bluuebunny/crossref_metadata_2025 huggingface dataset:
Minor Blood Group Antigen Disparities in Sickle Cell Disease Population
Authors: Kumar Mukesh , Shelia Palmer , Winkler Annie Marie , Eckman James R.
Published: October 2019

Abstract 4381

Introduction:

Red blood cell (RBCs) transfusion is the mainstay of treatment for sickle cell disease (SCD) patients and indications continue to expand. Differences in geographic origin between blood donors and sickle recipients has been posited as a cause of the high rate of alloantibodies in patients with sickle cell disease (Vichinsky et al. NEJM 1990; 322: 1617–21). Alloimmunization is one of the major limiting factors of obtaining compatible blood for transfusion and exchange transfusion compromising the management of patients with SCD. Mismatch in minor antigen distribution has been attributed as one of the main reasons for significantly high rate of alloantibodies in SCD patients. Here we describe the minor blood group antigen lacking in SCD patients and comparing with frequencies in white and black populations.

Methods:

Patients with diagnoses of SCD, confirmed by standard hemoglobin electrophoresis, who had at least one type and cross match done from 1995 through 2011 were included in this study. All patients are evaluated for K, C, E, S, Fya, Fyb, Jka and Jkb antigens in addition to ABO and RhD system before transfusion. Results were routinely recorded in the electronic database of the Georgia Comprehensive Sickle Cell Program at Grady Health System to improve patient transfusion management. The frequency of antigens was compared to published prevalence of antigens in Whites and African American population.

Results:

A total of 594 patients were identified with SCD who have been tested for antigens during this period, of these, 310 (52%) females and 284 (48%) males. Average age of the patients is 27 (range 3–84). Most common antigen lacking in SCD patients is from Kell blood group K (98%). About 9% of white population is positive for K. Frequency of E+ is 30% in white population and 2% in blacks while 83% of SCD patients lack E antigen. Overall antigen distribution and comparison included in Table 1.

Conclusion:

Mismatch of antigen expression in white population (mainly donors) especially for K, E, C and Fya; may explain why these are common antibodies detected in SCD patients Anti E (42.3%), Anti C (30.2%), Anti K (28.1%) and Anti Fya (18.3%) (Rosse et al, Blood, Vol 76, No 7). Given these antigen frequency differences, efforts should be made to increase blood donation in the African American population and at least limited phenotypic matching continues to be justified.

Disclosures:

No relevant conflicts of interest to declare.
Therapeutic Red Blood Cell Exchange in Sickle Cell Anaemia Using the Spectra Optia® Apheresis System
Authors: Sipurzynski-Budraâ Sabine , Sovinz Petra , Lanzer Gerhard , Schallmoser Katharina
Published: October 2019

Abstract 4383

Background:

Sickle cell disease, an autosomal recessive genetic blood disorder, is caused by abnormal hemoglobin (Hb) due to a substitution of valine by glutamic acid at β6 (HbS). Typical adverse events are vaso-occlusive crises due to obstruction of capillaries causing ischemia, pain and organ damage, as well as enhanced hemolysis and frequent hemolytic crises. Further complications may include acute chest syndrome and stroke. To prevent long-term sequelae, repeated blood transfusions are necessary to decrease erythropoiesis of sickle cells. We suggest that it may be more effective to reduce sickle cells by a red blood cell (RBC) exchange, which is advantageous in avoiding iron overload by transfusions.

Patient and Methods:

We report on a 10 year old boy suffering from the homozygote form of sickle cell anemia. RBC exchange was performed using the Spectra Optia® Apheresis System (Caridian BCT vers.5) when the patient's hematocrit (hct) was less than 26%. Traditional RBC exchange was modified by a depletion phase to improve efficacy. During this phase (= isovolemic hemodilution) HbS-containing red cells were removed without admixture of donor red cells by using plasma as replacement fluid. To prevent adverse side effects caused by adenine, the additive solution in the RBC concentrates was replaced by saline solution or plasma.

Results:

The therapeutic apheresis was feasible repeatedly via peripheral venous access (flow: 30 ± 3 mL/min, anticoagulant (AC) infusion rate 0.8 – 1.1mL/min/L total blood volume, inlet: AC ratio 13: 1) and no adverse side effects were observed. The median interval between the procedures was 42 ± 2 days. The depletion time (replacement fluid: plasma) was 25 ± 5 min, the exchange time 90 ± 10 min. The hct value at the end of the procedure was between 33 and 34%, the fraction of the patient's remaining cells (FCR) was 24 ± 2% (calculated by the device).

Conclusion:

RBC exchange with the Optia Spectra® System is a fast and safe method for reducing sickle cells, particularly efficient in combination with a depletion phase. Compared to blood transfusions alone, the HbS concentration declines rapidly by depleting and replacing sickle cells with normal RBCs without hyperviscosity or iron overload. However, only long-term surveillance and treatment of a larger patient collective will show whether it is possible to prevent vaso-occlusive crises and organ damage over a longer time period.

Disclosures:

No relevant conflicts of interest to declare.
Diminished Oxidative Stress Responses in Bone Marrow Stromal Cell Lines Derived From Fanconi Anemia (Fanc-D2−/−) Mice
Authors: Berhane Hebist , Epperly Michael W. , Cao Shaonan , Zhang Xichen , Shields Donna , Goff Julie , Sprachman Melissa , Wipf Peter , Li Song , Gao Xiang , Greenberger Joel S.
Published: October 2019

Abstract 4398

Fanconi Anemia (FA) is an inherited cancer susceptibility syndrome associated with a defect in one or more of the 15 FA pathway gene products leading to bone marrow failure, increased cellular sensitivity to DNA cross-linking agents and increased risk of myeloid leukemia. Underlying DNA repair vulnerabilities render FancD2−/−, a homozygous deletion recombinant negative mice, sensitive to whole body irradiation. We quantitated hematopoiesis in long term bone marrow cultures, and the radiosensitivity and oxidative responses of derived marrow stromal and IL-3 dependent hematopoietic progenitor cell lines derived from FancD2−/− mice on a C57BL/6 background (generously provided by Drs. A. D'Andrea and K. Parmar, Dana-Farber cancer Inst., Boston, MA). Long-term bone marrow culture stromal cell lines derived from FancD2−/− were compared to FancD2+/− or FancD2+/+. FancD2−/− long term marrow cultures showed decreased cobblestone islands, representative of hematopoietic stem cell generating foci, and production of non-adherent cells compared to wild type FancD2+/+ or heterozygous FancD2 +/− cultures (p = 0.0148) and produced fewer colony forming cells on day 7 and 14 compared to FancD2 +/− or FancD2 +/+ cell lines (p < 0.0001). FancD2−/− stromal cell lines showed decreased DNA repair by comet assay after irradiation to 5Gy (P<0.0001). Following irradiation to 5 or 10 Gy, FancD2−/− cells had an increased cell doubling time (44.1 + 0.7 and 54.2 + 7.8) compared to FancD2 +/+ cells (35.5 + 1.5hrs and 33.6 + 1.3,hrs p = 0.006 and 0.051, respectively). FancD2−/− cells had decreased baseline antioxidant levels compared to the FancD2 +/+ cells (0.55 + 0.11 and 0.91 + 0.17 trolox units, respectively). FancD2−/− cells at 24 hr following 10 Gy irradiation had 0.39 + 0.03 trolox units compared to 0.76 + 0.07 trolox units in FancD2 +/+ cells (p = 0.006). In clonogenic irradiation survival curve assays, FancD2−/− marrow stromal cells were radiosensitive (Do = 1.43 + 0.06 Gy, n = 4.98 + 0.65) compared to FancD2 +/+ (Do 1.70 + 0.09 Gy and n = 8.33 + 0.72, p = 0.0395 and 0.0040, respectively) or FancD2 +/− cells (Do = 1.67 + 0.08 and n = 3.63 + 0.44, p = 0.0348 and 0.1365, respectively). Furthermore, FancD2 +/− cells were more radiosensitive compared to FancD2 +/+ cells ((n = 3.63 + 0.44 or 8.33 + 0.72), p = 0.0003). Thus, the FancD2 gene controls several parameters of sensitivity to oxidative stress and ionizing radiation as demonstrated in mouse long-term marrow cultures and derived cell lines.

Disclosures:

No relevant conflicts of interest to declare.
FDG PET/CT May Overestimate Red Bone Marrow Volume
Authors: Spigelman Zachary Simon
Published: October 2019

Abstract 4405

Sambuceti, et al. (Eur. J. Nuclear Med Mol Imaging, Aug, 2012. 39: 1326–1338) used a computational FDG PET/CT model to estimate the volume of the bone marrow space and specifically “Red” or actively hematopoetic marrow in 102 nonmetastatic melanoma patients. 34.9 percent of all marrow space was estimated to be populated by “Red” Marrow. There was no associated pathologic bone marrow or peripheral blood count correlation of this FDG PET/CT model. In an attempt to define pathologic correlation of the findings of Sambuceti et al., 102 consecutive pathologic samples of femoral hip replacements were evaluated for their “Red” Marrow content and correlated with the patient's peripheral blood counts. The patients reviewed did not have active carcinoma or recent exposure to chemotherapeutic agents. 48/104(47.1%) of the samples obtained contained active “Red” Marrow”upon microscopic evaluation of femoral head and shaft samples stained with Hematoxylin and Eosin. Of these samples “Red' Marrow averaged 5.05 percent of the pathologic sample volume (range 0–40%) as opposed to 20–30 percent of the estimated bone marrow segmental space calculated to be populated by “Red” Marrow heads of long bones as defined by the FDG PET/CT model. In this study patient gender, height, BMI, weight, red marrow cellularity, peripheral white blood cell counts, platelets, hemoglobin, hematocrit, creatinine did not predict the extent of “Red” Marrow volume. Many variables may affect the results of these studies including: spotty metabolic activity of the marrow, clinical inflammatory effects of bone marrow in long bone heads, pathologic sampling errors, low study power, sample preparation and patient selection. Although there was no clear explanation for this overestimation of pathologic-FDG PET signal, the age of the populations were significantly different (55.9 years old in Sambuceti et al and 62.4 years old in this study). Age was found to have a significant correlation with “Red “Marrow volume in the Sambuceti et al study. The FDG PET scan evaluation may overestimate metabolic activity of “Red” Marrow and thus run the risk of obscuring other bone pathology. Therefore, prospective PET/CT-pathologic-clinical correlation studies are warranted in light of the prognostic, therapeutic, and diagnostic importance of “Red“Marrow activity in FDG PET/CT clinical staging of oncologic disease.

Disclosures:

No relevant conflicts of interest to declare.
Overexpression of the EphB4 Receptor Contributes to Imatinib Resistance in Chronic Myeloid Leukemia Through Regulating Mlcp and VAV1
Authors: Xiaoli Liu , Zhang Jinfang , Du Qingfeng , Xu Na , Xu Lulu , Xiao Xiaozhen , Zhou Xuan
Published: October 2019

Abstract 4421

Objective:

To study the role of EphB4 in imatinib (IM) resistant chronic myeloid leukemia (CML) and investigate the mechanism.

Methods:

We derived IM-resistant cells, K562-R cells, from wild K562 cells under gradually increasing IM concentrations. We analysed expression level of EphB4 in CML patients, wild K562 and K562-R cell lines by real-time reverse transcription PCR and Western blot analysis. Then we established stable under-expressing EphB4 cell (K562-R-EphB4-sh) lines. We analysed the sensitive for IM of K562, K562-R, K562-R-EphB4-sh cell lines by CCK8 assay. Microarray analysis was used to screen differential expression genes between K562-R and K562-R-EphB4-sh cell lines.

Results:

The mRNA and protein of EphB4 were significantly increased in IM resistant CML patients compared to IM sensitive CML patients (p<0.05). The Similar results were observed in K562-R and K562 cells (p<0.01). To analyze the role of EphB4 in IM resistance, EphB4 was knocked down with shRNA expressed by pLL3.7 lentivirus vector. We established stable under-expressing EphB4 cell line K562-R-EphB4-sh. RT-PCR and western blot analysis showed that mRNA and protein expression of EphB4 in K562-R-EphB4-sh cells were reduced (p<0.05). CCK8 assay found K562 cells (IC50 0.1207±0.0234μM), K562-R-EphB4-sh cells (IC50 0.7228±0.04752μM) were sensitive to IM but K562-R (IC50 2.8101±0.04674μM) still showed IM resistance (p<0.05). Those suggested K562-R-EphB4-sh cells resensitize to IM when the expression of EphB4 was down regulated. However, these cells were still less sensitive than K562 cells. Microarray analysis between K562-R and K562-R-EphB4-sh cell lines found 641 differential expression genes, most of them were related to cell adhesion and cell cytoskeleton. We confirmed MLCP and VAV1 were down regulated in K562-R-EphB4-sh cells compared to K562-R cell lines by western blot analysis.

Conclusion:

Our study suggest EphB4 receptor contributes to IM-resistant in CML through regulating cell adhesion molecular MLCP and VAV1, which may provide new biomarkers and contribute to] developping new drugs for the disease.

Disclosures:

No relevant conflicts of interest to declare.
Enhanced Adhesion, Migration and Pyk2 Expression in K562 Cells Following Imatinib Treatment
Authors: Ovcharenko Adelina , Granot Galit , Park Jeniffer , Shpilberg Ofer , Raanani Pia
Published: October 2019

Abstract 4426

Background/Aims:

Despite improved prognosis of CML patients with the use of imatinib (IM), its administration is associated with extramedullary disease (EMD) occurrence. We postulate that, like in the metastatic processes, changes in migration and adherence potential may enable leukemic cells to inhabit extramedullary sites. Focal adhesion complexes linking between extracellular matrix and the cell cytoskeleton are likely to play an important role in these processes.

Pyk2 is a tyrosine kinase highly expressed in hematopoietic cells, localized to focal adhesion complexes, and known to participate in adhesion and migration processes. We have previously shown that Pyk2 participates in NB4 (an acute promyelocytic leukemia [APL] cell line) cells' adhesion and migration following exposure to the APL targeted therapy ATRA.

We postulate that similar to the effect of ATRA on NB4 cells, IM being also a targeted therapy, might also be associated with enhanced adhesion and migration abilities of the CML cell line K562.

Our objectives were to identify the effect of IM administration on pyk2 expression and on K562 cell adhesion and migration ability and to establish the role of these changes in treatment-associated EMD.

Results:

We found a 2.6-fold increase in pyk2 mRNA expression in K562 cells following exposure to IM. We also found that 30% of IM-treated K562 cells adhered to fibronectin (FN) compared to untreated cells having no adhesion ability. In addition, a 3-fold induction in migration was seen in K562 cells following treatment. Furthermore, K562 cells treated with IM demonstrated a 2-fold increase in invasion potential as compared to untreated cells. In order to assess whether Pyk2 is essential for IM-dependent adhesion and migration of K562 cells, these cells were infected with pyk2 specific shRNAs. While 30% of the non-infected NB4 cells adhered to FN following IM treatment, only 12% of the pyk2-shRNA–infected K562 cells exhibited adhesion potential (Pvalue<0.002). In addition, we witnessed over a 3-fold reduction in the ability of pyk2-shRNA–infected K562 cells to migrate following exposure to IM when compared to parental K562 cells. These data support the role of Pyk2 in IM-mediated adhesion and migration.

Finally, we found that IM treatment induced an in-vivo increase in pyk2 mRNA expression level in leukocytes derived from 3 out of 5 CML patients studied.

Conclusions:

IM induces K562 cell adhesion, migration and invasion accompanied by increased pyk2 expression. Pyk2 is one of the key proteins regulating IM-induced cell migration and adhesion. Collectively our data suggest a critical role of Pyk2 in adhesion and migration initiated by the targeted therapy IM and a possible role in EMD development. These data support a common mechanism for the development of EMD in hematological malignancies treated by targeted therapies via pyk2 expression.

Disclosures:

No relevant conflicts of interest to declare.
Ectopic Expression of ADAMTS13 in Platelets As a Novel Therapeutic Approach for Arterial Thrombosis
Authors: Pickens Brandy , Jin Sheng-Yu , Li Dengju , Zheng X. Long
Published: October 2019

Abstract 491

Megakaryocytes and platelets have been shown to produce ADAMTS13 and its only known substrate, von Willebrand factor (VWF). However, the role of platelet expression of ADMTS13 in modulation of thrombus formation is not known. Previous studies have shown that platelet-targeted delivery of clotting factor VIII corrects bleeding phenotype in hemophilia A mice despite of inhibitors. These results suggest that platelet-delivery of ADAMTS13 may also be efficacious for anti-arterial thrombosis and perhaps for treatment of acquired idiopathic thrombotic thrombocytopenic purpura (TTP) with inhibitors. In the present study, transgenic mice (JAX B6SJL/F1 hybrid) carrying a human full-length ADAMTS13 gene under a platelet glycoprotein 1b alpha promoter were generated. The mice were crossed with Adamts13−/− and TTP-sensitive mice (CAST/Ei) for 4 generations. Plasma and platelet ADAMTS13 protein and proteolytic activity were determined. By Western blotting and the cleavage of a fluorescein-labeled VWF73 substrate, we were able to show that human ADAMTS13 protein (∼195 kDa) and activity were present in the platelet lysate of transgenic (A13-PltTG) mice, but not in adamts13−/− mice or wild-type mice. No proteolytic activity was detected in plasma of the transgenic mice. The platelet ADAMTS13 protein was releasable upon stimulation with various concentrations of thrombin (0.1–0.5 U/ml) and collagen (2.5–10 μg/ml). The released ADAMTS13 and VWF (as a positive control) were primarily associated with platelet membrane, demonstrated by surface biotinylation. However, a small fraction of the released ADAMTS13 and VWF proteins were detected in the releasate after stimulation. Moreover, the A13-PltTG mice exhibited systemic anti-thrombotic activity, which attenuated the rate of thrombus formation in the mesenteric arterioles induced by a topical application of 10% ferric chloride. The rate of arterial thrombus formation in the transgenic mice was significantly lower than that in Adamts13−/−mice and wild-type mice in the same genetic background. We conclude that we have generated transgenic mice overexpressing human ADAMTS13 metalloprotease in platelets. The platelet expressed ADAMTS13 is releasable upon stimulation by agonists. The platelet derived ADAMTS13 is biologically functional in cleaving VWF in vitro and in vivo, which attenuate systemic arterial thrombosis after oxidative injury. Our ongoing effort is to determine the efficacy of platelet delivered ADAMTS13 as a potential novel therapeutic for acquired TTP patients with inhibitors.

Disclosures:

No relevant conflicts of interest to declare.
Haploinsufficiency of RPS14 and RPS24 in 5q-Syndrome
Authors: Corinaldesi Giorgio
Published: October 2019

Abstract 4964

The 5q-syndrome is a subtype of myelodysplastic syndrome, a rare disorder caused by the loss of a part of the long arm of chromosome 5, being firstly described in 1974 by Van Den Berghe; the cytogenetic characterization reports 4 types of deletion of chromosome 5 (q13-q31), (q13-q33), (q22-q25), (q32-q33) in a critical deletion region (CDR 1 and 2), that shows a reduction in gene expression of 50–60%, the aploinsufficiency includes the tumor suppressor genes, the regulation of gene splicing and apoptosis, the growth inhibitory protein, and the PDGF and p53 pathway. The CDR1 deletion involves genes encoding the 40S ribosomal subunit (D55479-CDC23, EGR1, CDC25C), while the CDR2 deletion includes several genes (SPARC, WIG-1, BMI-1, MEGF-1, RPS14, RPS24) encoding the transcription factor Egr-1/K20×20, the cytoskeletal remodeling protein, and the alphacatenin.

The mechanism of the aploinsufficiency remains unresolved, but we have observed the characteristic changes Cys224-with Arg234, and the down-regulation of micro RNA-genes RPL28, EF1D, and up-regulation of several pro-apoptotic genes BAX and CAPS3, that is caused by a defect in ribosomal protein function, which is characterized by macrocytic anemia, thrombocytosis, megakaryocyte hyperplasia with nuclear hypolobation, erythroblastopenia associated with displastic abnormalities of hematopoietic stem cells/progenitor cells (HSCs/HPCs), hypogranular neutrophils or pseudo Chediak-Higashi large granules, ringed sideroblasts, excess of blasts, and increased malignancy (acute myeloid leukemia).

In accordance of what recent data are currently suggesting we have also observed the mutation of the JaK2 gene and FLT3; this means that many other candidate genes may play a role in this disease and in the risk for clonal evolution and in the progression of acute myeloid leukemia.

Disclosures:

No relevant conflicts of interest to declare.
Towards CD38-Targeted Immunochemotherapy of Multiple Myeloma: Significant Improvement of Anti-Myeloma Effects of Lenalidomide, Bortezomib and Dexamethason by CD38-Specific Antibody Daratumumab, Even in Chemotherapy Resistant/Intolerant Patients
Authors: Nijhof Inger S. , van Bueren Jeroen Lammerts , van Kessel Berris , de Weers Michel , Bakker Joost M , Parren Paul W. H. I. , Lokhorst Henk , Mutis Tuna
Published: October 2019

Abstract 4988

To date, multiple myeloma (MM) remains an incurable malignancy of antibody-producing clonal plasma cells. The introduction of a new generation of immunomodulatory agents, such as lenalidomide (LEN), and the potent proteasome inhibitor bortezomib (BORT), used alone or in combination with steroids (dexamethasone; DEX or prednisone; PRED) has significantly improved the overall survival of MM patients. Nonetheless, all chemotherapy strategies are eventually hampered by the development of drug-resistance. Towards a novel and effective targeted immunotherapy for MM, we have developed daratumumab (DARA), a CD38 human antibody with broad-spectrum killing activity. In vitro, DARA induces substantial anti-MM effects mainly via ADCC (antibody dependent cellular cytotoxicity) and CDC (complement dependent cytotoxicity). In ex vivo assays, which allowed us to address killing of MM cells in bone marrow aspirates isolated from MM patients, enhanced or even synergistic MM cell killing was observed when DARA was combined with LEN, or with cocktails of LEN/BORT/DEX and melphalan/BORT/DEX. We now extended these ex vivo analyses to evaluate whether DARA in combination with LEN, BORT and DEX could improve the lysis of MM cells in bone marrow aspirates derived from 22 patients of whom 9 became refractory for LEN and 6 for LEN and BORT. DARA significantly enhanced the lysis of MM cells when combined with LEN or BORT in virtually all patients, including the LEN- and LEN/BORT-refractory patients. The combination of DARA+BORT and DARA+DEX induced additive killing, suggestive of lysis by independent mechanisms. When combined with LEN, DARA improved the lysis of MM cells in a synergistic manner in both non-refractory and LEN-refractory patients. This is suggestive of killing by at least partly complementary mechanisms. Synergistic activity of LEN and DARA was attributable to LEN-induced activation of effector cells that were involved in DARA-mediated ADCC. In addition, enhanced/synergistic direct killing of MM cells was observed. Experiments are under way to further investigate the mechanism underlying synergistic activity of DARA and LEN. In conclusion, our results provide a rationale for clinical evaluation of DARA in combination with LEN, BORT and DEX including in patients refractory to these drugs.

Disclosures:

van Bueren: genmab: Employment. de Weers:genmab: Employment. Bakker:genmab: Employment. Parren:genmab: Employment. Lokhorst:genmab: Consultancy, Research Funding. Mutis:genmab: Research Funding.
Peripheral Blood Involvement with Minimal Disease Is Common in Multiple Myeloma but Not in Smoldering Myeloma
Authors: Tembhare Prashant Ramesh , Yuan Constance , Korde Neha , Manasanch Elisabet E. , Kwok Mary , Roschewski Mark , Maric Irina , Calvo Katherine R , Mulquin Marcia , Zuchlinski Diamond , Landgren Ola , Stetler-Stevenson Maryalice
Published: October 2019

Abstract 4994

Background:

Plasma cell dyscrasias (PCD) are characterized by the accumulation of clonal abnormal plasma cells (APC) in the bone marrow (BM) and excessive monoclonal proteins in the serum and/or urine that ultimately leads to end-organ damage. BM evaluation of plasma cells (PC) is an important criterion for the diagnosis and risk stratification schemes in patient with PCD. The percent APCs in BM determined by flow cytometry (FC) has been shown to be an independent risk factor for progression from myeloma precursor disease (monoclonal gammopathy of uncertain significance, MGUS; smoldering multiple myeloma, SMM) to multiple myeloma (MM). However, the role of FC evaluation of APC in peripheral blood (PB) is not well studied. In this study we evaluate FC characterization of circulating PCs in the PB from patients with SMM and MM.

Methods:

Both PB & BM aspirate specimens from 27 patients (15 SMM & 12 MM) were analyzed with 8-color multiparametric FC using a panel of antibodies (CD138, CD38, CD19, CD20, CD27, CD28, CD45, CD56, CD117, CD13, CD14, CD16, CD3, CD34 and intracellular kappa & lambda light chains) and acquiring over 3 million events. The pattern of surface antigen and intracellular light chain expression was utilized to determine the percent of all PCs that are APC (defined as monoclonal with aberrant antigen expression). The percentages of PB APCs were correlated with percentages of BM APCs and BM biopsy findings.

Results:

Of the 27 cases, PCs were detected in PB from 20 patients (10 SMM & 10 MM). Mean percentage of total PCs in PB from SMM was 0. 011% (range 0. 002–0. 01) and from MM patients 0. 099% (range 0. 006–0. 5). APCs were detected in 4/15 (26%) SMM cases (mean APC 34. 8% of PCs, range 7–99%) and 10/12 (83%) MM cases (mean APC 97. 6% of PC, range 90–99%). We assessed the immunophenotypic features of APCs from PB with APCs of BM and no immunophenotypic statistical differences were detected.

Conclusions:

FC evaluation of PB PCs from patients is useful in the differentiation between MM and the precursor disease SMM. Greater than 95% APC was observed in PB in some MM patients. Extent of PB involvement with APC may be an independent prognostic indicator in monitoring progression of SMM to MM.

Disclosures:

No relevant conflicts of interest to declare.
Lenalidomide Synergistically Enhances the Effect of Dendritic Cell Vaccination in Mouse Multiple Myeloma Model
Authors: Nguyen-Pham Thanh-Nhan , Jung Sung-Hoon , Vo Manh-cuong , Lee Hyun Ju , Hoang My-Dung , Choi Nuri , Kim Hyeoung-Joon , Lee Je-Jung
Published: October 2019

Abstract 5010

Background:

Multiple myeloma (MM) is a B-cell malignancy that has remained essentially incurable by conventional therapy, highlighting the urgent need for novel treatment strategies. Lenalidomide (LEN) is thalidomide analog belonging to the class of immunomodulatory drugs which the activity are related with immunomodulatory properties. LEN augments both the adaptive and innate immune system via the co-stimulation of T cells, NK and NKT cells. In addition, LEN can inhibit the frequency and function of immunosuppressor cells. Therefore, LEN could be used to enhance immune response against MM. Cellular therapy with dendritic cells (DCs) is emerging as a useful immunotherapeutic modality to treat MM. The purpose of this study was to investigate the immunomodulatory effects of lenalidomide in combination with DCs vaccine to treat MM in vivomouse model.

Methods:

We used the MOPC315 myeloma murin model to evaluate tumor-specific cytotoxic T lymphocytes responses by a DC vaccine in combination with LEN. After tumor growth, LEN (50 mg/kg/day) was injected intraperitoneally at three consecutive days to cover the DC vaccination. The tumor growth inhibition effect and the antitumor activity of splenocytes from vaccinated mice were evaluated to reveal the synergistic effect of DCs and LEN.

Results:

The combination of LEN and DC vaccine efficiently inhibited tumor growth in mouse MM model when compared to single therapeutic agent. These vaccinated mice exhibit the reduction of myeloid-derived suppressor cells (MDSC) and regulatory T cell (Treg) in spleen. Inhibition of MDSC and Treg resulted in the increasing proportion of CD4+ and CD8+T cell in the spleen. High ratio of Th1- to Th2-type cytokines was induced by LEN plus DC vaccine. LEN also enhance the innate immune response by modulating NK cell number and function. In addition, LEN also can enhance the population of effector memory T cells in the spleen of vaccinated mice. Furthermore, the treatment of LEN can down-regulate the levels of VEGF and TNF-a on tumor tissues of vaccinated mice.

Conclusion:

These results suggest that a treatment combining the immunomodulatory drug lenalidomide with DC vaccine can improve antitumor immunity in mouse MM model by inhibiting immunosuppressor cells and recovering effector cells, as well as superior polarization of the Th1/Th2 balance in favor of Th1 type immune response.

Disclosures:

No relevant conflicts of interest to declare.
Lenalidomide Suppression of Multiple Myeloma Cell Proliferation Is Associated with Downregulation of LEF/TCF Activity
Authors: Hu Bo , Barlogie Bart , Chen Yu , Epstein Joshua , Qiang Ya-Wei
Published: October 2019

Abstract 5014

Lenalidomide (Rev) is frequently used to treat multiple myeloma (MM). We reported that Rev promotes Dkk expression in MM cells. A recent study reported that resistance to Rev was associated with induction of Wnt/β-catenin signaling by increased β-catenin transcription and its decreased destruction (Bjorklund, JBC 2011). In this study, we evaluated whether these reported effects represent selection of pre-existing cell by exposure to Rev or regulation of the canonical Wnt signaling pathway by Rev, and whether the Wnt signaling pathway is associated with Rev's direct effect on MM cell survival.

To test the effect on Rev on proliferation of MM cells lines, the six MM cell lines H929, INA6, MM144, OPM-1, RPMI 8226, and U266 were cultured in growth media containing serial concentrations (0 to 1000 μM) of the drug for 24, 48 and 72 hours, and effect on proliferation measured by MTT assay. Rev diminished proliferation of these cell lines at concentrations between 50 to 1000 μM at 24 hours, and maximal inhibition occurred at 72 hours. Rev had little effect on the proliferation of the five MM cell lines at levels lower than 50 μM. Treatment with ≥5 μM Rev for 24, 48 and 72 hours resulted in increased DKK1 mRNA and Dkk1 protein levels as determined by qRT-PCR and by ELISA, respectively, in a dose dependent fashion, even at concentration that did not inhibit cell proliferation. These data suggest that Rev diminish MM proliferation is independent of its effects on Dkk1. We next examined the effect of Rev on β-catenin protein in cells treated with serial concentrations (0 to 1000 μM) of Rev for 6 hours. Immunoblotting analysis showed increased total β-catenin protein in 8226, OPM-2, H929, MM144 and U266 exposed to ≤100 μM, and no further increase in β-catenin levels when these cells were exposed to Rev concentrations higher than 100 μM. Rev did not affect changes in β-catenin levels in INA6. To determine the effects on Rev concentrations on TCF transcriptional activity, we infected cell lines with lentiviral particles containing the TCF reporter or with empty vector. Rev increased TCF activity at lower concentrations (10–20 μM) in all cells. As Rev concentration increased, TCF transcriptional activity gradually decreased, and was strongly inhibited (over 80%) at concentrations from 125 μM to 1000 μM, depending on the cell line; in this range, Rev suppressed MM proliferation.

These results suggest that at cytotoxic concentrations, Rev regulation of TCF transcriptional activity is independent of its effect on total β-catenin levels. It remains to be determined if Rev-mediated inhibition of TCF activity is the cause of the drug's cytotoxic effect, and the mechanism of the concentration dependent effects of Rev on TCF activity.

Disclosures:

No relevant conflicts of interest to declare.
TGR-1202: A Novel, Targeted PI3Kδ Inhibitor in Multiple Myeloma
Authors: Vakkalanka Swaroop , Viswanadha Srikant , Boise Lawrence H. , Sportelli Peter , Miskin Hari , Lonial Sagar
Published: October 2019

Abstract 5018

Background:

The bone marrow microenvironment contributes to the pathogenesis of Multiple Myeloma (MM) by promoting the oncogenic process including drug resistance. High expression levels of the PI3Kδ isoform in patient MM cells implicate this target as a novel and attractive intervention strategy aimed at attenuating the progression of the disease. Herein, we describe the biological and pharmacokinetic properties of TGR-1202, a novel and small molecule PI3Kδ inhibitor with scope to be developed as a clinical candidate for patients with relapsed and/or refractory MM.

Material & Methods:

Activity of TGR-1202 on individual PI3K isoforms was determined in enzyme, cell, and whole blood based assays. Potency of the compound was confirmed viaMM cell viability and apoptosis assays besides testing for inhibition of pAkt, a downstream kinase regulating cell survival and growth. Additionally, the compound was tested for potency in viability, apoptosis, and Akt phosphorylation assays using immortalized and MM patient derived primary cells. ADME and pharmacokinetic properties of the molecule were also determined.

Results:

TGR-1202 demonstrated significant potency against PI3Kδ (22. 2 nM) with several fold selectivity over the α (>10000), β (>50), and γ (>48) isoforms. Additionally, the compound inhibited B-cell proliferation (24. 3 nM) and FcεR1 induced CD63 expression in human whole blood basophils (68. 2 nM) indicating specificity towards the delta isoform. The addition of 5 μM TGR-1202 to dexamethasone (0. 55 μM) caused a dramatic shift in GI50 of MM-1S cells compared to that of either TGR-1202 (9. 7 μM) or dexamethasone (18. 3 μM) alone. Further viability testing demonstrated that the compound caused a dose-dependent inhibition (>50% @ 1–3 μM) in growth of immortalized (U266B1 and MM-1R) as well as patient-derived MM cells. Reduction in viability was accompanied by a reduction in pAKT (>80% @ 3 μM) along with induction of apoptosis in both cell lines and patient samples. Pharmacokinetic studies across species indicated good oral absorption (>40% bioavailability for mice, rat, and dog) with favorable plasma concentrations (3–10 μM @ 20 mg/kg for mice, rat, and dog) relevant for efficacy.

Conclusions:

Data demonstrate the therapeutic potential of TGR-1202 in MM viainhibition of the PI3Kδ pathway. Data from immortalized as well as primary patient-derived cells justify further evaluation of this molecule in MM. Combination therapy of TGR-1202 and dexamethasone in a mouse MM-1S xenograft model is currently ongoing.

Disclosures:

Vakkalanka: Rhizen Pharmaceuticals S A: Employment, Equity Ownership. Viswanadha:Incozen Therapeutics: Employment. Sportelli:TG Therapeutics, Inc.: Employment, Equity Ownership. Miskin:TG Therapeutics, Inc.: Employment, Equity Ownership.
Bortezomib with Dexamethasone in Newly Diagnosed Patients with Primary Systemic Light Chain Amyloidosis or Multiple Myeloma-Associated AL Amyloidosis
Authors: Huang Beihui , Li Juan , Liu Junru , Zheng Dong , Chen Mei , Zhou Zhenhai , Xu Duorong , Zou Waiyi
Published: October 2019

Abstract 5036

Objective:

To assess the efficacy and tolerability of bortezomib with dexamethasone for patients with primary systemic light chain (AL) amyloidosis or multiple myeloma-associated AL amyloidosis.

Methods:

Twelve newly diagnosed patients with primary systemic AL amyloidosis and six patient with multiple myeloma-associated AL amyloidosis were treated with a combination of bortezomib (1. 3 mg/m2 d1, 4, 8, 11) and dexamethasone (20 mg d1–4).

Results:

Sixteen patients was evaluable. 12/16 had a hematologic response and 6/16 (37. 5%) a hematologic complete response. Median cycles to response was 1 cycle and median cycles to best response was 2 cycles. In patients with primary AL amyloidosis, 8/10 (80. 0%) had a hematologic response and 5/10 (50. 0%) a hematologic complete response. In patients with myeloma-associated AL amyloidosis, 7/10 (70. 0%) had a hematologic response and 1/6 (16. 7%) a hematologic complete response. Twelve patients (75. 0%) had a response in at least one affected organ, in which 7 in patients with primary AL amyloidosis and 5 in myeloma-associated AL amyloidosis. Person correlation between hematologic response and organ response was 0. 667 (p=0. 005). Fatigue, diarrhea and infection were the most frequent side effects. Three patients developed herpes zoster and had to stop chemotherapy.

Conclusions:

VD produces rapid and high hematological responses in the majority of patients with newly diagnosed AL regardless of primary or associated with myeloma. It is well tolerated with few side effects. This treatment may be a valid option as first-line treatment for newly diagnosed patients with primary systemic AL amyloidosis and multiple myeloma-associated AL amyloidosis.

Disclosures:

No relevant conflicts of interest to declare.
Lymphocytopenia Is Associated with an Increased Risk of Severe Infections in Patients with Multiple Myeloma Treated with Bortezomib-Based Regimens
Authors: Jung Sung-Hoon , Ahn Jae-Sook , Kang Seung-Ji , Yang Deok-Hwan , Kim Yeo-Kyeoung , Kim Hyeoung-Joon , Lee Je-Jung
Published: October 2019

Abstract 5042

Bortezomib is a proteasome inhibitor with potent antimyeloma activity in relapsed/refractory multiple myeloma (MM) patients. We evaluated the type and factors affecting the onset of infectious complications and mortality owing to infection in MM patients treated with bortezomib-based regimens. We reviewed 139 patients with MM treated with regimens containing bortezomib in order to assess the type and factors affecting the development of severe infections. Infections occurred in 56 (40. 3%) of 139 patients and 83 (7. 8%) cases of the 1, 069 evaluable cycles. Severe infections developed in 43 (30. 9%) patients and ten patients (7. 1%) died during bortezomib-based treatment. Multivariate analysis determined lymphocytopenia grade 3–4 (OR 3. 17, 95% CI 1. 38–7. 31, p = 0. 007) and number of cycle ° Â 8 cycles (OR 3. 91, 95% CI 1. 39–11. 02, p =0. 010) as risk factors associated with increased severe infection. This study showed that MM patients who received bortezomib-based regimens are at a higher risk of severe infections within eight cycles of treatment during especially severe lymphocytopenic period. MM patients treated with bortezomib-based regimens should be closely monitored for the development of infectious complications during lymphocytopenia.

Table I. Multivariate analysis of factors affecting severe infection development Parameter OR (95% CI) p-Value Age (> 65) 1.78 (0.77–4.13) 0.180 Immunophenotype IgA 1.51 (0.60–3.77) 0.379 Lymphocytopenia (grade 3–4) 3.17 (1.38–7.31) 0.007 Neutropenia (grade 3–4) 1.25 (0.55–2.85) 0.600 Number of cycles (°Â 8 cycles) 3.91 (1.39–11.02) 0.010

Disclosures:

No relevant conflicts of interest to declare.
Time to Treatment of Greater Than 1 Year Optimally Stratifies Patients with Improved Survival in Mantle Cell Lymphoma
Authors: Shah Bijal D. , Martin Peter , Cultrera Jennifer L. , Bello Celeste M. , Sokol Lubomir , Sotomayor Eduardo M.
Published: October 2019

Abstract 5082

Introduction:

Retrospective analyses suggest an improved outcome for patients with mantle cell lymphoma (MCL) in whom safe watchful waiting can be employed. We explored our own cohort of patients treated at the H Lee Moffitt Cancer Center in an effort to validate these data.

Methods:

We retrospectively identified 280 patients with MCL treated at our institution, for whom treatment data are available among 260. Patients were stratified according to whether they were treated within 3 months, within 3–12 months, and beyond 12 months. Treatment includes radiation, surgery, chemotherapy, immunotherapy, or any combination there-of. Median overall survival (OS) is calculated from date of diagnosis to death. Median progression free survival (PFS) is calculated from time of treatment start to progression or death.

Results:

Of 260 evaluable patients 191 were treated within 3 months of diagnosis, 33 were treated between 3–12mo, and 36 had received no treatment beyond one year. The median OS for each group were approximately 62mo, 74mo, and 130mo, respectively (Figure 1A). Median PFS following treatment initiation was nearly indistinguishable between the three groups, at approximately 24mo, 26mo, and 19mo, respectively (Figure 1B).

Conclusions:

Time to treatment of greater than 12 months may identify those with a more indolent course. However, once treatment is required, patients appear to experience similar treatment benefit.

Disclosures:

No relevant conflicts of interest to declare.
Adult T-Cell Leukemia/Lymphoma in Peru: A Report of 120 Cases
Authors: Beltran Brady E , Cotacallapa Erick , Castillo Jorge J
Published: October 2019

Abstract 5110

Background:

Adult T-cell leukemia/lymphoma (ATLL) is an aggressive subtype of peripheral T-cell lymphoma associated with the human T-cell lymphotropic virus type-I (HTLV-1). ATLL follows a specific geographic pattern and is more frequently seen in Southern Japan, the Caribbean basin, the Middle East and South America. This retrospective study attempts to describe the characteristics and define prognostic factors for patients with ATLL in Peru.

Methods:

We included consecutive cases of ATLL identified at our institution between January 1997 and January 2012. Diagnosis of ATLL was based on clinical history and histological findings consistent with ATLL, and either a positive HTLV-1 serology or evidence of HTLV-1 integration in the pathological sample. We used the Shimoyama classification to differentiate among ATLL subtypes. Patients' characteristics will be presented descriptively. For the univariate analysis, overall survival (OS) curves were estimated using the Kaplan-Meier method, and compared using the log-rank test.

Results:

We have included 120 patients who met the criteria for ATLL. The median age was 61 years (range 23–92 years), and 46% of patients was >60 years. The male-to-female ratio was 1. 2:1. ATLL types were acute in 49 (40%), lymphomatous in 53 (44%), cutaneous in 12 (10%), smoldering in 3 (3%) and chronic in 3 (3%) patients. An ECOG performance status >1 was seen in 51%, B symptoms were reported in 63%, stage III/IV in 85%, bone marrow involvement in 46%, and LDH was increased in 87% of the cases. An IPI score 0–2 was reported in 31% and score 3–5 in 69% of patients. Lymphocyte count <1000/uL occurred in 38% and monocyte count >1000/uL was in 33% of the cases. Chemotherapy was administered in 82% of patients, 18% could not receive treatment due to a bad performance status. The median OS for the entire group was 5. 5 months. Acute ATLL had a significantly shorter OS than lymphomatous (p<0. 001). A high IPI score (score 3–5) was associated with a shorter OS (p=0. 01).

Conclusions:

ATLL is a heterogeneous disease with distinct clinical features and outcomes. Based on our series, which represents the largest Latin-American cohort, acute had a worse prognosis than lymphomatous ATLL, and the IPI score should be used for risk-stratification.

Disclosures:

No relevant conflicts of interest to declare.
Clinicopathologic Characteristics and Clinical Outcomes in Patients with Testicular Non-Hodgkin Lymphoma
Authors: Kim Jeongseok , Yoon Dok Hyun , Park Ji Hyun , Jang Il Young , Kim Shin , Lee Kyoungmin , Kang Eun Hee , Huh Jooryung , Park Chan-Sik , Suh Cheolwon
Published: October 2019

Abstract 5111

Purpose

We aimed to evaluate the clinicopathologic characteristics and clinical outcomes in patients with testicular non-Hodgkin lymphoma.

Material and Methods

We reviewed the medical records of 24 patients with testicular non-Hodgkin lymphoma diagnosed at the Asan Medical Center between November, 2000 and June, 2012.

Results

Median age of the patients was 52 years (23–79 years). Histopathologic subtypes were as follows: DLBCL (n=18, 75%), Burkitt's lymphoma (n=2, 8. 3%), extranodal natural killer/T-cell lymphoma (NKTCL) (n=2, 8. 3%), chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) (n=1, 4. 2%) and T-cell lymphoblastic lymphoma (T-LBL) (n=1, 4. 2%). Ten patients (41. 7%) were in stage I, 1 patient (4. 2%) in stage II, and the other 13 patients (54. 2%) were in stage IV. Eastern Cooperative Oncology Group Performance Status (ECOG PS) was 1 in 22 patients (87. 5%) and >1 in 5 patients (20. 8%). Serum LDH levels were elevated in 14 patients (58. 3%). International Prognostic index (IPI) score was low (0–1) in 6 patients (25%), low-intermediate (2) in 8 patients (33. 3%), high-intermediate (3) in 7 patients (29. 2%), high (4–5) in 3 patients (12. 5%). B-symptoms were present in 4 patients (16. 7%). Bilateral testicular involvement was observed in 5 patients (20. 8%).

Fifteen patients (62. 5%) underwent orchiectomy as an initial therapeutic and diagnostic procedure. All the patients underwent chemotherapy: R-CHOP (n=16, 66. 6%), CHOP (n=2, 8. 3%), and other regimens (n=6, 25%). None received intrathecal prophylaxis just except a T-LBL patient. Prophylactic radiotherapy to contralateral testis was given in 12 patients (50%).

Twenty-one patients (87. 5%) achieved complete response. At a median follow-up duration of 22 months (1–139 months), 2 patients (8. 3%) showed disease progression and 7 patients (29. 2%) experienced disease recurrence; in the central nervous system (n=2, 8. 3%), regional lymph nodes (n=3, 12. 5%), bone marrow (n=1, 4. 2%), nasopharynx (n=1), skin (n=1), and testicular bed (n=1). Five patients (20. 8%) died of sepsis (n=3, 12. 5%) or progression of disease (n=2, 8. 3%). Median progression free survival and overall survival were 20 months (1–139 months) and 22 months (1–139 months), respectively.

ECOG PS >1 (p=0. 015) and bilateral testicular involvement (p=0. 000) were associated with a significantly short progression free survival (PFS). ECOG PS >1 (p=0. 001), high-intermediate or high risk of IPI (p=0. 010), presence of B symptoms (p=0. 035), and bilateral testicular involvement (p=0. 001) were associated with a significantly short overall survival.

Conclusions

Testicular lymphoma is a rare but aggressive extranodal lymphoma. High ECOG PS, high IPI, B symptom, and bilateral testicular involvement were associated with poor prognosis.

Disclosures:

No relevant conflicts of interest to declare.