genomic (mRNA) signatures were investigated to determine if they (i) were known regulators of the genes used in MyPRS and (ii) in a cross-validated analysis, were able to predict the patients MyPRS risk group and molecular subtype.

An additional series of paired peripheral blood and bone marrow aspirates (isolated CD138+ plasma cells) were also microRNA and MyPRS profiled, to further investigate the role and potential clinical utility of extracellular microRNAs in multiple myeloma. Results will be presented which describe the abundance of individual microRNA's in realtion to disease status, including microRNA's previously linked to the regulation of mRNA's contained in the GEP70 signature.

The ability to predict MGUS progression risk or active myeloma prognosis using molecular signatures in peripheral blood may increase access to, and adoption of, genomic technologies in the diagnosis and management of these related conditions.

Reference:

[1] van Laar R, Flinchum R, Brown N, et al. Translating a gene expression signature for multiple myeloma prognosis into a robust high-throughput assay for clinical use. BMC Medical Genomics. 2014;7(1):25.

Disclosures

Van Laar: Signal Genetics: Employment, Equity Ownership. Flinchum:Signal Genetics: Employment. Nathan:Signal Genetics: Employment. Ramsey:Signal Genetics: Employment. Shaughnessy:Signal Genetics: Consultancy.
Oxymatrine Induces Apoptosis in Human Myeloma Cells By Cell Cycle Arrest and Activation of Caspase-Dependent Apoptosis
Authors: Wu Wenjun , Wu Cai , Zi Fuming , Li Yi , Yang Li , He Jingsong , He Donghua , Yang Yang , Zhao Yi , Han Xiaoyan , Zheng Gaofeng , Huang He , Cai Zhen
Published: October 2019

Background : Multiple myeloma (MM) is a B cell malignant hematologic cancer. Despite the introduction of new drugs and improvement of chemotherapy, MM is still an incurable disease. Oxymatrine (OMT), the active ingredients of traditional Chinese herbal medicine sophora, has been reported to have antitumor activity. This study was to estimate the therapeutic efficacy of OMT in MM.

Methods: The growth inhibition of myeloma cell lines (RPMI8226, U266, ARP-1) or primary cells by OMT was assessed by MTT assay. Apoptosis of MM cells was examined by annexin V-FITC using flow cytometry analysis. DNA content was analyzed by flow cytometry. RT-PCR and western-blot analysis were used to assess the expression of Bcl-2 family proteins and the IAP family proteins. Western blotting was also used to elucidate the signaling pathway that may mediate OMT-induced apoptosis of MM cells.

Results: OMT treatment resulted in cell growth inhibition and apoptosis in primary MM cells and all tested MM cell lines in a dose-dependent manner (P <0.05). To elucidate OMT -induced MM cell apoptosis, MM cell lines were treated with or without OMT for 24h and assessed for caspase activation and signaling pathway by Western blotting. The results showed the cleavage of PARP, caspase-3, and caspase-9, and p-AKT were down-regulated after OMT treatment. The mRNA expression of survivin and HIAP by RT-PCR was down-regulated. OMT treatment at 5mM for 48h resulted in increased G-phase cells and decreased S-phase cells in MM cell lines (P <0.05). Cell cycle repressor P21 protein was up-regulated while CDK4, CDK6 and CyclinD1 expression was down-regulated. Our finding also showed a synergistic anti-MM activity of OMT and dexamethasone or adriamycin at a low does (CI<1). In addition, LC3-II expression was significantly increased both in RPMI8226 and U266 cells after treatment with OMT. However, treatment with different doses of OMT and 5 mM autophagy inhibitor 3-MA, significant increased cell apoptosis (P <0.05).

Conclusion: Our findings demonstrate the anti-MM activity of OMT and indicate that OMT alone or together with other MM chemotherapeutics may be a prospective treatment for MM.

Disclosures

No relevant conflicts of interest to declare.
Ocular Complications of Bortezomib Therapy in Multiple Myeloma
Authors: Puri Sonam , Joshi Jitesh , Derman Olga , Kornblum Noah , Verma Amit , Braunschweig Ira , Battini Ramakrishna
Published: October 2019

Introduction:

Bortezomib (Velcade¨; V), a proteasome inhibitor, is currently FDA (Food and Drug Administration) approved for the treatment of multiple myeloma (MM) and relapsed mantle cell lymphoma. Common side effects reported with its use include thrombocytopenia, fatigue, peripheral neuropathy and neutropenia. Ocular complications associated with bortezomib are less well described. We describe 6 patients with multiple myeloma who developed meibomitis, multiple chalazions and blepharitis after treatment with bortezomib containing regimens, resulting in delay and in some cases termination of the therapy.

Methods:

We reviewed the charts of forty patients who received induction chemotherapy for multiple myeloma between June 2013-June 2014 at Montefiore Medical Center, New York. Charts were reviewed for data pertaining to demographics, chemotherapy regimen and schedule as well as follow up of ocular symptoms.

Results:

Six of these forty (15%) patients complained about bilateral eye soreness, itching and redness. They did not have any evidence of viral or upper respiratory infections. Ophthalmology evaluation showed blepharitis, meibomitis and multiple chalazion. Majority of the patients were 60 years or greater, with 50% African-Americans. Half of the cases had stage 3 MM with a median duration of chemotherapy 8 weeks prior to onset of ocular symptoms. 4 of these 6 patients received VCD regimen (Bortezomib via subcutaneous route and Cyclophosphamide/ Dexamethasone as tablets) while the remaining 2 patients received VD and VCD-R (addition of Lenalidomide) via intravenous and subcutaneous route respectively. Ocular symptoms led to a 1-3 week delay in the next cycle of chemotherapy. These symptoms promptly responded to withdrawal of chemotherapy and other conservative measures. Chemotherapy was resumed in 4 out of 6 patients, with recurrence of chalazion in two patients within 3 weeks of starting Bortezomib.

Conclusions:

Ocular symptoms are commonly seen but rarely reported with bortezomib therapy. They can adversely affect patient's quality of life as well as lead to interruption in chemotherapy. Although these symptoms respond well to withdrawal of chemotherapy and conservative measures, the rate of recurrence is high once bortezomib is resumed. Awareness of these complications and early intervention can potentially avoid treatment delay.

Figure 1 Figure 1. Figure 2 Figure 2.

Disclosures

No relevant conflicts of interest to declare.
Severe Diffuse Alveolar Hemorrhage Associated with Bortezomib Administration in Patients with Multiple Myeloma
Authors: Ayed Ayed O , Moreb Jan S , Hsu Jack W. , Hiemenz John W , Wingard John R , Norkin Maxim
Published: October 2019

Background: Bortezomib, a proteasome inhibitor, is frequently used in treatment of patients with multiple myeloma (MM). Bortezomib is generally well tolerated with gastrointestinal symptoms and peripheral neuropathy being the most common adverse effects. Here we report cases of severe diffuse alveolar hemorrhage (DAH) associated with bortezomib administration.

Methods and Results: We identified 3 cases of severe DAH that were associated with bortezomib administration in MM patients treated at our institution between 2010 and 2014 (Table 1). All 3 patients presented with fever and worsening hypoxia shortly after initiation of bortezomib therapy and later developed progressive respiratory failure due to DAH. None of 3 patients had any respiratory symptoms or preexisting pulmonary conditions prior to bortezomib initiation. In one patient respiratory symptoms developed after 4 doses of bortezomib, improved off therapy, however rapidly progressed to a respiratory failure with a subsequent bortezomib treatment. Imaging studies showed diffuse areas of ground-glass opacities, apparent prominence of segmental and subsegmental bronchi and interlobular septal thickening. Infectious workup, which included bronchoscopy with bronchoalveolar lavage and respiratory viral studies, was completely negative in all 3 patients. All patients received supportive care, empiric broad-spectrum antimicrobial therapy and high-dose glucocorticoids, which were started within 4 days of hospital admission. Two patients died from complications of DAH and 1 patient recovered without pulmonary sequelae.

Conclusion: Bortezomib-associated DAH is a rare, but serious and potentially fatal adverse event. Pathogenesis remains unclear and is likely related to impaired regulation of the inflammatory response. Early recognition of bortezomib associated DAH is essential for immediate discontinuation of the drug and prompt initiation of high dose steroids. More reports are needed to provide further insights about the pathogenesis and most optimal management of bortezomib induced DAH.

Abstract 5761. Table 1: Patients who developed DAH after bortezomib administration Pt Age/ Gender/Race Smoker Sx Bor dose Bor schedule # of Bor doses Time of onset after last bor dose (days) Platelet count at the onset (103/mm3) Imaging/BAL findings Time to initiation of high dose steroids Outcome 1 67/M/C Never SOB, fever 1.3 mg/m2 IV/2Xwk 8 5 157 Bilateral infiltrates and GGO/DAH 2 Death 2 72/M/C Yes Fever, cough 1.3 mg/m2 IV/2Xwk 2 3 108 Bilateral infiltrates/DAH 4 Recovery 3 55/M/C Yes Fever, chills 1.5 mg/m2 IV/2Xwk 1 2 144 Bilateral diffuse airspace disease/DAH 4 Death

Abbreviations: Pt, patient; M, male; C, Caucasian; Sx, symptoms; Bor, bortezomib; BAL, broncho-alveolar lavage; GGO, ground-glass opacities.

Disclosures

No relevant conflicts of interest to declare.
Survival Outcomes of Double and Triple-Only Combination Anti-Myeloma Chemotherapeutic Regimens on 26 Multiple Myeloma Patients Seen in a Sub-Saharan African Country
Authors: Nwabuko Ogbonna Collins , Nnoli Martin Anazodo , Igbigbi Elizabeth Eneikido , Okoh Dorathy Adaunwa , Chukwuonye Ijezie Innocent
Published: October 2019

Backgound: Multiple myeloma (MM) is one of the commonest haematological malignancies of public health importance in low-income countries of sub-Saharan Africa. Though primarily a disease of the bone marrow, it often poses a diagnostic dilemma for the orthopaedic surgeons because of the frequent skeletal manifestations. Consequently, misdiagnosis and late presentation are often common contributory factors to the poor prognosis and survival of victims in this environment. This retrospective study aimed at finding out the therapeutic challenges of MM in developing countries such as Nigeria.

Methodology: A-10-year multi-centered retrospective analysis of 26 patients diagnosed and managed in three major centers between 2003 and 2013. Informations on the clinical, laboratory, radiological datas and therapeutic interventions were obtained at presentation until patients were lost to follow-up.

Result: The median age of diagnosis was 60.6 years with M:F ratio of 2.3:1.(p<0.05). 61.5%, 30.8% and 7.7% presented in Durie Salmon (DS) stages III, II and I diseases respectively. The mean survival interval was 39.2 months (95% CI, 32.0-47.2 months). 84.5% and 8% were on Melphalan plus Prednisolone (MP) and Cyclophosphamide plus prednisolone (CP) combination chemotherapies respectively. The longest survival interval of 80 months was recorded by MP plus Bortezomib (V) triple regimen.There was no significant difference between the outcome of MP and VMP (p=0.33).

Conclusion: Triple combination regimens (i.e,VMP, MPT) are superior to standard MP in terms of overall survival (OS),and quality of life of MM patients in developing countries, although this is not statistically significant.

Disclosures

No relevant conflicts of interest to declare.
Branched Polyethylenimine-Superparamagnetic Iron Oxide Nanoparticles (bPEI-SPIONs) Improve Immunogenicity of Myeloma Tumor Antigen to Enhance Th1 Polarization of Dendritic Cells
Authors: Hoang My-Dung , Lee Hwa-Jeong , Lee Hyun-Ju , Jung Sung-Hoon , Choi Nu-Ri , Vo Manh-Cuong , Nguyen-Pham Thanh-Nhan , Park In-Kyu , Kim Hyeoung-Joon , Lee Je-Jung
Published: October 2019

Introduction: The application of nanoparticles in dendritic cell (DC)-based cancer immunotherapy represents a promising strategy to enhance antigen-specific T cell immune responses. This study was to investigate the effect of bPEI-SPIONs on antigen-specific T cell responses elicited by DCs loaded with apoptotic U266 myeloma tumor antigen in the presence or absence of bPEI-SPIONs.

Materials and Methods: The myeloma tumor antigens were prepared as following: 1) apoptotic U266 cells by UBV-irradiation and overnight incubation (16 h irradiated cells) and 2) apoptotic U266 cells by UVB-irradiation and 2 h incubation in the absence (2 h irradiated cells) or 3) presence of bPEI-SPIONs (bPEI-SPION 2 h irradiated cells). Monocyte-derived immature DCs were activated with TNF-α, loaded with several kinds of myeloma tumor antigens 2 h after TNF-α stimulation, and cultured for 2 days.

Results: Optimal concentration of bPEI-SPIONs was 16 µg/mL to uptake into tumor cells and the bPEI-SPIONs render U266 cells sensitive to UVB-irradiation through reactive oxygen species (ROS) induction pathway hence accelerated the apoptotic cell death. 2 h irradiated cells and bPEI-SPION 2 h irradiated cells released immunogenic proteins, including HSP70, HSP90, and HMGB1. bPEI-SPION 2 h irradiated cells were easily up-taken by DCs without alteration of surface phenotypes and migration capacities. DCs loaded with bPEI-SPION 2 h irradiated cells showed highest IL-12p70 production level and Th1 polarization compared to other DCs.

Conclusion: These results suggest that bPEI-SPIONs are a promising tool to improve immunogenicity of myeloma tumor cells and to enhance Th1 polarization of DCs loaded with these tumor cells.

Disclosures

No relevant conflicts of interest to declare.
Targeting on the Stem Cell Niche Can Protect Hematopoietic Stem Cell from Chemotherapy and G-CSF
Authors: Li Sidan , Zhai Qiongli , Zou Dehui , Li Changhong , Qiu Lugui
Published: October 2019

The majority of hematopoietic stem/progenitor cells (HSPCs) reside in the bone marrow surrounded by specialized bone-shielded environment. The specialized microenvironment or niche not only provides a favorable habitat for HSPC maintenance and development but also governs stem cell function.

Here we investigated the potential role of bone remodeling osteoblasts and osteoclasts in homeostasis and stress-induced mobilization of hematopoietic progenitors, then further tested the hypothesis that targeting the niche might improve stem cell–based therapies using six mouse models to mimic the multiple rounds of chemotherapy followed by autologous hematopoietic stem cells (HSCs) transplantation in a clinical setting.

Herein, we show that multiple rounds treatment of cytotoxic drugs influence niche. Serum osteocalcin level declined obviously (22.19 ± 1.08 ng/mL, before treatment vs 16.08 ± 2.12 ng/mL, steady state, P=0.01) in autologous HSPCs transplant patients. In mouse models, the number of CD45- Ter119- OPN+ osteoblast was significantly reduced (untreated, 3993 ± 129 cells/femur; CTLs, 1937 ±196 cells/femur; Gs, 1055 ± 43 cells/femur; P<0.01). Pharmacologic use of parathyroid hormone (PTH) or receptor activator of nuclear factor kappa-B ligand (RANKL) increases the number of HSC mobilized into the peripheral blood for stem cell harvests and protects stem cells from repeated exposure to cytotoxic chemotherapy. Ttreatment with granulocyte colony stimulating factor (G-CSF) plus PTH led to relative preservation of the HSC pool (G vs PTH, P<0.01; CTL vs PTH, P<0.05). Recipient mice transplanted with circulation HSPCs of P+R and P+R+G groups also showed more robust myeloid and lymphatic cell engraftment than did HSCs from either CTL or G group.

These data provide evidence that targeting the HSPC niche may improve the efficacy of HSPC mobilization.

Disclosures

No relevant conflicts of interest to declare.
­Impact of Porcine-Human Mixed Hematopoietic Chimerism on Human NK Cell Response to Porcine Cells
Authors: Vishwasrao Paresh , Markus Holzl , Li HaoWei , Choi Goda , Megan Sykes
Published: October 2019

Mixed hematopoietic chimerism permits durable tolerance of T, B and NK cells to xenoantigens in a rat to mouse bone marrow transplant model. However, it is unclear whether tolerance of human NK cells to pig xenoantigens can be induced by mixed hematopoietic chimerism. We assessed the tolerance of human NK cells towards pig cells in a humanized mouse model with established pig and human mixed xenogeneic chimerism. Pig and human mixed chimeras (MCs) were generated by injection of pig bone marrow cells to irradiated pig cytokine (IL3, GMCSF and SCF) transgenic NOD-scid common gamma chain knockout (NSG) mice followed by injection of human fetal liver CD34+ cells 3 day later. In the control group, only human CD34+ cells were transplanted. 12 weeks post-transplant, hydrodynamic injection of plasmid encoding human Flt3L followed by injection of three rounds of recombinant IL15/IL-15 receptor alpha Fc complex was given to promote human NK cell reconstitution. The control non–mixed chimeric group (Non-MC) received the same treatment without pig cells. 12 days following induction of human NK cell reconstitution, human NK cells from both MC and Non-MC mice were isolated from the spleen and their cytotoxic responses in vitro to pig cells were determined in a chromium release assay. In addition, the presence of pig cells in various tissues of the chimeric mice was studied.

While human NK cells were usually undetectable in peripheral blood prior to the injection of human Flt3L plasmid and IL-15/IL-15 receptor alpha-Fc complex, they were detected by 5 days post-injection of IL-15 protein. 12 days post-induction of human NK cell reconstitution, pig cells remained detectable in peripheral blood, spleen, bone marrow and liver in the chimeric mice together with human NK cells. The co-existence of human NK cells and pig cells suggested that human NK cells in MCs might be tolerant to pig cells. Consistent with this notion, cytotoxicity assays showed that human NK cells from MCs demonstrated decreased killing of pig PBMC blasts compared to NK cells from Non-MC mice. Importantly, killing of K562 cells by NK cells from MCs mice was also decreased compared to that of Non-MC mice, suggesting that human NK cell tolerance to pig cells induced by mixed chimerism was associated with global hyporesponsiveness, as we have previously observed in a rat-to-mouse bone marrow transplantation model. Moreover, higher percentages of CD56highCD16low and lower percentages of CD56lowCD16high human NK cell subsets were observed in bone marrow of chimeric mice than in non-chimeric mice, indicating that the development of human NK cells in bone marrow might be altered by the presence of pig cells. In summary, our data suggest that mixed xenogeneic chimerism may induce tolerance of human NK cells towards porcine cells, but the tolerance may be associated with global hyporesponsiveness.

Disclosures

No relevant conflicts of interest to declare.
Thymus Transplantation Plus Allogeneic Bone Marrow Transplantation Can Induce Strong Graft-Versus Leukemia Effects with Low Risk of Graft-Versus-Host Disease
Authors: Zhang Yuming , Feng Xiaoqing , Wu Cuiling , Guo Wenling , Li Huiping , Yuan Shanshan
Published: October 2019

Abstract: [Objective] Allogeneic bone marrow transplantation (allo-BMT) has been used for the treatment of refractory leukemia and donor lymphocyte infusion (DLI) was used for the purpose of graft versus leukemia (GVL) effects. However, DLI is associated with a risk of graft versus host disease (GVHD). Thus, new cellular-based methods are desired. In the present study, we performed thymus transplantation (TT) plus allo-BMT to explore it’s anti leukemia effects. [Methods] Recipient B6 mice (H-2b) bearing leukemia (EL-4 cells, H-2b) were irradiated 8 Gy. The next day, bone marrow cells from BALB/c mice (H-2d) were transplanted into the B6 mice. Simultaneously, DLI and thymus transplantation from the same donor were carried out. The survival period of the recipient B6 mice were examined, histological studies were performed in the liver, intestine, and the engrafted thymus from the recipients 4 weeks after the BMT. Surface markers on lymphocytes from the spleen were analyzed by 3-color fluorescence staining using a FACScan system to determine chimerism. [Results]. All mice treated with BMT showed fully donor-derived chimerism(H-2d). The survival rate in mice treated with BMT plus TT was significantly prolonged compared with those treated with BMT alone or BMT plus DLI. Histologically, both the cortex and medullar areas of the engrafted thymus under the renal capsule were clearly shown. Normal T-cell differentiation was also observed in the engrafted thymus. Microscopic founding of small intestine and liver in the BMT plus TT group indicated mild GVHD, whereas those treated with BMT plus DLI showed moderated to serious GVHD. The number of the CD4+ cells was significantly greater in the mice treated with BMT+TT compared with those with BMT alone or those with BMT + DLI. The percentage of FoxP3+ regulatory T cells among CD4+ cells was higher in the mice treated with BMT + TT comparable to those treated with BMT + DLI. The results for CD8+ T cells were similar to those for CD4+ cells. [Conclusion]. Allo-BMT combined with TT induces high thymopoiesis, and can elicit strong GVL effects with mild GVHD reaction. We thus found that donor-derived T cells play an important role in the treatment of leukemia. Also the details of the mechanisms are still unknown, one possibilities may be the continuous supplementation of T cells from the allogeneic thymus. The results of the present study suggest this strategy will become a new way for the treatment of refractory or relapse leukemia in human.

Disclosures

No relevant conflicts of interest to declare.
Acute Graft Versus Host Disease after Donor Lymphocyte Infusion: A Single Center Analysis
Authors: He Fiona C. , Weisdorf Daniel J. , Warlick Erica D. , Miller Jeffrey S. , Holtan Shernan G. , Verneris Michael R. , Cao Qing , MacMillan Margaret L.
Published: October 2019

Donor lymphocyte infusion (DLI) is an option for relapsed hematologic malignancies following allogeneic hematopoietic cell transplantation (HCT). We analyzed the incidence and manifestations of acute graft versus host disease (GVHD) in patients with malignant and non-malignant conditions treated with DLI. At the University of Minnesota, we gave 171 DLI to 120 patients from 1995-2013. The cumulative incidence of grade II-IV acute GVHD was 31.6% (CI 25-42%,n = 40); grade III-IV 23.3% (CI 16-32%,n = 29). GVHD after DLI (n = 46) included involvement of skin in 70% (n = 32), lower gastrointestinal (GI) 65% (n = 30), upper GI 43% (n = 20), and liver 35% (n = 16). Patients receiving chemotherapy prior to DLI (chemo-DLI) had more frequent acute GVHD and GI GVHD. Significant risk factors for grade II-IV acute GVHD included: age > 40, chemo-DLI, malignant disease, and time from HCT to DLI < 200 days. Response to treatment of acute GVHD at 8 weeks was complete in 40% and complete/partial in 52%.

Patients developing GVHD had frequent disease response. In chronic myelogenous leukemia (CML) patients, responses were excellent (80%) with or without GVHD. The CR rate was 34% for non-CML malignancies; only 9% achieved CR without acute GVHD. Non-malignant diseases showed poor prognosis following acute GVHD and good prognosis without. Overall survival at 2 years for CML patients was similar (83% vs 79%, p = 0.89) with or without grade II-IV acute GVHD, but in non-CML malignancies survival was better in absence of acute GVHD (41% vs 22%, p = 0.04).

We observed frequent, yet therapy-responsive acute GVHD following DLI. DLI often induced remission in CML, but less so for non-CML malignancies without chemo-DLI, particularly in absence of acute GVHD. Improvements in DLI efficacy and GVHD management are still needed.

Disclosures

No relevant conflicts of interest to declare.
CD40L+ CD8+ T-Cell Dependent Antitumor Immunity
Authors: Thiel Andreas , Frentsch Marco , Listopad Joanna , Stark Regina , Sada Japp Alberto , Matzmohr Nadine , Meier Sarah , Taniuchi Ichiro , Blankenstein Thomas
Published: October 2019

CD40 is frequently expressed on malignant cells of different origin. Due to the observed antitumorigenic effects induced after CD40 engagement it represents an attractive target for immunotherapies. We demonstrate here that CD40L expressing tumor-specific CD8+ T-cell population can act as potent physiological CD40 agonist against CD40 expressing tumor cells. We demonstrate that in the course of cancer cell rejection high frequencies of tumor-specific CD40L+ CD8+ T cells are induced. Strikingly, in contrast to wild-type (wt), CD40L deficient CD8+ T cells were unable to prevent tumor formation in lymphopenic as well as in fully immunocompetent hosts. Apparently, in our setting CD40L expressed by CD8+ T cells is essential for cancer cell rejection. CD40L-mediated rejection does not depend on interaction with CD40+ host cells such as antigen-presenting cells but on CD40 expression by cancer cells. Our findings disclose CD40L expression by CD8+ T cells as a new antitumor effector function that should be implemented in future adoptive T-cell therapies against cancer.

Disclosures

No relevant conflicts of interest to declare.
Cytopenia at Diagnosis Impairs Stem Cell Mobilization Among Patients with Multiple Myeloma
Authors: Toprak Selami Kocak , Uslu Atilla , Atilla Erden , Topcuoglu Pervin , Gurman Gunhan , Ilhan Osman , Beksac Meral
Published: October 2019

Introduction:

The GIMEMA group has recently developed a risk score predicting mobilization failure (Musto et al, EHA 2014). The risk score included age, cytopenia at diagnosis, induction therapy and their toxicity. In this study we aimed to evaluate the impact of these factors on peripheral blood stem cell (PBSC) mobilization among patients with multiple myeloma (MM).

Patients and methods:

125 newly diagnosed patients with MM (M: 76, F: 49) planned for autologous stem cell transplantation; median age 58 years (range, 30-67) were included in the analysis. This retrospective study examined the impact of age (> 60 years), gender of the patients, cytopenia (Hb<10 gr/dL, absolute neutrophil count < 1x109/L, thrombocyte count < 100x109/L) at diagnosis, and severe hematological toxicity during induction therapy, the type of induction and mobilization methods, the presence of neuropathy, comorbid diseases such as diabetes mellitus, hypertension and renal failure (creatinine > 2 mg/dL), and the use of beta blocker drugs. Patients with CD34+ levels of <20/μL in peripheral blood at maximum stimulation were considered to be Poor Mobilizers. The total amount of PBSC <5x106/kg after a single mobilization procedure was defined as sub-optimal collection. Poor mobilization was observed in only two patients (<2x106/kg). Statistics: Comparison of categorical variables was evaluated by chi-square test or Fisher exact test. Nominal variables were compared with non-parametrical test, Mann-Whitney U or Kruskal Wallis test. P value below as 0.05 was accepted as significance.

Results:

Optimal mobilization with median two apheresis (1-4) sessions for PBSC was obtained in 85.6% of the patients (n=107). Median CD34+ cells in this group were 8.33x106/kg (5-27x106/kg). The presence of cytopenia at diagnosis was the only significantly detrimental factor on mobilization (90% vs 77.1%, p=0.04). When the patients was scored in four groups as having single or combined variables (age, cytopenia at the diagnosis or during induction therapy), we were not able to develop a risk score.

Conclusions:

In our experience, 14.4% of the myeloma patients showed suboptimal or poor mobilization. Use of bortezomib, age, presence of neurotoxicity or hematological toxicity at mobilization did not significantly impair mobilization. We were able to confirm only cytopenia at diagnosis, from the four factors reported by Musto et al, as a detrimental factor impairing the stem cell mobilization.

Disclosures

No relevant conflicts of interest to declare.