bluuebunny/crossref_metadata_2025 huggingface dataset:
Prognostic Utility of Immunophenotyping in Diffuse Large B-Cell and Follicular Lymphomas
Authors: Abdel-ghaffar Hasan A. , Degheidy Heba A. , Shamaa Sameh S. , Abd-El Aziz Sherin M.
Published: April 2020

Patients with Diffuse Large B-Cell Lymphoma (DLBCL) and Follicular Lymphomas (FL) exhibit marked variability in survival, the international prognostic index (IPI) predicts the cure with chemotherapy as IPI is a well established predictor of outcome in DLBCL and FL. Biological prognostic markers including expression on of BCL2, BCL6, CD5, CD10, and HLA-DR have been described as IPI independent prognostic factors. The purpose of this study is to high light how these biomarkers might be incorporated into current risk – adjustment models for prognosis. Biological parameters were evaluated by flowcytometric immunophenotyping of 96 patients with DLBCL and FL.

In our study, Superior overall survival (OS) was associated with high expression of HLA-DR in DLBCL either do novo or on top of FL (p < 0.001 for both group), and with low expression of CD 5 and BCL2 in de novo DLBCL (p < 0.001 for both ) independently of the IPI. On the other hand Inferior overall survival was established with high expression of BCL6 in DLBCL. However Inferior progression free survival (PFS) was independently correlated with high expression of BCL2 (p = 0.007 and 0.018 ) and lower expression of HLA-DR ( p=0.003 and 0.03 ) in both de novo DLBCL and on top of follicular, but not in FL and with high expression of CD10 in de novo DLBCL only ( p<0.001 ).

In 96 patients with DLBCL and FL treated with risk adapted therapy, low HLA-DR expression is associated with poor outcome in DLBCL either de novo or on top of follicular but not in a FL group. the time is right to begin to consider how these biomarkers should be incorporated into current prognostic models to move beyond the clinically based IPI.
Effects of Anti-Retroviral Drugs to Expression of Cell Surface Adhesion Molecules and Hypermutation of Immunoglobulin Heavy Chain Variable Gene of HIV-Related Lymphoma
Authors: Sasakawa Atsushi , Maeda Yasuhiro , Hirase Chikara , Kanamaru Akihisa
Published: April 2020

It has been known that non-Hodgkin’s lymphomas (NHL) are a major complication in human immunodeficiency virus (HIV) infection, and a major cause of death in HIV infected patients. Recently, it has been reported that highly active anti-retroviral therapy (HAART) declines the incidence of HIV-related lymphoma. In this study, we tried to make in vitro model of HIV-related lymphoma by Epstein-Barr virus transformation, and to examine the cellular and molecular characteristics of the HIV-related lymphoma cells derived from patients with HIV infection. PBMCs from 10 patients HIV infection and 10 HIV-negative normal individuals were obtained by Ficoll-Paque (Amersham Biosciences Corp. NJ) density gradient centrifugation. Lymphoblastoid cell lines (LCL) were obtained from each patient through transformation with Epstein-Barr virus (B95-8). Cells were cultured with RPMI 1640 medium (GIBCO, Grand Island, NY) supplemented with 10% fetal calf serum (FCS) (JRH Bioscences, Inc. Lenexa, Kansas), antibiotics, and L-glutamine and maintained at 37°C in an atmosphere containing 5% CO2. FACS analysis revealed the cells to be CD3−, CD4−, CD8−, CD5−, CD19+, CD20+, HLA-class I+, -class II+. After the establishment of LCLs, to obtain a highly purified population, CD19+LCLs were sorted on a FACS Vantage SE (Becton Dickinson, San Jose, CA). The purity of each CD19+LCLs population evaluated by FACS analysis was always higher than 98%. After growth advantage of LCLs, growth ability of the LCLs from HIV patients was examined by MTT assay. In results, there is no significant difference of growth ability between LCLs derived from HIV patients and those derived from normal individuals. Furthermore, the expression of cell adhesion molecules (CD11a, CD11b, CD18, CD50, and CD54) of LCLs was observed by FACS. The results showed that the expression of CD18 on LCLs derived from HIV patients was enhanced significantly in compared to those of normal LCLs. This phenomenon seems to be consistent with development of primary central nervous system (CNS) lymphoma. Furthermore, effects of anti-retroviral drugs to the expression of the adhesion molecules are examined. Somatic hypermutation of immunoglobulin heavy chain variable gene (IGHV) has been detected over 90% of HIV-related lymphoma. The effects of HAART to hypermuation of IGHV of LCL derived from HIV patients will be addressed.
Double Hematological Malignancy: An Unusual Presentation in Three Cases
Authors: Haddad Rami Y. , Attri Navneet , Kawar Yaser
Published: April 2020

The occurrence of more than one hematological malignancy in the same patient is an unusual pathologic condition and may pose a difficult challenge during decision to start various chemotherapy regimens. Cases of solid tumors of lung and gastrointestinal tract have been noted secondary to treatment of hematological malignancies but the occurrence of two hematological malignancies concomitantly is a rare presentation. We describe three cases of coexistent hematological malignancies at our institution.

First case describes a 77 yo male with synchronously occurring B cell Non Hodgkin Lymphoma: marginal zone lymphoma (main bone marrow population); Chronic Lymphocytic leukemia (Fluorescent-in–situ hybridization test positive for trisomy 12) and a Monoclonal Beta, elevated IgM, elevated B2. BM evaluation revealed involvement by both processes. The patient has been started on Rituximab recently. The second case is an 85 yo male with findings with peripheral blood flow cytometry consistent with chronic lymphocytic leukemia of B-cell immunophenotype and lymph node biopsy consistent with Follicular Lymphoma. He was found to be BCL2 + on BM and had a normal Karyotype: 46, XY. He was treated with Rituximab ×8 cycles. A follow up PET scan showed partial response. Our third case was an 83 yo man with simultaneous presentation of myelodysplastic syndrome (MDS) and multiple myeloma (MM). This patient had MDS (Refractory anemia with Ring sideroblasts RARS type) and smoldering Multiple myeloma (monoclonal plasma cells 10–15%) bone marrow infiltration which over a course of 3 years transformed into full blown Multiple Myeloma with bone marrow revealing 30–40% plasma cells and osteolytic lesions on skeletal survey. Cytogenetic were normal. He was treated with Lenalidomide (after failure of ESA) and became transfusion in dependent for one year (Hgb rose from baseline of 6–7 to 13 g/dL), after progression to active multiple myeloma he was treated with Thalidomide and Dexamthesone. He achieved a partial response on SPEP. Subsequently he was treated for MDS progression with Azacytidine for 5 cycles with minor hematological benefit (transfusion was less frequently), he recently succumbed to his disease, he was transfusion dependent and became acutely ill after an acute episode of diverticulitis. Patients with MM, MDS have been reported after chemotherapy but few cases documenting the coexistence of MDS and MM at diagnosis have been reported in the literature.

Conclusion: In this report, we describe a three cases of double hematological clonal processes or malignancy, all diagnosed at same time, without preceding hematological disorder or chemotherapy, and all required treatment.
Heparin Cofactor II (HCII) Levels and the Risk of Ischemic Stroke. No Deleterious Role of HCII Deficiency but Potential Protective Role of High Plasma Level
Authors: Toulon Pierre A. , Zuber Mathieu , Adda Raphael , Mas Jean-Louis
Published: April 2020

Background: Heparin cofactor II (HCII) is a thrombin inhibitor which activity is dramatically enhanced by heparin and more specifically by dermatan sulfate, another glycosaminoglycan. Its pathophysiological role remains unknown even if cases of HCII deficiency were reported in patients with a history of venous or arterial thrombosis. Recently, it was suggested that high plasma HCII level could play a protective role against atherosclerosis in elderly individuals and protect from restenosis after femoro-popliteal or coronary artery stenting. The aim of this case-control study was to investigate the significance of plasma HCII level in the development of ischemic stroke.

Methods: 81 cases (44 M and 37 F, mean age of 43 years, range: 22–63) and 80 age-and sex-matched healthy controls of the same ethnic origin. HCII activity was evaluated as antithrombin dermatan sulfate cofactor activity using a chromogenic assay. Test results were expressed in arbitrary unit per mL: 1 U/mL is the activity found in 1 mL of pooled normal human plasma.

Results: Plasma HCII activity was not significantly different in cases and in controls (1.04±0.17 vs. 1.00±0.23). The same applied after stratification in males and in females. The normal range for HCII, previously defined in a large cohort of healthy individuals, was between 0.60 and 1.40 U/mL. The frequency of HCII deficiency was not significantly different in cases and in controls [n=3/81 (3.7%) vs. n=2/80, (2.5%); p=1.00] and the same applied to the frequency of HCII level above 1.40 U/mL [n=0/81 (0%) vs. n=2/80 (2.5%); p=0.25]. Interestingly, HCII activity was found above 1.30 U/mL in 7 controls and in only 1 case (p<0.05), none presenting with any of pathophysiological state usually associated with increased HCII levels such as pregnancy, oral contraceptive, inflammation, infection, or acute phase of illness.

Conclusions: These results suggest that HCII deficiency is not in itself a risk factor for the development of ischemic stroke. Even if the frequency of HCII level above the normal range was not statistically different in cases and in controls, the significance of a higher proportion of controls than cases presenting with a HCII level in the high end of the normal range deserves to be further evaluated in connection with a potential protective role against ischemic stroke.
Activated Akt Is Associated with Hyperglycemia and Platelet Hyperactivity in a Mouse Model of Diabetes
Authors: Lu Lin , Woulfe Donna
Published: April 2020

Platelet hyperactivation has been reported in patients with both Type I and Type II diabetes mellitus and likely contributes to the increased prevalence of thrombotic complications in diabetic patients. However, the mechanisms leading to platelet hyperactivity in diabetes are not fully understood. Our previous studies showed that the Akt signaling pathway positively regulates platelet activity. Here, we evaluate the effect of hyperglycemia on Akt phosphorylation and platelet activation using a Type I diabetic mouse model. Hyperglycemia was induced by intraperitoneal injection of streptozotocin (STZ 50mg/kg for 5 days) in C57BL/6 mice. Blood glucose levels were elevated in 12 mice 4 weeks after STZ injection (mean ± SD: 410 ± 70 mg/ml) compared with 11 mice without STZ injection (212 ± 50 mg/ml). Platelets from hyperglycemic mice showed enhanced agonist-dependent aggregation (max % aggregation: 58% ± 10% in hyperglycemia versus 0% in normoglycemia at 0.6mM AYPGKF, n=3 each), fibrinogen binding (62.2% fibrinogen-bound cells ± 5.4% in hyperglycemic conditions versus 46.1% ± 16.3% in normoglycemia at 2mM AYPGKF) and P-selectin binding (42.1% cells with surface P-selectin ± 3.7% in hyperglycemic platelets, 25.3% ± 5.9% in normoglycemia at 2mM AYPGKF) compared with platelets from mice with normal glucose levels. The blood glucose levels were directly correlated with Alexafluor-fibrinogen binding (r=0.65, p=0.042) when platelets were stimulated with thrombin receptor agonist peptide AYPGKF (1.5mM), and also directly correlated with P-selectin surface exposure (r=0.95, p=0.003) after platelets were stimulated with 1.5mM AYPGKF. To determine whether the Akt pathway is involved in enhanced platelet activation in diabetes, we tested phosphorylation of Akt ser473 by immunoblotting. Akt phosphorylation of this residue was increased (35% ± 4%) in platelets from hyperglycemic mice compared with platelets from nondiabetic mice. In conclusion, platelets from hyperglycemic mice are more sensitive to PAR4 agonist-induced fibrinogen binding and P-selectin exposure compared with nondiabetic platelets. Enhanced activation of Akt in platelets under hyperglycemic conditions may play a role in platelet hyperactivation in diabetes.
Palmitoylation Augments the Association of Tetraspanin CD63 with the αIIbβ3-CD9 Complex and the Actin Cytoskeleton in Thrombin-Activated Platelets
Authors: Israels Sara J. , McMillan-Ward Eileen M.
Published: April 2020

CD63 and CD9 are members of the tetraspanin superfamily of integral membrane proteins that function as organizers of multi-molecular signaling complexes involved in cell morphology, motility and proliferation. In resting platelets, CD63 is located in the membranes of lysosomes and dense granules, and CD9 complexes with the αIIbβ3 integrin on the platelet surface. Following platelet activation and granule exocytosis, CD63 translocates to the plasma membrane, where it co-localizes with the αIIbβ3-CD9 complex and is incorporated in the Triton-insoluble actin cytoskeleton. Tetraspanin complexes cluster dynamically in unique cholesterol-rich membrane microdomains (tetraspanin-enriched microdomains, TEMs) that differ from prototypic lipid rafts. The assembly and maintenance of TEMs depends on the palmitoylation of both tetraspanins and some of their partner proteins. Protein palmitoylation most commonly involves the thioester linkage of palmitate to a cysteine residue and, because the process is reversible, can regulate cellular functions. In cell lines, palmitoylation of tetraspanin juxta-membrane cysteine residues affects subcellular distribution, complex stability, cellular signaling and motility. Recently we demonstrated that tetraspanins and their partner proteins form TEMs in platelets, however the role of palmitoylation in platelet TEMs assembly and maintenance has not been studied. 3[H]-palmitate-labeled, washed human platelets were studied at rest, or following activation with thrombin (0.1U/ml). CD63 and CD9 were isolated by immunoprecipitation, separated by density gradient centrifugation, and 3[H]-palmitate quantitated. Palmitate levels increased in all fractions however the relative inter-fraction distribution did not change, consistent with previous results showing that the distribution of CD63 and CD9 does not change following platelet activation. 2-bromopalmitate (2- BP), which blocked palmitoylation as demonstrated by decreased 3[H]-palmitate-labeling of platelets, inhibited both thrombin-induced platelet aggregation and platelet spreading on immobilized fibrinogen, in a dose and time-dependent manner. 2-BP also inhibited the activation-dependent association of CD63 with CD9 and the incorporation of CD63 into the Triton-insoluble actin cytoskeleton in thrombin-activated platelets. In contrast 2-BP had minimal effect on either the integrity of the α IIb β3-CD9 complex or its agonist-induced association with the cytoskeleton. In summary, inhibition of palmitoylation blocked the activation-dependent association of CD63 with the αIIbβ3-CD9 complex and with the actin cytoskeleton but did not alter the tetraspanin-integrin association present in resting platelets. The effect of 2-BP on platelet spreading on fibrinogen mirrored that previously seen with anti-CD63 MoAbs, and supports the hypothesis that the association of CD63 with αIIbβ3-CD9 modulates outside-in signaling in adherent platelets.
UCP2 Modulates Cell Proliferation through the MAPK/ERK Pathway during Erythropoiesis and Has No Effect on Heme Biosynthesis
Authors: Elorza Alvaro A , Hyde Brigham B , Mikkola Hanna , Collins Sheila , Shirihai Orian S
Published: April 2020

UCP2, an inner membrane mitochondrial protein, has been implicated in bioenergetics and Reactive Oxygen Species (ROS) modulation. UCP2 has been previously hypothesized to function as a facilitator of heme synthesis and iron metabolism by reducing ROS production. While UCP2 has been found to be induced by GATA1 during erythroid differentiation its role in erythropoiesis in vivo or in vitro has not been reported thus far. Here we report on the study of UCP2 role in erythropoiesis and the hematologic phenotype of UCP2 deficient mouse. In vivo we found that UCP2 protein peaks at early stages of erythroid maturation when cells are not fully committed in heme synthesis and then becomes undetectable at the reticulocyte stage. Iron incorporation into heme was unaltered in erythroid cells from UCP2 deficient mice. While heme synthesis was not influenced by UCP2 deficiency, mice lacking UCP2 had a delayed recovery from chemically induced hemolytic anemia. Analysis of the erythroid lineage from bone marrow and fetal liver revealed that in the UCP2 deficient mice the R3 (CD71high/Ter119high) population was reduced by 24%. The count of BFU-E and CFU-E colonies, scored in an erythroid colony assay, was unaffected, indicating an equivalent number of early erythroid progenitor cells in both UCP2 deficient and control cells. Ex-vivo differentiation assay revealed that UCP2 deficient c-kit+ progenitor cells expansion was overall reduced by 14% with population analysis determining that the main effect is at the R3 stage. No increased rate of apoptosis was found indicating that expansion rather than cell death is being compromised. Reduced expansion of c-kit+ cells was accompanied by 30% reduction in the phosphorylated form of ERK, a ROS dependent cytosolic regulator of cell proliferation. Analysis of ROS in UCP2 null erythroid progenitors revealed altered distribution of ROS resulting in 14% decrease in cytosolic and 32% increase in mitochondrial ROS. Restoration of the cytosolic oxidative state of erythroid progenitor cells by the pro-oxidant Paraquat reversed the effect of UCP2 deficiency on cell proliferation in in vitro differentiation assays. Together, these results indicate that UCP2 is a regulator of erythropoiesis and suggests that inhibition of UCP2 function may contribute to the development of anemia.
Rational Anemia Diagnostics and Therapy Control of Pediatric Patients Employing the Novel Hemaplot and Thomasplot
Authors: Weimann Andreas , Lun Andreas , Gaedicke Gerhard
Published: April 2020

“Anemia” is one of the most frequent diagnoses during childhood. For the choice of the right therapy an exact diagnosis is a prerequisite. Due to numerous and rather diverse pathophysiological etiologies of anemia and different mechanisms of compensation of the organism, establishing the correct diagnosis might turn out to be rather difficult for the general practioner as well as for the hospital doctor. Within a prospective study the routine blood counts of 100 anemic pediatric patients were measured at ten time points per patient with SYSMEX XE-2100 blood analyzer including the parameters Hb, MCV, MCH, Ret-Hb, delta-Hb and RPI. Plasma samples were checked for transferrin, iron binding capacity, sTfR, Ferritin and CRP levels. The EXPERTviewer was used for transmitting the numerical and graphical data to the clinician. The “Hemaplot” (Ret-Hb versus delta-Hb) as well as the “Thomasplot” (Ret-Hb versus Ferritin-Index) and all regular scatterplots and histograms were conveyed. Preliminary data show that the Thomasplot and Hemaplot help to distinguish hyperregenerative anemias and nutritive or deficiency types. The plots also proved to be helpful in identifiying hyporegenerative as well as congenital and aquired forms of anemia. Thomasplot and Hemaplot might help to better understand pathophysiology of anemias and thus to make rational therapeutic decisions easier and more efficient.
Rituximab Therapy for Treatment of Chronic Warm Autoimmune Hemolytic Anemia
Authors: Sreenivasappa Shylendra B , Telfer Margaret Clare
Published: April 2020

Autoimmune hemolytic anemia (AIHA) is a disorder in which auto antibodies are directed against red blood cells resulting in severe anemia. It is a relatively uncommon disorder with an estimated incidence of 1–3 cases per 100,000 persons per year. These disorders are classified as either warm AIHA or cold AIHA depending on the temperature at which the auto antibodies show maximal binding. First line therapy for patients (pts) with warm AIHA is corticosteroids, 20% achieve complete remission most patients require maintenance treatment. Splenectomy is the usual second line treatment. Cytotoxic and immunosuppressive medications can be tried. Rituximab a monoclonal antibody against CD 20 has also shown efficacy in pt with chronic AIHA.

In this study we retrospectively studied 2 cases of chronic warm AIHA. Case 1 was a female diagnosed with AIHA at the age of 15 years with a history of vasculitis. AIHA had a relapsing and a remitting course. She was treated with steroids, splenectomy, cytotoxic therapy with cyclophosphamide and immunosuppressive therapy with azathioprine with no long-term remission. At 36 yr of age she had a relapse, steroids induced a minimal response, cyclophosphamide induced a partial response. She developed hemorrhagic cystitis hence cyclophosphamide was discontinued. Rituximab was administered at 375mg/m2 weekly for 3 doses with minimal response. After 4 weeks she was given 3 more doses of weekly rituximab with minimal response. 2 weeks after the last Rituximab dose, pt developed pancytopenia, gram-negative sepsis and died.

Case 2 was also a female who was diagnosed with AIHA at the age of 21 years, etiology was considered idiopathic. She had a relapsing remitting course as well. She had been treated with steroids, splenectomy, cytotoxic therapy and immunosuppressive therapy. At 41 years of age she relapsed, this episode was, not responsive to steroids, Rituximab 375mg/m2 was infused weekly for 3 weeks with minimal improvement, after 6 weeks, 3 more doses of Rituximab were infused. She had a gradual improvement of her hemoglobin over 4 months and pt has been in remission since. She has had a sustained response for over 24 months.

There have been a few case series published about the effects of Rituximab in pt with warm AIHA, with an estimated complete response ranging from 40 – 100%. The toxicities of this therapy in this pt population has been not been well documented. We conclude that Rituximab must be used cautiously in pt with AIHA. Prospective Randomized clinical trails are needed to assess the efficacy of Rituximab in pt with warm AIHA.
HDAC Inhibitors, FK228 and Valproic Acid, Enhance the Effects of IL-3 or GMCSF on the Generation of Early Megakaryocytic as Well as Erythroid Precursors from Human Hematopoietic Precursors
Authors: Ohishi Kohshi , Ryuu Hyou , Yamamura Kentaro , Masuya Masahiro , Heike Yuji , Takaue Yoichi , Katayama Naoyuki
Published: April 2020

We previously reported that HDAC inhibition by FK228 (depsipeptide) stimulates the proliferation of early erythroid precursors in the presence of IL-3. Here, we examined the effects of HDAC inhibition on human megakaryopoiesis. When G-CSF-mobilized peripheral blood CD34+ cells were incubated in serum-free liquid cultures containing SCF+IL-3 for 8–9 days, cultured cells were subdivided into 4 fractions with the expression of CD61 (GPIIIa) and glycophorin A (GPA). In the semisolid cultures containing SCF+TPO+EPO, CD61+GPA− cell population mainly gave rise to megakaryocytic colonies, while both of erythroid and megakaryocytic colonies were formed from CD61+GPA+ cell population. CD61−GPA+ cells exclusively generated erythroid colonies. After CD34+ cells were cultured with IL-3 or SCF+IL-3 for 8–9 days, addition of FK228 (0.4 ng/ml) to the cultures significantly increased the numbers of CD61+GPA− and CD61+GPA+ cells. Nevertheless, differentiation into CD61+CD42b+ more mature megakaryocytic cells was repressed by FK228. As expected, when cultured cells were replated in the secondary cultures containing SCF+TPO for another 7 days, higher number of CD61+CD42b+ mature megakaryocytic cells were developed in the cultures that had contained FK228 during the initial culture period. Stimulatory effects by FK228 on the generation of early megakaryocytic and erythroid precursors were also seen in the presence of GM-CSF. Moreover, we obtained similar findings with pharmacological concentration (100μg/ml) of valproic acid, which is also class I HDAC inhibitor. These data suggest a modulatory role for HDAC in the IL-3- or GM-CSF-mediated megakaryopoiesis and that the combinatory treatment with HDAC inhibitors and IL-3 or GM-CSF would be useful for stimulating early megakaryopoiesis as well as erythropoiesis in clinical settings.
X-Linked Inhibitor of Apoptosis Protein (XIAP) Supports Urokinase (uPA)-Induced Endothelial Cell Survival
Authors: Prager Gerald W , Mihaly Judit , Brunner Patrick , Zielinski Christoph , Binder Bernd
Published: April 2020

High uPA expressing tumors are associated with poor prognosis. While a direct effect on tumor cell behavior is described, uPA has especially been shown to mediate (tumor-) angiogenesis. Originally, the urokinase system has been implicated to assist the angiogenic process by it’s proteolytic activities. It is now becoming increasingly evident that uPA additionally elicits a whole array pro-angiogenic responses like differentiation, proliferation and cell migration, independent of its proteolytic activity by inducing intracellular signal transduction.

Here we show that uPA induces upregulation of inhibitor of apoptosis proteins (IAPs), which protects endothelial cells against apoptosis. Thereby, uPA-induced endothelial cell survival is mediated by transcriptional upregulation the X-linked inhibitor of apoptosis protein (XIAP), because downregulation of XIAP by small interfering RNA techniques significantly reduces uPA mediated cell survival efficiencies of uPA in endothelial cells. The antiapoptotic activity of uPA was dependent on the presence of uPAR and LRP, but independent of the PI3kinase pathway, while VEGF-dependent antiapoptosis is mainly PI3kinase dependent. uPA-induced cell survival is dependent on the type of extracellular matrix on which cells are attached used indicating the involvement of integrin adhesion receptors. TherebyConsistently, uPA induces phosphorylation of the CDC42 downstream effector p21-activated kinase 1 (PAK1), which leads to IkappaB kinase alpha (IKKa) phosphorylation, a prerequisite for NFkappaB activation. As a consequence, p52/p50 but not p65 is are translocated into the nucleus. Blocking NFkappaB by using the specific NFkappaB inhibitor BAY 11–7082 or by adenoviral-mediated overexpression of its inhibitor, IkB, inhibits uPA-induced XIAP expression as well as uPA-induced cell survival.

From these data we conclude that uPA, which is a main player in endothelial cell migration and invasion, provides an additional, PI3-kinase independent but NFkappaB dependent cell survival mechanism.
Serum VEGF Per Platelet Count as a Prognosis Predicting Factor in Advanced Gastric Cancer Patients
Authors: Kim Byung Soo , Choi Chul Won , Kim Seok Jin
Published: April 2020

Background: New blood vessel formation is a crucial step in the process of tumor growth and systemic metastasis. Recent studies have shown that VEGF expression not in tissues but in serum sample is correlated with tumor vascularity, and the high serum VEGF levels could predict poor prognosis in cancer patients. However there has been no data regarding the clinical and prognostic significance of serum VEGF levels per platelet count in advanced gastric cancer. In this study, we conducted a study to evaluate the prognostic implication of serum VEGF per platelet count in the patients with advanced gastric cancer.

Methods: 111 patients with histologically confirmed gastric cancer, 35 patients with early gastric cancer were included and control serum samples were acquired from 25 healthy volunteers. The levels of VEGF were measured using human VEGF quantitative enzymelinked immunosorbent assay (ELISA). Survival curves were calculated using the Kaplan-Meier method and survival comparisons were made by the log rank test in metastatic gastric cancer. The Cox proportional hazards regression model was utilized for multivariate analyses after univariate analysis defined relevant prognostic variables.

Results: The mean serum VEGF level was higher in the patients of AGC compared to those with EGC and controls (AGC 465 ± 315.8pg/ml; EGC 306 ± 97.8 pg/ml controls 230.8 ± 53.2 pg/ml, P< 0.033). A trend toward a significant positive correlation between serum VEGF and platelet counts was observed in patients of AGC (r = 0.477, P = 0.000, Fig 2) and there was a significant correlation between serum VEGF levels and differentiation of tumor (p = 0.014), stage (p = 0.036). The overall survival (log rank, p =0.0432) and the progression free survival (median 4.5 vs. 8.9 months; log rank, p =0.0116) were significantly shorter in patients with high VEGF per platelet count (≥1.626 pg/106). In the multivarivate analysis, performance status (P=0.025), the presence of peritoneal carcinomatosis (P=0.006), serum VEGF per platelet (P=0.005) were found to be significantly associated with the short progression free survival

Conclusions: This study demonstrated that serum VEGF per platelet count is correlated with short overall survival and progression free survival in advanced gastric cancer patients. Therefore, serum VEGF per platelet may be a useful marker for predicting the prognosis of advanced gastric cancer patients.
Factors Determining Survival after Unrelated Donor Stem Cell Transplantation in Primary Refractory Acute Myeloid Leukemia
Authors: Craddock Charles F. , Labopin Myriam , Finke J. ürgen , Ehninger Gerhard , Bunjes Donald , Kolb Hans-Jochem , Greinix Hildegard , Zander Axel , Polge Emmanuelle , Schmid Christoph , Beelen Dietrich , Rocha Vanderson
Published: April 2020

Allogeneic stem cell transplantation using an HLA identical sibling donor has the capacity to produce long term disease free survival in a significant number of patients with primary refractory acute myeloid leukaemia (AML). The increased availability of volunteer unrelated donors has resulted in allogeneic transplantation becoming a realistic option in large numbers of patients lacking a sibling donor. However the outcome of patients with primary refractory AML after unrelated donor transplantation has not been systematically examined before. We have therefore studied the outcome of 186 adults with primary refractory AML who underwent an unrelated donor transplant from 1995 to 2006. All patients were refractory to induction chemotherapy having received a mean of 2.2 courses of chemotherapy (range 1 to 6). Cytogenetic status at diagnosis is available in 96 patients: 3 patients had good risk cytogenetics, 60 intermediate risk and 33 adverse risk cytogenetics by MRC criteria. The median interval from diagnosis to transplant was 4.2 months (range 2–6 months). 141 patients underwent transplantation from a 6/6 matched unrelated donor and 45 from a mismatched unrelated donor. 150 patients were transplanted using GCSF mobilised peripheral blood stem cells. 136 patients were transplanted using a myeloablative conditioning (MAC) regimen. 50 patients were transplanted using a reduced intensity conditioning (RIC) regimen of whom 30 received a low dose TBI based regimen. 137 patients achieved a complete remission (CR) post-transplant. The 2 year overall survival for the whole group was 31% and 43% for patients who achieved a CR post-transplant. The day 100 non-relapse mortality was 16% (19% for MAC allografts v 9% for RIC allografts). In univariate analysis time to transplant (<4.2 months), presentation white blood cell count, the absence of circulating blasts at the time of transplant, and the use of a RIC regimen were associated with improved survival. Adverse risk cytogenetics at diagnosis was associated with a decreased OS. In multivariate analysis a short time from diagnosis to transplant (p=0.05), the absence of adverse risk cytogenetics (p=0.02) and the use of a RIC regimen (p=0.036) were associated with improved survival. The improved survival in patients receiving a reduced intensity transplant occurred despite their increased age at the time of transplant (54 years v 40 years). On the basis of this study we conclude that there is an important role for unrelated donor transplantation in primary refractory AML-particularly if performed early after diagnosis. The encouraging results obtained using a RIC require further examination.
Epigenetic Manipulation of Cancer Testis Antigen (CTA) Expression: A Strategy for Manipulating the Graft-Versus Leukaemia Response in Patients Allografted for Haematological Malignancies
Authors: Stankovic Tatjana , McLarnon Andrew , Agathanggelou Angelo , Goodyear Oliver , Craddock Charles , Moss Paul
Published: April 2020

An immunologically mediated graft-versus-tumor (GVT) underlies the curative effect of reduced intensity allografts in acute myeloid leukaemia (AML) and other haematologic malignancies. Cancer testis antigens (CTA) represent a family of immunodominant proteins that are variably expressed in haematological malignancies and represent a potential target of a GVT response. Importantly a number of CTA genes demonstrate promoter hypermethylation in solid tumours which can be reversed using demethylating agents such as azacitidine. We have therefore examined whether donor T cell responses to CTAs are observed in patients allografted for AML and multiple myeloma (MM) and whether epigenetic therapies can be used to manipulate T cell mediating killing of haemopoietic targets.

We screened 37 patients with AML and 8 patients with MM, who had not received demethylating agent treatment, for T cell responses to 25 peptides derived from 10 CTA genes, including BAGE, LAGE, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-C2, RAGE-1, by interferon-γ cytokine secretion assay (IFN-γ CSA). We found CTA specific T cells in 11.1% of patients (5 of 45) with frequencies ranging from 0.0005% to 0.2% (median 0.024%). Subsequently, we studied expression of 15 cancer-testis antigens (CTA) at the RNA and protein levels in AML, MM and Hodgkins’ lymphoma cell lines before and after exposure to demethylating agent 5-aza-2′-deoxycytidine (Aza) and histone deacetylase inhibitor sodium valproate (VPA), as single agents and in combination. We found that expression of CTAs HAGE, SACA3, SPANXB, LAGE, XAGE, MAGEA3 could be induced in a dose dependent manner by Aza alone but not with VPA alone. We also observed that expression induced by Aza treatment was increased further by combination treatment with VPA. Induction of CTAs was confirmed in vitro in primary AML cells and in vivo in 2/6 patients on VPA/Aza trial. Furthermore, using interferon-γ ELISA assay we observed that Aza-induced expression of the CTA MAGEA3 in MM cell lines was accompanied by increased recognition by MAGEA3-specific T cells.

These studies confirm the importance of members of the CTA family as targets of a T cell mediated immune response. Our data demonstrate that the expression of these putative immunodominant antigens in haematological malignancies in disease such as AML and myeloma, can be significantly up-regulated by epigenetic therapies with functional increases in target cell killing and support the use of adjunctive epigenetic therapies after allogeneic transplantation with the aim of augmenting a GVL response. A Phase II clinical trial combining post transplant azacitidine with a reduced intensity allograft is currently ongoing in patients with AML.
MicroRNA Signature in Waldenstrom Macroglobulinemia
Authors: Roccaro Aldo M , Sacco Antonio , Chen Changzhong , Leleu Xavier , Runnels Judith , Azab Feda , Azab Abdel Kareem , Jia Xiaoying , Ngo Hai T , Melhem Molly R. , Burwick Nicholas , Quang Phong , Varticovski Lyuba , Novina Carl D , Rollins Barrett J. , Anderson Kenneth C. , Ghobrial Irene M.
Published: April 2020

Background. MicroRNAs (miRNAs) constitute a class of small, non-coding, 18–24 nucleotide RNAs, that act as negative regulators of gene expression. By repressing several target mRNAs, mature miRNAs play a pivotal role in regulating development, cell differentiation, apoptosis, and cell proliferation. Moreover, miRNAs have been described to play roles in both solid tumors and hematologic malignancies, however the role of miRNAs in WM has not been yet elucidated.

Methods. We performed miRNA expression profiling of bone marrow-derived CD19+ WM cells, compared to their normal cellular counterparts and validated data by qRT-PCR. In vitro and in vivo functional studies were performed on miRNA-155 knockdown WM cells. Effect of miRNA-155 on signaling cascades have been evaluated by western blot and immunofluorescence. NF-kB activity has been investigated using a DNA-binding enzyme-linked immunosorbent assay-based assay. Gene expression profile analysis has been performed on both miRNA-155 knockdown- and control probe-transfected WM cells in order to identify potential miRNA-155 targeted genes. Finally correlation between miRNA signature and prognosis has been evaluated.

Results. We identified a WM specific miRNA signature characterized by increased expression of miRNA-363*/-206/-494/-155/-184/-542-3p; and decreased expression of miRNA-9* (ANOVA;P< 0.01). Our data showed that miRNA-155 regulates proliferation and growth of WM cells in vitro and in vivo, by inhibiting MAPK/ERK, PI3/AKT, and NF-kB signaling pathways. Potential miRNA-155 target genes were identified using gene expression profiling and included genes involved in cell cycle progression, adhesion, and migration. Importantly, increased expression of the 6 miRNAs significantly correlated with a poorer outcome predicted by the International Prognostic Staging System for WM (P<0.01). We further demonstrated that therapeutic agents commonly used in WM (rituximab, perifosine, bortezomib) alter the levels of the major miRNAs identified, by inducing down-modulation of five increased miRNAs (all but miR-206) and upmodulation of patient-downexpressed miR-9* microRNA.

Conclusion. These data indicate that miRNAs play a pivotal role in the biology of WM; represent important prognostic markers; and provide the basis for the development of new miRNA-based targeted therapies in this disease.
Aberrant Overexpressions of MicroRNA-21 and MicroRNA-155 Activate AKT Signaling Via Downregulation of Tumor Suppressors in NK-Cell Lymphoma/Leukemia.
Authors: Tagawa Hiroyuki , Yamanaka Yasuo , Watanabe Atsushi , Takahashi Naoto , Sawada Ken-ichi
Published: April 2020

Abstract 1917

Poster Board I-940

Background:

Natural Killer (NK) cell lymphomas/leukemias are characterized groups of highly aggressive lymphoid malignancies, which are comprised of “extranodal NK/T cell lymphoma, nasal type” and “aggressive NK-cell leukemia”. Notably, these two subtypes show many similarities in their morphologic features, immmunophenotypes and genotypes, and are invariably associated with Epstein-Barr virus (EBV), which suggests they may share the same genetic alterations. The gene(s) responsible for natural killer (NK)-cell lymphoma/leukemia have not been identified.

Materials and Methods:

In the present study, we used Northern and quantitative PCR analyses to screen for and quantitatively assess miRNA expression in NK-cell lymphomas/leukemias using 66 probe sets of miRNAs. For function analysis, we conducted antisense oligonucleotides and lentivirus transfection assays using NK-cell lymphoma cell lines.

Results:

We found that in NK-cell lymphoma lines (n=10) and specimens of primary lymphoma (n=10), levels of miR-21 and miR-155 expression were inversely related, and were significantly higher than those seen in normal natural killer (CD3-CD56+) cells (n=8). To determine the functions of these microRNAs in lymphomagenesis, we examined the effects of antisense oligonucleotides targeting miR-21 (ASO-21) and/or miR-155 (ASO-155) in NK-cell lymphoma lines overexpressing one or both of these miRNAs. Conversely, cells showing little endogenous expression of miR-21 or miR-155 were transduced using lentiviral vectors, leading to their overexpression. Reducing expression of miR-21 or miR-155 led to upregulation of phosphatase and tensin homologue (PTEN), programmed cell death 4 (PDCD4), or Src homology-2 domain-containing inositol 5-phosphatase 1 (SHIP1). ASO-21- and ASO-155-treated cell lines all showed downregulation of phosphorylated AKTser473 (pAKT). Moreover, transduction with either miR-21 or miR-155 led to downregulation of PTEN and PDCD4, or SHIP1 with upregulation of pAKT.

Conclusions:

Because recent reports suggest that EBV infection can lead directly to upregulation of miR-21 and miR-155, aberrant overexpressions of these miRNAs via EBV infection could be the first-hit genetic alterations during NK-cell lymphomagenesis. Collectively, these results provide important new insight into the pathogenesis of NK-cell lymphoma/leukemia and suggest targeting miR-21 and/or miR-155 may represent a useful approach to treating NK-cell lymphoma/leukemia.

Disclosures:

No relevant conflicts of interest to declare.
The Lymphoproliferative Disease of Granular Lymphocytes: Updated Criteria for Diagnosis
Authors: Semenzato Gianpietro , Zambello Renato , Starkebaum Gordon , Oshimi Kazuo , Loughran Thomas P.
Published: April 2020

The lymphoproliferative disease of granular lymphocytes (LDGL), also referred to as LGL leukemia, is a heterogeneous disorder, but is clinically, morphologically, and immunologically distinct. Although LDGL has recently been included in the revised classification of lymphomas as an independent clinical entity, no consensus exists on the criteria to establish the diagnosis. The aim of this report was to refine the parameters needed to make the diagnosis of LDGL. We studied 11 patients with chronic granular lymphocytosis selected from among 195 cases observed by our institutions from three different geographic areas (North America, Europe, and Asia). These cases did not meet the current criteria for inclusion in LDGL, since all patients had less than 2,000 GL/μL. However, in each of these patients, we found evidence for expansion of a discrete GL population. Clonal rearrangement of the T-cell receptor (TCR) β gene was found in peripheral blood mononuclear cells (PBMC) of all nine patients with CD3+ LDGL. Using recently generated monoclonal antibodies (MoAbs) against the TCR Vβ gene regions, we identified a unique TCR Vβ on GL from each of three patients studied. In two patients with CD3− LDGL, we also identified a restricted pattern of reactivity, by staining with MoAbs against p58 antigen found on normal natural killer (NK) cells. The clinical features of these 11 patients with relatively low absolute number of GL were similar to those reported previously for patients with greater than 2,000 GL/μL. These data demonstrate that newer techniques such as MoAbs against Vβ gene regions and p58 molecules and molecular analyses are useful to identify expansions of discrete GL proliferations. Demonstration of an expansion of a restricted GL subset is evidence for the diagnosis of LDGL, even in patients with a relatively low GL count. Our results also contribute to distinguish between the end of normality and the beginning of pathology in the broad spectrum of GL lymphocytoses.
Recombinant Methionyl Human Stem Cell Factor and Filgrastim for Peripheral Blood Progenitor Cell Mobilization and Transplantation in Non-Hodgkin's Lymphoma Patients — Results of a Phase I/II Trial
Authors: Moskowitz Craig H. , Stiff Patrick , Gordon Michael S. , McNiece Ian , Ho Anthony D. , Costa John J. , Broun E. Randolf , Bayer Robert A. , Wyres Melody , Hill Jerome , Jelaca-Maxwell Kathy , Nichols Craig R. , Brown Sherri L. , Nimer Stephen D. , Gabrilove Janice
Published: April 2020

To examine the safety and efficacy of recombinant-methionyl human stem cell factor (r-metHuSCF), 38 patients with intermediate-grade or immunoblastic high-grade non-Hodgkin's lymphoma who were eligible for autologous transplantation were randomized to receive r-metHuSCF (5, 10, 15, or 20 μg/kg/d) plus Filgrastim (10 μg/kg/d) or Filgrastim (10 μg/kg/d) alone to mobilize peripheral blood progenitor cells. Subcutaneous administration of r-metHuSCF was well tolerated in conjunction with a multi-agent pre-medication regimen; local injection site reactions were the most commonly seen adverse event. The total mononuclear cell count, CD34+ cell content, granulocyte-macrophage colony-forming cells (GM-CFC), and burst-forming units-erythroid (BFU-E) per kilogram in the apheresis product was similar when all patients were analyzed by treatment cohort and mobilization regimen (Filgrastim or r-metHuSCF in combination with Filgrastim); however, when prior chemotherapy was taken into account in a supplementary analysis, clinically important differences were observed. Extensive prior therapy was defined as the amount of exposure to specific stem cell toxic chemotherapeutic agents that patients received. These agents include procarbazine, nitrogen mustard, melphalan, nitrosoureas (≥2 cycles of any of these drugs) or greater than 7.5 g of cytosine arabinoside. In these patients, there was an increased number of CD34+ cells (1.76 v 0.28 × 106/kg), GM-CFC (20.5 v 5.0 × 104/kg), and BFU-E (36.9 v 8.9 × 104/kg) in patients receiving r-metHuSCF and Filgrastim (N = 18) compared with Filgrastim alone (N = 5). These patients also had a decreased time to an untransfused platelet count of 20 × 109/L that was 10.5 days shorter in the patients who received r-metHuSCF and Filgrastim (12.5 v 23 days). These differences were not found to be statistically significant, possibly because of small size, but are clinically important.
An Experimental Model of Human Leukemic Meningitis in the Nude Rat
Authors: Barone Tara A. , Plunkett Robert J. , Hohmann Philip , Lis Agnieszka , Glenister Norman , Barcos Maurice , Ostrow Peter T. , Spence Phyllis M. , Greenberg Steven J.
Published: April 2020

An experimental animal model of meningeal leukemia was developed in the nude rat, rnu/rnu, using the human-derived acute lymphoblastic leukemia cell line HPB-ALL. Anesthetized rats were placed in a modified stereotaxic frame and then injected intrathecally, at the level of the cisterna magna, with human leukemic cells. Cerebrospinal fluid and tissue samples from brain, spinal cord, heart, liver, kidney, spleen, bone marrow, and cervical lymph nodes were subjected to histopathologic examination and molecular genetic screening by clonotype primer-directed polymerase chain reaction (CPD-PCR). Ninety-three percent of animals (n = 14) developed signs of meningeal irritation leading to death 30 to 63 days postinjection (median, 36.0 days, mean, 38.7); death occurred between 30 and 39 days in 77% of all animals. Leukemic cells progressively infiltrated the pericerebellar and pericerebral subarachnoid space and infiltrated the Virchow-Robin (perivascular) space. The infiltrating meningeal leukemia closely resembled the pathologic presentation in the human condition. By CPD-PCR, leukemic cells were first detected in cerebrospinal fluid (CSF ) on day 4 postinjection, were variably present over the ensuing 17 days, and were consistently detected after day 21. At terminal stages, CPD-PCR tissue surveys showed leukemic DNA in all brains and spinal cords and rarely in cervical lymph nodes, but leukemic DNA was not detected in any other tissue screened. Leukemic meningitis was reliably produced with a predictable survival time. Intrathecal administration of leukemic cells was an efficient means of transmitting leukemic meningitis and it compartmentalized the disease to the central nervous system (CNS), eliminating potential complications of systemic illness. The use of human-derived cell lines may render this model more relevant to the development of future therapeutic strategies to treat leukemia and lymphoma that invade the CNS.