bluuebunny/crossref_metadata_2025 huggingface dataset:
Pharmacokinetics of Dasatinib in a Patient with Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia During Treatment with Anti-Tuberculosis Therapy
Authors: Zeller Michelle P. , Beumer Jan H , Christner Susan M , Xenocostas Anargyros
Published: October 2019

Abstract 5006

Objectives:

Dasatinib is a second generation protein-tyrosine kinase inhibitor (TKI) indicated for first line treatment of chronic myeloid leukemia (CML) and second line treatment of Philadelphia chromosome-positive acute lymphoblastic leukemia (ALL) in patients who are imatinib resistant or intolerant.

In this study, we present the case of a patient with Philadelphia chromosome- positive ALL, intolerant to imatinib, who developed reactivated tuberculosis (TB) while receiving dasatinib during maintenance chemotherapy. The purpose of our study was to determine the effects of concomitant anti-TB therapy on dasatinib exposure by measuring plasma dasatinib levels using three different oral doses of the drug in this patient.

Methods:

The patient was treated with isoniazid, rifampin, ethambutol, pyrazinamide and pyridoxine for 2 months, followed by isoniazid, rifampin and pyridoxine for an additional 4 months. Dasatinib therapy was continued at the initiation of TB therapy using 100 mg po od, and titrated by 50 mg increments to 200 mg po od. Dasatinib plasma levels were measured prior to initiating anti-TB treatment and at regular intervals during concomitant treatment using three different dasatinib doses.

Results:

Eight days after initiation of anti-tuberculosis therapy, the apparent clearance of dasatinib had increased from 1410 to 2174 L/h at 100 mg. However, at 150 mg and 200 mg doses, apparent clearance had decreased to levels lower than baseline. As expected, the direction of peak plasma concentration changes was inverse to the changes in apparent clearance: Cmax values were 30 at baseline, and 22, 46, 102 ng/mL for the 3 doses of dasatinib during anti-TB therapy, respectively.

Conclusions:

Our findings suggest dasatinib clearance is increased by approximately 50% shortly after initiating anti-tuberculosis treatment and increasing the dose of dasatinib during anti-TB therapy was able to restore dasatinib plasma levels to those observed before initiation of anti-TB therapy.

(This work was supported by an unrestricted grant from Bristol Myers Squib)

Disclosures:

No relevant conflicts of interest to declare.
Nelfinavir Activates Epstein-Barr Virus and Kaposi's Sarcoma Herpesvirus Lytic Cycle by Inducing ER Stress
Authors: Shirley Courtney , Kalu Nene , Ambinder Richard F
Published: October 2019

Abstract 5011

Epstein-Barr virus (EBV) and Kaposi's Sarcoma herpesvirus (KSHV) are associated with lymphomas and other malignancies. We previously demonstrated that the proteasome inhibitor, bortezomib, leads to ER stress, induction of the unfolded protein response (UPR), and activation of EBV lytic gene expression.1 Here we investigate nelfinavir, an HIV protease inhibitor that has been reported to induce the UPR.2 Nelfinavir treatment of EBV Burkitt's lymphoma (BL) and KSHV primary effusion lymphoma (PEL) cell lines resulted in changes indicative of ER stress: elevated levels of ATF4, XBP1(s), and CHOP10 (Figure 1), as well as the EBV and KSHV immediate early proteins ZTA and RTA (Figure 2), respectively. The appearance of these UPR markers preceded expression of viral lytic RNAs. Regulated knockdown of Bip, an ER-stress sensor and activator of the UPR, by shRNA inhibited viral lytic RNA induction. These effects were observed using drug levels at or just above the levels achieved with standard clinical dosing of nelfinavir. Gantt et al. have reported that nelfinavir inhibits herpes virion production, including KSHV, in vitro.3 The ability to activate viral gene expression in combination with inhibition of virion production may identify nelfinavir as an especially promising agent for virus-targeted cancer therapies.Figure 1Figure 1. Figure 2Figure 2.

Analysis of nelfinavir (NFV) induced ER stress markers by RT-qPCR in Burkitt's lymphoma (Akata) and primary effusion lymphoma (BCBL-1) cells.

Analysis of nelfinavir (NFV) induced lytic activation by RT-qPCR in Burkitt's lymphoma (Akata) and primary effusion lymphoma (BCBL-1) cells.

Disclosures:

No relevant conflicts of interest to declare.
Elevated Chemokine Expression in the Bone Marrow of Patients with Myeloma and MGUS Is Associated with Marked Alterations in the Distribution of CD4+ and CD8+ T Cell Subsets within the Blood and Bone Marrow
Authors: Goodyear Oliver , Essex Sarah , McGettrick Helen , Basu Supratik , Anandram Seetharam , Buckley Christopher D , Filer Andrew , Khanum Rahela , Piper Karen P , Moss Paul AH , Pratt Guy
Published: October 2019

Abstract 5076

Multiple Myeloma (MM) is a malignant disease of bone marrow plasma cells characterised by immune dysfunction and increased chemokine levels. We investigated chemokine receptor expression on T cells, particularly the Th1 and Th2-associated receptors, CXCR3 and CCR4, in patients with paraproteinaemia and compared this to controls and correlated this to the level of related chemokines in blood and bone marrow.

The frequency of CCR4+ T cells within the bone marrow was markedly increased in patients with MM and the normal distribution between blood and marrow was reversed. CXCR3+ Th1 Cells which were rare within normal marrow were increased relative to blood. Th17 CD4+ T cells were markedly increased within marrow with increased Th17 CD4+ CCR4+ and Th17 CD4+ CXCR3 populations and the suppressor CD25highCd127low FoxP3positive subset reduced within blood. The expression of the ligands for CCR4 and CXCR3 was markedly increased within the marrow of patients with MGUS and MM. Mesenchymal stem cells isolated from the bone marrow of myeloma patients supported T cell adhesion and migration with a preferential recruitment of CCR4 and CXCR3-positive cells. Chemokine gradients within the tumour microenvironment alter the distribution of major T cells subsets between blood and marrow. This pattern is established early in the development of paraproteinaemia and may offer a potential target for new forms of treatment.

Disclosures:

No relevant conflicts of interest to declare.
Abrogation of Niche Signals Discovered New Genes Regulating Homing, Expansion and homeostasis of Multiple Myeloma Cells
Authors: Cohen Yossi , Garach-Jehoshua Osnat , Adina Bar-Chaim , Kornberg Abraham
Published: October 2019

Abstract 5080

Introduction:

Multiple myeloma (MM) cells depend on the bone marrow (BM) niche signals for their viability. Thus, tracing the earliest molecular changes in response to the lost of niche signals can disclose new homing related interactions.

Methods:

BM aspirates were collected in heparinized syringes and samples were flesh frozen at different time points. GEP (Affymetrix 1.0 st) was performed for 8 cases with massive BM infiltration with plasma cells. The GEP was also analyzed at different time points following spreading of cultured BM cells from the same patients (maintained in culture for several days).

Results:

Spontaneous induction of genes started within few minutes following aspiration, peaking in hours and persisting for at least 12 hours (Tables 1). RT-PCR results along several time points following BM aspiration from a representative case are presented in Table 2. Interestingly, the induction of genes reverted entirely in cultured conditions imposing cell-cell-matrix contact. However, spreading of the cultured cells re-induced these genes similar to that followed BM aspiration. Furthermore, in comparison of the GEP of cultured cells (frozen immediately after spreading) Vs the GEP from samples taken immediately after BM aspiration, an absolutely different repertoire of genes showed significant changes (Table 3):

Conclusions:

1. BM cells from MM patients respond immediately to changing microenvironment, with prompt upregulation of genes active in tumor expansion (FOS, AREG, JUN, PTGS2). However, such constitutive upregulation plus elevation of tumor suppressor genes (like NR4A2) is intolerable if not reverted by cell-cell-matrix contact. 2. In culture, BM cells from MM patients respond with dramatic upregulation of genes supporting neovascularisation (IL8, PLA27, MMP12, CXCL5), matrix (SPP1, FN1) and osteoclasts. 3. Most interestingly, in culture there is also downregulation of 20 genes critical to mitosis, especially relating to spindle machinery (KIF members, NUF2, TUBB1, ASPM, CENPE, CENPF) suggesting that the failure of long-term culture of primary MM cells results from mitotic crisis. This may also explain the lobulation of primary MM cell nuclei observed. 4. The unannotated gene MOP-1 has a role in MM homing.

Disclosures:

No relevant conflicts of interest to declare.
Anti-Myeloma Activity of LDC-000067, a Novel Selective Inhibitor of Cyclin Dependent Kinase 9: A Targeted Approach
Authors: Niepoth Kathrin , Gustavus Dirk , Wenning Doris , Meisterernst Michael , Kienast Joachim , Berdel Wolfgang E. , Bisping Guido
Published: October 2019

Abstract 5110

Background:

Cyclin-dependent kinase (CDK) inhibitors are reported to induce apoptosis in myeloma cells (Gojo et al., 2002; MacCallum et al., 2005). Here we investigated pro-apoptotic and anti-proliferative effects of LDC-000067, a novel selective CDK9-inhibitor, in three multiple myeloma (MM) cell lines (L-363; RPMI-8226; U-266). In addition, we studied consequences of selective CDK 9-inhibition regarding transcriptional activity (RNA-polymerase II phosphorylation) and related anti-apoptotic pathways (Mcl-1 and NFkB).

Methods:

L-363, RPMI-8226 and U-266 cells were exposed to either DMSO (vehicle control) or serial dilutions of LDC-000067 [0.5–20μM]. Apoptosis was quantified by flow cytometry analyses (FITC Annexin V / propidium iodide staining) after 24 hours in corresponding samples. To further investigate mechanisms of induced apoptosis by LDC-000067 we examined corresponding protein extracts by immunoblot analyses.

Results:

We found that LDC-000067 showed anti-myeloma activity in all myeloma cell lines tested. The proportion of induced apoptosis by exposure to LDC-000067 was equipotent (RPMI-8226) or higher (L-363 and U-266) compared to Seliciclib, a selective CDK 2-, 7- and 9-inhibitor. The IC50 for LDC-000067 was 4.0 μmol/L for L-363, 6.9 μmol/L for U-266 and 11.5 μmol/L for RPMI-8226. LDC-000067 inhibited phosphorylation of RNA polymerase II at Ser2 of the carboxyl-terminal domain in all MM lines tested. In addition, exposure to LDC-000067 resulted in a rapid decline of the protein levels of Mcl-1, known as an anti-apoptotic protein in MM. Furthermore, it is reported that in 40% of MM cell lines and 17% of MM patients the NFkB pathway is constitutionally activated by mutations (Demchenko et al., 2010) and inhibition of the NFkB activity causes induction of apoptosis (Hongyu et al., 2001). In our study we showed that LDC-000067 potently inhibited the NFkB pathway. Immunoblot analyses revealed a decreased phosphorylation and degradation of the NFkB inhibitor IkBa and an inhibition of phosphorylation of the NFkB subunit RelA (p65). Moreover, our data demonstrated that exposure to LDC-000067 caused a significant reduction of the protein levels of cyclin D1 and cyclin D2 strongly correlated to cell cycle arrest.

Conclusion:

In conclusion, our data corroborate that LDC-000067, a novel selective CDK 9-inhibitor, shows anti-myeloma activity by targeting three key pathways in the molecular pathogenesis of MM: firstly, it induces consecutive decline of Mcl-1 protein and inhibition of RNA polymerase II phosphorylation; secondly, it inhibits constitutively activated NFkB pathway; and thirdly, it reduces levels of D-type cyclins.

Disclosures:

No relevant conflicts of interest to declare.
A Phase I Study of Arsenic Trioxide (Trisenox), Ascorbic Acid, and Bortezomib (Velcade) Combination Therapy in Patients with Relapsed/Refractory Mulitple Myeloma
Authors: Held Lauren , Gockerman Jon P. , Diehl Louis F. , de Castro Carlos Manuel , Moore Joseph O. , Rizzieri David A. , Long Gwynn D. , Horwitz Mitchell E. , Chao Nelson J. , Gasparetto Cristina
Published: October 2019

Abstract 5129

Purpose:

Bortezomib is a well recognized standard therapy, however it is not curative for multiple myeloma. New agents and approaches are needed to overcome resistance in multiple myeloma. Arsenic trioxde (ATO) induces apoptosis of plasma cells through a number of mechanisms, including down regulation of gene overexpression, activating cell cycle arrest by inducing p21 cyclin-dependent kinase inhibitor protein, and by triggering apoptosis through caspase-3 in a dose dependent manner. This phase I study assessed the feasibility and tolerability of concomitant administration of arsenic trioxide (ATO), ascorbic acid (AA), and bortezomib (VelcadeÔ) (AAV) in patients with relapsed/refractory multiple myeloma.

Experimental Design:

A standard dose of ATO (0.25 mg/kg IV infused over 1–2 hours) and AA (1g IV infused over 15 minutes after infusion of ATO) were given once weekly × 2 with an escalating dose of bortezomib (cohort 1: 1 mg/m2 or cohort 2: 1.3 mg/m2 IV bolus on days 1, 8) of a 21 day cycle). ATO was given at least 1 hour prior to bortezomib and patients were allowed up to a maximum of 6–8 cycles.

Results:

A total of ten patients (median age, 62 years old) were enrolled with a median follow up of survivors of 25 months. Patients had an average of 4 prior failed therapies. Seven (70%) patients were refractory to bortezomib when previously administered. Despite our patient population being heavily pre-treated, objective responses were observed, with one partial response (Cohort 2), two minimal response (Cohort 1 and 2), and one stable disease (Cohort 2). To date, three of the 10 patients are continuing maintenance therapy 13–43 months from initiating this study.

Conclusion:

Of the patients that completed the treatment, objective responses were observed despite suboptimal dosing and previous bortezomib treatment failure. Tolerability was observed in most patients as discontinuation was not due to treatment toxicities, but due to aggressiveness of disease. Further studies are warranted with a larger patient population to effectively determine the effectiveness of AAV in relapsed/refractory multiple myeloma.

Disclosures:

Rizzieri: Cephalon: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Long:Millennium: Speakers Bureau. Gasparetto:Millennium: Speakers Bureau.
Early Response to Botezomib Combined Chemotherapy Can Be Helpful for Prediction of Progression Free Survival in Patients with Multiple Myeloma Who Were Ineligible for Stem Cell Transplantation
Authors: Lee Ho Sup , Kim Yang Soo , Kim Kihyun , Kim Jin Seok , Kim Hyo Jung , Min Chang-Ki , Suh Cheolwon , Eom Hyeon-Seok , Yoon Sung-Soo , Lee Jae Hoon , Kim Min Kyoung , Kim Sung Hyun , Bae Sung Hwa , Mun Yeung-Chul , Jo Deog Yeon , Chung Joo Seop
Published: October 2019

Abstract 5138

Background:

Response to treatment has been associated with improved survival in multiple myeloma (MM). The use of novel agents changed achievement of CR rates compared with conventional chemotherapy. The purpose of our study is to show influence of early response after treated with bortezomib combined chemotherapy to survival in patients with newly diagnosed MM who are ineligible for stem cell transplantation.

Methods:

We assessed response at least forth cycles before next chemotherapy by international myeloma working group response criteria. The median duration from starting chemotherapy to initial response date was 2.3 month (range; 0.9 – 3.7 months). The enrolled cases were all received bortezomib combined chemotherapy as front line therapy. We divided into good response group (A group) which were included showing more than very good partial response (VGPR) and poor response group (B group) which were partial response (PR) or less than PR. Endpoints were comparision of progression free survival (PFS) and overall survival (OS) between A and B groups.

Results:

We retrospectively analyzed 129 patients registered data for our study from the Korean Multiple Myeloma Working Party (KMMWP) performed a nationwide registration of MM patients. In our results of initial response, 16 patients were in CR (12.4%), 50 were in VGPR (38.8%), 40 were in PR (31.0%), 14 were in SD (10.9%), and 9 were in PD (7.0%). However, in results of best responses, 46 patients were in CR (35.7%), 31 were in VGPR (24.0%), 32 were in PR (24.8%), 11 were in SD (8.5%) and 9 were in PD (7.0%). 3 years PFS of A group and B group were 55.6% and 18.4%, respectively (p-value < 0.001). 3 years OS of A and B group were 65.3% and 52.9%, respectively (p-value 0.078).

Conclusion:

Early response at least forth cycles before next chemotherapy might be helpful for prediction of PFS in patients who were ineligible stem cell transplantation.

Disclosures:

No relevant conflicts of interest to declare.
Selective Accumulation of CD45RO+CCR7- in the Marrow Aspirates in Patients with Essential Thrombocythemia
Authors: Lange Andrzej , Sedzimirska Mariola , Klimczak Aleksandra , Skrzypek Daria Drabczak
Published: October 2019

Abstract 5155

Patients with newly diagnosed essential thrombocythemia (ET) were investigated for selective accumulation/expansion of CD4+ lymphocytes subpopulations in the marrow, to verifie the hypothesis that the neoplastic process associated factor(s) are recognized by the immune system.

Sixteen patients (F/M: 12/4, age 28–72 - median 60 yrs) with ET diagnosed according to the WHO criteria were studied. They had platelets from 496–809 x 10̂3/ul (median 644 × 10̂3 ), (12 heterozygotes, 2 homozygotes). Cytological evaluation revealed normal differentiation of erythroid and myeloid lineages, in contrast an increased number of megakaryocytes often enlarged, hyperlobulated and in clusters were seen in all patients. Trephine biopsy revealed activation of megakaryopoiesis with enlarged numerous megakaryocytes with normal representation of other lineages and a lack of an increase in the reticulin fibers. Marrow cellularity was from 9 – 126 x10̂3/ul (median 42 × 10̂3/ul).

The whole populations of marrow and blood cells were labeled for a purpose of this study with the use CD4, CD25, CD45RO and CCR7 MoAb (Becton Dickinson, San Jose, CA, UmarSA) followed by detection and analysis with the use of flow cytometry (FACS Calibur, Becton Dickinson) equipped with PCLysis programe. Immunostaining of trephine biopsies was used for detection of FOXP3 cells.

It was found:

(i) Lymphocytes in the marrow were in proportions from 4,5%-15,7% (median 7,25%), in numbers from1377/ul to 8245/ul (median 3870/ul) what was significantly higher as compared to blood from 1300/ul to 3900/ul (median 2100/ul) (Wilcoxon Matched Pairs Test, p=0.010).

(ii) CD45RO+CCR7- lymphocytes were higher in numbers in the marrow aspirates as compared to blood (median 1281/ul vs 631,2/ul, p=0,04 Wilcoxon Test for pairs).

(iii) In contrast numbers in the marrow aspirates of CD45 RO-CCR7+ (median 297,4/ul vs median 238,2/ul), as well as CD45 RO+CCR7+ (median 149,6/ul vs 146,4/ul) did not differ from those in blood.

(iv) It was a significant increase of CD4+CD25high+ cells in the marrow aspirates as compared to blood (median 36,2/ul vs 9,1/ul; p=0,02 Wilcoxon Test for pairs). Indeed in 11 out of 14 trephine biopsies investigated FoxP3+ cells were identified.

In conclusion an increase, in the marrow of ET patients, of effector/memory CD4 lymphocytes (CD45RO+CCR7-) but not those of naïve and central memory lymphocytes phenotypes points out on the selective accumulation of these cells in the marrows. Concomitant increase of T regulatory cells (CD4+CD25+high) suggests that an active immunological is taking place at the site. It is possible that effector memory cells are attracted to come to the marrows by ET process associated factors and if so the present observation constitute the primary observation building the rationale behind immunotherapy in ET patients

Disclosures:

No relevant conflicts of interest to declare.
Clinico-Pathological Characteristics of RAS Associated Autoimmune Lymphoproliferative Syndrome Like Disease (RALD), and RAS Mutated Juvenile Myelomonocytic Leukemia (JMML)
Authors: Takagi Masatoshi , Morio Tomohiro , Koike Kenichi , Mizutani Shuki
Published: October 2019

Abstract 5169

Autoimmune lympho proliferative syndrome (ALPS) is a disease characterized by dysfunction of the FAS-mediated apoptotic pathway, currently categorized as Type Ia, germline TNFRSF6/FAS mutation; Type Ib, germline FAS ligand mutation; Type Is, somatic TNFRSF6/FAS mutation; and Type II, germline Caspase 10 mutation. Patients exhibit lymphadenopathy, hepatosplenomegaly, and autoimmune diseases such as immune cytopenia and hyper-γ-globulinemia. An additional subclassification has been proposed that includes Types III, which has been defined as that with no known mutation but with a defect in FAS-mediated apoptosis. Juvenile myelomonocytic leukemia (JMML) is a chronic leukemia in children. Patients show lymphadenopathy, hepatosplenomegaly, leukocytosis associated with monocytosis, anemia, thrombocytopenia. About 80% of patients with JMML have been shown to have a genetic abnormality in their leukemia cells including mutations of NF1, RAS family, CBL, or PTPN11. HSCT is currently considered the only curative treatment. However, some patients have been shown to achieve remission without HSCT. Interestingly, there are several cases of JMML reported simultaneously having clinical and laboratory findings compatible with autoimmune disease. Germline RAS pathway mutations cause Costello (HRAS), Noonan (PTPN11, KRAS, and SOS1), and cardio-facio-cutaneous (CFC) syndromes (KRAS, BRAF, MEK1, and MEK2). Patients with Costello and Noonan syndromes have an increased propensity to develop solid and hematopoietic tumors, respectively.

We have recently reported a unique disease which lies between ALPS and JMML. The patients were characterized by prominent autoimmune cytopenia, lymphoadenopathy/splenomegaly and moderate monocytosis resembling to ALPS and JMML, but they did not satisfy the diagnostic criteria for ALPS or JMML. KRAS mutation in hemopoietic precursors including T and myeloid linage is the common molecular feature, and we have recently proposed these cases to be defined under a new disease entity of RAS associated ALPS like disease (RALD). These patients were characterized by activated T cells resistant to IL-2 depletion but not to FAS-dependent apoptosis associated with the decreased expression of pro-apoptotic protein BIM.

Since somatic RAS mutation has been identified in classical JMML and RALD, it is tempting to speculate that the developmental stage dependent RAS mutation of hemopoietic stem/precursors may determine the clinical features of JMML and RALD. Clinico-pathological characteristics of three RALD cases and three JMML cases cured without SCT will be presented and discussed.

Disclosures:

No relevant conflicts of interest to declare.
Inflammation and Thrombosis In Essential Thrombocythemia
Authors: Cacciola Rossella , Di Francesco Ernesto , Ferlito Carmen , Pezzella Francesca , Seria Elisa , Cipolla Antonino , Cacciola Emma
Published: October 2019

Abstract 5175

The essential thrombocythemia (ET) is a myeloid neoplasm characterized by an inflammatory component responsible for coagulant activation and angiogenesis. Therefore, we evaluated platelets, interleukin-8 (IL-8) and interleukin-15 (IL-15), as inflammatory indicators, tissue factor (TF), as marker of coagulative activation, and vascular endothelial growth factor (VEGF), as indicator of angiogenesis, in patients with ET and thrombosis. We recruited 25 thrombocythemic patients (10 males and 15 females, mean age 58 years) who fulfilled WHO.criteria. Their mean duration of disease was 8 years (range, 5–21 years). All patients were on aspirin. None of the patients had thrombotic risk factors. Of 25 patients, 10 developed thrombosis including 5 episodes of microvascular thrombosis of extremities (erythromelalgia), 2 of transient ischemic attack (TIA), 2 of myocardial infarction (IMA) and 1 of deep vein thrombosis (DVT)) whereas 15 did not. Platelets were measured by automated analyser. IL-8, IL-15, and VEGF and DD were measured by ELISA and immunoturbidimetric assay, respectively. All patients had thrombocytosis (961±235×109/L). IL-8, IL-15, TF and VEGFPLT were higher in patients with thrombosis than patients without thrombosis (27±14 pg/ml and 14±4 pg/ml and 214±266 pg/ml and 2±1 pg/106l vs 14±7 pg/ml and 26±6 pg/ml and 108±64 μg/l and 1±0.9 pg/ml, respectively) (p=0.009 and p<.0001 and p=0.005 and p=0.059, respectively). A positive correlation there was between IL-8 and IL-15 (p<.0001) and between IL-15 and TF (p=0.046). A tendency towards correlation was found between IL-15 and VEGF (p=0.082). These findings suggest a inflammatory IL-8-IL-15 interplay responsible for coagulation and angiogenesis activation that might mean increased thrombophilia in ET.

Disclosures:

No relevant conflicts of interest to declare.
Early Macrophage and Mast Cell Responses in Diffuse Large B-Cell Lymphoma After Immunochemotherapy
Authors: Taskinen Minna , Raunio Anna , Pöyhönen Maria , Karjalainen-Lindsberg Marja-Liisa , Leppä Sirpa
Published: October 2019

Abstract 5206

Background:

The prognostic impact of tumor microenvironment on the survival of lymphoma patients has recently been reported. However, early molecular and cellular responses to immunochemotherapy are unknown. Here, we have compared the tumor-associated macrophage (TAM) and mast cell (MC) contents in the lymphoma tissue in vivo before and after the first immunochemotherapy course in a small cohort of aggressive B-cell lymphoma patients.

Patients and methods:

The population of this pilot study consisted of seven diffuse large B-cell lymphoma (DLBCL) and three grade IIIB follicular lymphoma (FL) patients treated with rituximab in combination with CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone)-like regimen (immunochemotherapy). Paired tumor samples were collected before and a day after the first course of therapy, and evaluated immunohistochemically for CD68+, CD163+ macrophages and tryptase+ MCs. Freshly frozen lymphoma tissue containing enough material for paired mRNA analyses was available from 8 patients.

Results:

Comparing pre- and post-treatment tissue samples, an increase in the number of CD68+ TAMs was observed (p=0.023), whereas no variation in MC contents was found. If the patients were grouped according to response, i.e. remission (n=7) vs relapse (n=3), the most significant increase after therapy was observed in M2-type CD163+ TAM content (p=0.001). In the exon array analyses, the mRNA levels of both CD68 (p=0.052) and CD163 (p=0.023) genes increased after therapy.

Conclusions:

Our preliminary data suggest significant changes in macrophage content and their relative subsets in the lymphoma microenvironment after the first course of immunochemotherapy.

Disclosures:

No relevant conflicts of interest to declare.
Clinical, Phenotypic, and Genetic Characteristics of Non-CLL Type Monoclonal B Lymphocytosis (MBL)
Authors: Piran Siavash , Amato Dominick , Mullaithilaga Nisa , Wang Chen
Published: October 2019

Abstract 5209

Monoclonal B-cell lymphocytosis (MBL) has been established as a diagnostic category to denote the finding of clonal B lymphocytes in otherwise healthy and asymptomatic individuals. MBL likely represents early lesions in lymphoid neoplasms. Immunophenotypically, MBL is divided into CD5+ CLL-type and CD5- non-CLL type MBL, respectively. While most studies have focused on CLL-type MBL and provided evidence of progression of MBL to clinical CLL, the biological nature and clinical relevance of non-CLL type MBL remain unclear. In this study, we have identified and evaluated a series of 18 patients with non-CLL type MBL. This group comprised 10 males and 8 females with a median age of 74 years (range 53–95). All the patients presented initially with a mild to moderate absolute lymphocytosis. Clinical and laboratory evaluations were performed with a follow-up period of 1–20 years (median 8.5). Clonal B lymphocyte populations were confirmed by flow cytometry analysis. All the MBL clones showed negative for CD5, CD10 and CD103, positive for CD19 and CD20, and heterogeneous for CD23, CD25, CD79b, FMC7 and CD38. The phenotypic findings excluded CLL, mantle cell lymphoma and hairy cell leukemia, but may overlap with marginal zone lymphoma or lymphoplasmacytic lymphoma. Cytogenetics were available for 9 patients. Of the cytogenetic abnormalities, 7q deletion was found in two clones, 17 q deletion in two other clones, 11q deletion in one clone and t(2;7) in another clone. An overall tendency was shown toward clonal expansion with an increase in absolute lymphocyte count (ALC) over time (initial mean ALC of 4.9 ± 0.46 × 109/L to a recent mean ALC of 8.3 ± 2.18 × 109/L, p = 0.151). Three patients had an initial ALC >8 × 109/L which remained stable in one patient and decreased to <6 × 109/L in 2 patients. The patient with the longest follow-up of 20 years underwent a completely benign course, while the CD5- clonal B cell population was persistent but non-progressing. Of the progressive clones, three patients had an increase in ALC >10 × 109/L; one patient's ALC progressed over a period of 9 years to 35 × 109/L that required treatment. Clinically, three patients developed splenomegaly and one patient progressed with lymphadenopathy. Unlike the findings from population screening, this study represents a series of clinical MBL of non-CLL phenotype, and our results indicate the heterogeneity in genetic changes and clonal progression.

Disclosures:

No relevant conflicts of interest to declare.
Prevalence and Prognostic Molecular Profiles in Hepatitis B Virus Infected Patients with Diffuse Large B Cell Lymphoma: A Retrospective Study From Chinese Population
Authors: Wang Huijie , Cao Junning , Hong Xiaonan
Published: October 2019

Abstract 5211

Introduction:

Meta-analysis demonstrated that B cell lymphoma appears to be at high risk of infection by Hepatitis B Virus (HBV). Recently, cohort study showed HBV infection is associated with the increasing risk of B cell type lymphoma. Retrospective studies demonstrated high prevalence of HBV infection in Chinese lymphoma patients, with HBsAg-positive rates ranging from 23.5% to 30.5%. HBsAg-positive Diffuse Large B Cell Lymphoma (DLBCL) patients are associated with younger age and more advanced stage. However, the relation between HBV infection and poor prognosis in DLBCL remains uncertain. Molecular markers based on Immunohistochemical (IHC) expression were widely used as prognostic markers in DLBCL. Our study aimed to investigate the prevalence of HBVinfection in the different subtypes of lymphoma, and the molecular prognostic profiles in HBsAg-positive DLBCL.

Patient and Methods:

From Jan 2008 to Dec 2010, a total of 672 patients diagnosed as lymphoma were tested for HBsAg. Of these, 399 were male and 273 were female; 426 were B-NHL, 216 were T-NHL and 30 were HL. The expressions of BCL-2, BCL-6, CD10, MUM-1 were tested in patients diagnosed with DLBCL.

Results:

We found the overall HBsAg-positive rate was 15.9% (107/672) in total lymphoma patients. The positive rate of HBsAg was higher in B-NHL patients (20%,85/426) than T-NHL(9.7%,21/216) and HL (3.3%,1/30) (P<0.001).The lower prevalence were observed several subtypes of lymphoma including mantle cell lymphoma (0/20, 0%), Burkett's lymphoma (0/13, 0%), HL (1/29, 3.3%) and PTCL (4/53, 7.5%).The rate of HBV infection was higher in DLBCL patients (20.9%, 68/326). Among prognostic molecular profiles in DLBCL, the positive expression rates of BCL-2, BCL-6, CD10 and MUM-1 were 65.4% (134/205), 51.3% (116/226), 17% (39/230) and 61.5% (139/226), respectively. The non-GCB subtype accounted of 68.7% of DLBCL. Between HBsAg negative and positive group of DLBCL, the positive expression rates of BCL-2 were 64.1% vs 69.2% (P=0.498), CD 10 were 16.6% v 18.2%(P=0.781), MUM-1 were 60.5% vs 64.8%(P=0.567). However, the BCL-6 positive rate was slight higher in HBsAg negative group compared with positive group (55% vs40%, P=0.053). Finally, non-GCB subtype rates were 67.7% vs 68.7% in HBsAg negative and positive group DLBCL.

Conclusion:

Our date demonstrated that the infection rate of HBV was higher in DLBCL, whereas lower in the subtypes of mantle cell lymphoma, burkitt's lymphoma and HL. Based on IHC analysis, non-GCB subtype accounted of two-third of DLBCL in our series. Patients with HBsAg-positive DLBCL had lower positive rate of BCL-6. However, the expression of Bcl-2, CD10, MUM-1 seems no significant difference between patients with HBsAg-positive and negative DLBCL.

Disclosures:

No relevant conflicts of interest to declare.
Thromboelastography - Viscoelastic Haemostatic Assay (VHA) Versus Routine Plasma Based Coagulation Parameters in Trauma Patients to Detect Coagulopathy within 24 Hrs After Injury
Authors: Shah Bhaumik Arvindkumar , Subramanium Arulselvi , Sharma Subhadra , Agrawal Deepak , Chhabra Gaurav , Mukhopadhyay Asok Kumar
Published: October 2019

Abstract 5248

In India trauma related deaths occur every 1.9 minutes. Mortality in severe traumatic injury (ISS>16) is six times higher in developing country like India. Coagulopathy is observed in almost 25– 30% of trauma patients which itself is an independent risk factor for haemorrhage. Coagulopathy detected early after injury is indicative of injury severity and itself is a prognostic factor for mortality.

Aim

To find out the usefulness of thromboelastography (TEG) in detecting coagulopathy in contrast to conventional methods of plasma based standard coagulation parameters (PT, aPTT, TT, fibrinogen, D-dimer)

Objective

To detect coagulopathy early by TEG in trauma patients within 24 hrs after injury which can be useful to guide haemostatic therapies to reduce mortality.

Materials and methods

Patients admitted to trauma casualty were studied within 24 hrs after injury. Native whole blood was withdrawn through venepuncture appropriately in syringe using 21G needle and TEG was performed within 2 mins. Blood was also collected in citrated tube to assess standard coagulation parameters (prothrombin time, activated partial thromboplastin time, thrombin time, fibrinogen, D-dimer) and also by means of thromboelastography.

Results

Patients (n=87,New ISS-24.78(mean)) admitted to J.P.N Apex trauma centre casualty from 1st April,2011 to 31st July,2011 were studied. The cases included in the study were isolated head injury (n=40, NISS-25.87(mean)), multiple trauma with head injury (n=13,NISS – 30.69 (mean)) and trauma other than head injury (n=34, NISS-21.24 (mean)).Thromboelastography was performed using whole blood (n=69) and citrated blood (n=18). Coagulation tests were performed on all 87 patients using both TEG and conventional coagulation parameters. Total 52 patients showed coagulopathy by TEG and only 14 patients showed coagulopathy by standard coagulation parameters (prothrombin time, activated partial thromboplastin time). Only in 10 cases coagulopathy was detected by both methods. 4 patients showed coagulopathy only by conventional methods while 42 patients showed coagulopathy by only Thromboelastography (TEG). To find out whether there is any stastistical significance in the observed apparently better result by TEG, McNemar Test was carried out and P value was <0.0001.

Conclusion

Thromboelastography could be a better technique as compared to conventional measurements of PT, aPTT, TT, Fibrinogen, D-dimer in early detection of coagulopathy in trauma patients.

Disclosures:

No relevant conflicts of interest to declare.
Venous Thromboembolism (VTE) In Hospitalized Patients: What Proportion Is Attributable to Malignancy?
Authors: Akwaa Frank , Khorana Alok
Published: October 2019

Abstract 5250

Background:

VTE is a public health burden in hospitalized patients, affecting the length of hospital stay and overall cost of care. Recent studies suggest increasing incidence of VTE among cancer patients, but contemporary data regarding proportion of VTE attributable to cancer and non-cancer populations are lacking.

Methods:

Hospital-acquired VTE is a reportable event to New York State and we studied all reported VTE events among patients hospitalized at the University of Rochester Medical Center from January 2003 through April 2009. We utilized electronic medical records to identify additional information including demographics and diagnosis of malignancy.

Results:

We identified 2031 patients with 2185 documented VTE events during the study period. Of these, 1102 (54.3%) were men and 929 (45.7%) were women. VTE events included deep vein thrombosis (DVT) (N=1428, 65 %), pulmonary embolism (PE) (N=757, 34.6%), and concurrent DVT and PE (N=153, 7 %). Of the 2031 hospitalized patients with VTE, 492 (24.2%) had a concurrent diagnosis of cancer and 1539 (75.8%) did not. Of 492 cancer-related VTE, 167 (34%) were associated with hematologic malignancies, and 324 (66%) with solid tumors. The proportion of cancer-associated VTE varied by year, ranging from 21% to 31%.

Conclusions:

Approximately one-fourth of all VTE in hospitalized patients occurred in patients with cancer, including a substantial proportion with hematologic malignancy. Public health efforts to reduce hospital-associated VTE should focus on improving compliance with thromboprophylaxis for this population.

Disclosures:

No relevant conflicts of interest to declare.
Advanced Age As a Risk Factor for Folate-Induced Cobalamin (Cbl) Deficiency
Authors: Solomon Lawrence R.
Published: October 2019

Abstract 5268

Folate therapy can induce or exacerbate neuro-cognitive changes in patients with clinically significant Cbl deficiency. High serum folate (sFol) values in subjects with low Cbl levels have also been associated with increases in the Cbl-dependent metabolite, methylmalonic acid (MMA) as well as with hematologic, neuro-cognitive and fetal abnormalities, raising concerns about the use of both folate supplements and folate-fortified grains. However, in a more recent study of young adults, MMA values were unaffected by high sFol values. Significantly, advanced age is also linked to increased MMA values even when Cbl values are normal. Thus, a retrospective study was performed of 91 non-diabetic subjects with normal renal function and low-normal Cbl values (201–300 pg/ml) who were evaluated for Cbl deficiency in an ambulatory care setting over a 10 year period to determine if age is a significant determinant of the metabolic interaction of sFol and Cbl when Cbl levels are within the normal reference range. In older individuals (≥60 yrs) with low-normal serum Cbl values, the geometric mean MMA value was significantly higher (401 nmol/l; n=11) when sFol values were elevated (>20 ng/ml) than when sFol values were normal (298 nmol/l; n=35)(p<0.025). In contrast, in younger subjects, mean MMA values were similar in both high and normal sFol groups (302 nmol/l; n=15 vs 270 nmol/l; n=30)(p=0.52). There was no correlation between MMA and sFol when sFol values were within the normal range in either age group (r≤0.07). Finally, when sFol levels were elevated, Cbl therapy significantly decreased MMA values in 8 of 9 younger subjects (89%) and in all 7 older individuals (100%) studied. It is concluded that: 1) MMA values are increased when sFol levels are elevated and Cbl values are low-normal but this effect is age-dependent; 2) the effect of sFol on MMA is not progressive but occurs only at sFol values above the normal range; and 3) Cbl therapy decreases MMA values in almost all subjects regardless of pretreatment sFol values. Since these findings may have clinical significance, caution in the use of folate supplements is advised.

Disclosures:

No relevant conflicts of interest to declare.
Molecular Epidemiology of β-Thalassemia in Pakistan: Far Reaching Implications
Authors: Ansari Saqib Hussain , Shamsi Tahir S , Bohray Muneera , Khan Mohammed Tahir , Farzana Tasneem , Perveen Kousar , Erum Sajida
Published: October 2019

Abstract 5309

Introduction:

β -Thalassaemia, an autosomal recessive hemoglobinopathy, is one of the commonest genetically transmitted disorders throughout the world. Collective measures including carrier identification, genetic counseling and prenatal diagnosis are required for preventing β -thalassemia. To achieve this objective, Identification of the spectrum of genetic mutations, especially for various ethnic backgrounds in Pakistan, since the country is a High Burdon Country. Therefore, we designed a cross sectional study to identify the frequency of various gene mutations in different ethnic groups of Pakistan.

Methodology:

Over a period of five years, venous blood samples were collected from 466 individuals belonging to different ethnic groups residing in Karachi, having at least one affected family member known to have β-thalassemia major/ HbE- β-thalassemia/ HbE homozygotes/ β-thalassemia trait. Chorionic villus sampling at 11 to 15 weeks gestational age for 143 couples referred by thalassemia clinics as also used to obtain allele information. In all, 648 mutated alleles were identified. The diagnosis of β-thalassemia trait, β-thalassemia major and Hb E thalassemia were established from clinical data, hematological indices and hemoglobin electrophoresis by cellulose acetate method. DNA was extracted from whole blood for detection of mutations. Primers were designed for simultaneous detection of the following previously described mutations in a single reaction: IVS 1–5 (G-C), Fr 8–9, IVS 1-1 (G-T), Cd-30 (G-C), Cd-5 (−CT), Del 619bp, Cd-15 (G-A), Fr 41–42, Fr 16 (−C) and Cap +1 (A-C) along with two Hb variants: HbS and HbE.

Results:

The genetic heterogeneity in Karachi is reflected by the identification of all the common β-thalassemia alleles and two Hb variants but following eight mutations were more common: IVS 1–5 (G-C), Fr 8–9, Del 619 bp, IVS 1-1 (G-T), Fr 41–42, Cd-30 (G-C), Cd-5 (−CT) and Cd-15 (G-A), accounting for 93.9% of the β-thalassemia alleles. However, the distribution was uneven.

Although IVS 1–5 (G-C) was the most common mutation (40.89% of the sample), its frequency varied from 20% in the immigrant (from India) population to 76.9% in the Balochis. The second most frequent mutation was Fr 8–9, constituting 15.7% of the allele pool. Fr 8–9 was the most common mutation in the Pathans (31.3%) as it was in people of Saraikee origin (47%).

Discussion:

IVSI-5 (G-C),(40.89%), Fr8-9(15.7%), & IVSI-I(G-T),(8.17%), were the most common genetic mutations identified in Pakistan. Knowledge of the predominant mutation in a given ethnic group will not only help in developing a short panel of (population-specific) primers of mutations thereby providing a cost-effective method for prenatal diagnosis and also help the clinicians for genetic counseling and pregnancy termination.

Disclosures:

No relevant conflicts of interest to declare.
Decreased NO Bioavailability Leading to Endothelial Dysfunction in Children with ALL Results From Increased eNOS Inhibiting by Asymmetrical Dimethylarginine and May Be Associated with Increased Mortality
Authors: Porwolik Julita , Niedzielska Ewa , Doroszko Adrian , Piasecka-Grzeszek Alicja , Solska Urszula , Chybicka Alicja , Szuba Andrzej
Published: October 2019

Abstract 5318

Background

Endothelial dysfunction worsens the prognosis in numerous severe diseases. Impaired vascular reactivity, increased procoagulative and proinflammatory action are commonly observed when the nitric oxide bioavailability is limited. Asymmetrical dimethylarginine (ADMA) is a competive inhibitor of the endothelial nitric oxide synthase (eNOS). ADMA is produced during proteolysis of methylated proteins, especially histones. The aim this study was to determine if ADMA may play important role in pathogenesis of endothelial dysfunction in children with ALL.

Material and Methods

N=14 children at age of 4–18 years with ALL treated with the ALLIC protocol were investigated. Plasma levels of the NO pathway metabolites (L-Arginine, ADMA) were analyzed at baseline, then during the 33rd and 78th day of protocol. The control group constituted of N=14, age-matched healthy children.

Results

Plasma ADMA levels were significantly higher in children with ALL at baseline as compared to the control group (1.92±0.42 vs. 0.56±0.1 ng/ml, p<0.05), and were significantly decreasing to 0.63±0.15ng/ml, p<0.05 following the steroid therapy. During the 78th day the ADMA levels were maintained at similar levels as compared to the 33rd day. Opposite trends were observed with the L-Arginine levels and the L-Arg/ADMA ratio reflecting the NO synthesis. L-Arg and L-Arg/ADMA ratio were significantly lower in children with ALL at baseline vs. control group (41.65±4.12vs.52.4±2.31pg/ml and 28.7±6.61vs.83.41±14.13, respectively, p<0.05). Moreover, the L-Arg/ADMA ratio at the 78th day was significantly higer in cases treated unsuccesfully in comparison with those who survived (88.1±21.3vs.43.0±10.2, p<0.05).

Conclusions

Decreased NO bioavailability in children with ALL at baseline results from the eNOS inhibiting by ADMA and may be associated with increased mortality.

Disclosures:

No relevant conflicts of interest to declare.
Effects of Low ADAMTS 13 Levels and the Role of Von Willebrand Factor in Cardiovascular Disease
Authors: Corinaldesi Giorgio
Published: October 2019

Abstract 5320

Von Willebrand factor (vWF) is a large multimeric glycoprotein that plays an important role as a co-factor in platelet adhesion and aggregation and as the circulating carrier for factor VIII; increased vWF levels, and the persistence of unprocessed ultra-large vWF multimers (ULVWF) that are hyperactive to binding GPIb-IX-V complex with high-strength bonds, precipitate platelet clumping in arterioles and capillaries, causing platelet aggregation and thrombotic microangiopathy resulting in tissue ischemia with an increased risk of CAD and stroke. vWF is the major ligand binding the subunit of trombospondin, P-selectin, collagen and SLAM family (signaling lymphocytic activation molecule), mediates platelet translocation, tethering and rolling under high shear stress and adhesion.

The activity of VWF is modulated by ADAMTS-13 a Zn2+/Ca2+ dependent metalloprotease that cleaves vWF to smaller and less active forms at the tyr842/met843 within the A2 domain, and cleaves the tyr 1605/met 1606; this mechanism reduces the molecular weight and platelet-tethering function of vWF to the sub endothelium at high shear stress. ADAMTS-13 levels also correlate positively with cholesterol, triglycerides and body mass index (BMI). In addition, we have observed a negative association with fibrinogen, C-reactive protein, and ion trasport K+dependent Na+/Ca2+ exchange (SLC24A3); the phosphorylation of tyrosine 1605 and threonine/serine kinase inhibitors have negative effects on integrin GPIIb-IIIa that showed decrease expression.

In patients with a level of ADAMTS-13 below 38%, the presence of ultra large vWF multimers is high and the relative risk of ischemic heart disease was 18.2% (95% CI, 0.8 to 4.2), and the relative risk of stroke was 11.5 % (95% CI, 0.6 to 3.9).

We investigated the relation between the low activity of ADAMTS-13 and AMI or stroke; we measured VWF and ADAMTS-13 antigen levels in 16 patients with AMI, in 10 patients with stroke, and in 16 control subjects.

We demonstrated that vWF levels are significantly higher in patients with AMI or stroke, while a severe decrease of ADAMTS-13 activity augments the risk of cardiovascular disease, and cerebral ischemia; measurement of both VWF and ADAMTS-13 may provide a good indicator for the likelihood of AMI and stroke.

Disclosures:

No relevant conflicts of interest to declare.