bluuebunny/crossref_metadata_2025 huggingface dataset:
Rituximab and 2-Chlorodeoxyadenosine in Hairy Cell Leukemia as First Line Treatment: A Faster Way to Remission. Report of Five Cases.
Authors: Farina Giuliana , Giordano Giulio , Papini Stefano , Zappacosta Bruno , Carbone Arnaldo , Guerriero Massimiliano , Storti Sergio
Published: October 2019

Background Hairy-Cell leukaemia (HCL) is an uncommon B-cell chronic lymphoproliferative disorder that accounts for about 2% of all leukaemias. Although the disease is generally indolent in its natural course, the majority of patients requires treatment for life-threatening infections due to pancytopenia or symptomatic splenomegaly. Remarkable progresses have been made in the treatment of HCL. Prior to the advent of nucleoside analogues, which are currently the standard initial treatment, interferon and splenectomy were the most effective therapies. Treatment with purine analogs (PNA) such as 2-chlorodeoxyadenosine (2-CdA) is associated with excellent remission rate and long-term survival. Recently, immunotherapeutic approaches which use monoclonal antibodies, like rituximab, have increased the number of therapeutic options for HCL and offer promising salvage strategies for patients who relapse or who are refractory to treatment with purine analogues. Previously rituximab, used in patients relapsing or refractory to CdA and for elimination of MRD after treatment with PNA, showed hematological CR and good MRD eradication. Patients and treatment We analysed, in a retrospective study, five consecutive patients M/F 4/1, 4 with HCL and one with HCLVariant treated at the diagnosis with one rituximab administration at the dose of 375mg/mq and 2CdA at the dose of 0.14 mg/mq/day s.c. for 5 days. Their median age was 70 years (range 41–71). All patients showed splenomegaly and thrombocytopenia (median platelet count was 65000 with range 49000–88000), four of these presented neutropenia (median count was 435/mmc with range 430–800/mmc) and one patient was severely anemic (Hb < 8 g/dl).

Results None of the patients had severe therapy-related side effects. Four patients showed febrile cutaneous rash, although received prophyilaxis with acyclovir. The median time of recovery for neutrophil count >1500/mmc was 20 days (range 15–38) for platlets count> 100000/mmc was 21days (12–21), and hemoglobin level> 8g/dl was 31.5 days(27–36). Our median follow up at today was 264 days(range 40–570). At this time all patients are in hematological and morphologic complete remission.

Conclusions The use of Rituximab together with 2CDA in first line therapy is safe and feasible. Moreover when Rituximab is used in such setting, median time of recovery in neutrophils and platelets count is significatively shorter than that obtained after therapy with 2CdA alone (8 and 4 weeks respectively). A longer follow-up is needed to evaluate the long-term results of the treatment.
Monoclonal Immunoglobulins of Alpha-2 Mobility on Protein Electrophoresis: A Case Series.
Authors: Chari Ajai , Eisenberger Andrew , Pesce Michael
Published: October 2019

Protein electrophoresis (PEP) separates proteins based on mobility through support media such as agarose into five general regions: albumin, alpha-1, alpha-2, beta, and gamma. Monoclonal and polyclonal immunoglobulins typically migrate in the gamma or beta regions. Between April 1990 and July 2007 at Columbia University Medical Center, we identified and then retrospectively reviewed 15 patients with monoclonal proteins of alpha-2 mobility repeatedly on serum PEP (see figure). Immunofixation identified the paraprotein as IgA kappa in 7 patients, IgA lambda in 2, IgG lambda in 2, free kappa light chain in 4, free lambda light chain in 2, and one IgG lambda. In the urine, of the 14 patients who had PEP performed, 6 patients had serum concordant light chains (5 kappa and 1 lambda) also migrating in the alpha 2 region, however, for 5 other patients migration was either in the beta or gamma regions. Published case reports of monoclonal proteins with atypical mobility patterns are reviewed. The median age of our patients was 66 years and there were 9 males. 10 patients had multiple myeloma, 5 had monoclonal gammopathy of undetermined significance (MGUS), and 1 had lambda AL amyloidosis. This is the largest case series ever described of patients with monoclonal proteins migrating in alpha 2 region and illustrates the importance of immunofixation in screening patients for plasma cell dyscrasias. Possible explanations for this unusual migration pattern include protein complex formation with other plasma components, high carbohydrate content, or given the IgA predominance noted, properties of the IgA immunoglobulin such as polymerization.

Figure Figure
Safety and Efficacy of Bortezomib and Dexamethasone (BD) in Multiple Myeloma as First-Line, Second-Line or Third-Line Treatment.
Authors: Li Juan , Huang Beihui , Zhao Ying , Zheng Dong , Luo Shaokai
Published: October 2019

Purpose: To compare efficacy and safety of BDbortezomib and dexamethasone in multiple myeloma as first-line, second-line or third-line treatment.

Patients and methods: 18 patients(pts) treated with BD (bortezomig 1.3mg/m2 on d 1,4,8,11 and dexamethasone 20mg on d 1–4 in a 21-day cycle). Responses were evaluated by the criteria of EBMT but a nCR was included. Adverse events were graded by the WHO criteria.

Results: The median age was 52.5 years, and 13 (72.2%) were male. Ig subtypes were IgG, (8 pts, 44.4%), IgA (4 pts, 22.2%), light chain (2 pts, 11.1%), and IgD (1 pts, 5.9%). Durine/Salmon staging: IIA 5.9% (1/18), IIB 5.9% (1/18), IIIA 72.2% (13/18), IIIB16.6% (3/18). BD was administered in first-line (4 pts, 22.2%), second-line (7 pts 38.9%) and third-line (7 pts, 38.9%). The median number of BD cycles was 2.5 (range, 1–6 cycles). 17 pts could be evaluated, overall response was 88.3%, including CR in 2 pts (11.7%), nCR in 5 pts (29.4%), PR in 7 pts (41.2%), MR in 1 pts(5.9%), NC in 1 pts(5.9%), PD in 1 pts(5.9%). Pts in the first-line group received a response of 100%(4/4), including nCR in 1 pts (25.0%), PR in 2 pts (50.0%), MR in 1 pts(25.0%). The second-line group received a response of 100%(7/7), including CR in 1 pts(14.3%), nCR in 3 pts (42.9%), PR in 3 pts (42.9%). Those received BD as third-line or more got a response of 66.7% (4/6), including CR in 1 pts(16.7%), nCR in 1 pts (16.7%), PR in 2 pts (33.3%). Aderse events included diarrhea 44.4% (8/18; grade IV: 2/18), thrombocytopenia 44.4% (8/18; grade III–IV: 7/18), fatigue 44.4% (8/18; grade IV: 1/18), peripheral neuropathy 33.3% (6/18, grade II:1/18), pyrexia 22.2%, infection 16.7% (3/18), headache 11.1%(2/18), parageusis 11.1%(2/18), hypotension 5.9%(1/18), hypoglycemia 5.9%(1/18) while combining with glipizide. The median time to minimum counts of platets was 14 days. The median time to most serious peripheral neuropathy appeared after treatment of 3 cycles. In 1 pts the therapy was disrupted by toxicity.

Conclusion: BD demonstrated better response as first-line or second-line therapy than as third or more line therapy. Toxicities were torelated and manegable. There were no treatment related deaths.
Combined Cyclophosphamide, Fludarabine, Alemtuzumab, and Rituximab (CFAR), an Active Frontline Regimen for High-Risk Patients with CLL.
Authors: Wierda William G. , O’Brien S. , Ferrajoli A. , Faderl S. , Koller C. , Estrov Z. , Garcia-Manero G. , Verstovsek S. , Browning M. , Lerner S. , Keating M.
Published: October 2019

Patients (pts) with CLL have diverse clinical features, including time to first treatment (Rx), response to Rx, remission duration, and outcomes with salvage Rx. We identified serum beta-2 microglobulin (B2M) of 4 mg/L or greater as a high-risk feature, predicting for lower complete remission (CR) rate and shorter progression-free survival (PFS), with frontline chemoimmunotherapy. For pts <70 years old and B2M≥4 treated with the fludarabine (F), cyclophosphamide (C), rituximab (R)-based combination, the CR rate and estimated median PFS were 59% and 60 mo, compared to 81% CR and 80 mo PFS for similar aged pts with B2M<4. We combined FCR with alemtuzumab (A) in the CFAR regimen that was well-tolerated and had activity in treating relapsed/refractory pts. The CFAR regimen is being evaluated in higher-risk pts with an NCI indication for frontline therapy. Frontline CFAR consists of C-200mg/m2 d3–5; F-20mg/m2 d3–5; A-30mg IV d1,3,5, and R-375–500mg/m2 d2, each 28 days for 6 intended courses. Tumor lysis prophylaxis was allopurinol 300 mg/d for 7d, course 1 only. Neulasta 6mg d6 was routine. Antibiotic prophylaxis was TMP-SMZ DS twice daily 2–3 d/wk and valacyclovir (VA) or valgancyclovir (VG) during and for at least 2 months after completion of treatment. CMV antigen in blood was monitored before each course. There currently are 40 pts registered on this trial. The median age for the 21 pts currently evaluable for response was 63 yrs (43–69), B2M was 5.1mg/L (4–9.8), ALC was 72 K/μL (6–355), HGB was 10.9gm/dL (7.9–13.5), and PLT was 158 K/μL (65–390); 13 were Rai high-risk. The median time from diagnosis to Rx was 35 (2–117) mo. The median number of cycles administered was 4 (3–6). Reasons for not completing 6 courses included delayed recovery of counts (6), pt choice (3), AIHA (2), infection (1), and Rx failure (1). CR was achieved in 71%, nPR in 5%, PR in 19%, and 1 pt of 21 did not respond. All pts in CR and nPR and 3 of 4 in PR were free of disease in the bone marrow by 3-color flow cytometry. There was no significant correlation between CR or OR and Rai stage, IgVH mutation status, FISH status, or ZAP70 or CD38 expression. Grade 3 or 4 neutropenia and thrombocytopenia were seen in 27% and 7% of courses, respectively. Major and minor infections were seen in 2% and 8% of courses and FUO in 34% of courses. These FUO included A-infusion related fevers. CMV reactivation occurred in 2/8 pts who received VA and none of 13 pts who received VG prophylaxis during treatment. Two pts developed CMV reactivation 3–6 months after they switched from VG to VA after completing CFAR. In conclusion, CFAR is an active and promising frontline regimen in higher-risk pts with CLL. Accrual and treatment continue on this clinical trial.
Assessment of Psychological Distress in Patients Suffering from Hematological Disorders.
Authors: Cole Craig E. , Ballandby Rebecca , Schroeder Jonean E. , Lee Jennifer A. , Mathiason Michelle A. , Hoeg Rasmus T. , Bottner Wayne A. , Farnen John P. , Ettinger Robert S. , Peters Antoinette M. , Dawson Janette M. , Johnston Kaye L. , Go Ronald
Published: October 2019

Background: Distress is an unpleasant psychosocial response that may impede the capacity of patients to cope successfully with a disease, its physical symptoms, and its treatment. Measurements of distress have been extensively evaluated in patients with solid tumor malignancies. The National Comprehensive Cancer Network (NCCN) has recommend standards of care for distress management. However the degree of distress in patients with hematologic diseases (both malignant and non-malignant) is under-investigated.

Aim: This study was designed to assess and compare distress in patients with malignant or non-malignant hematological disorders.

Methods: Of 617 adult hematology patients enrolled between July 2006 and December 2006, 572 reported distress scores and were prospectively followed, while 45 patients who did not rate their distress were excluded. The NCCN Distress Thermometer (scale of 0–10) accompanied by a modified problem checklist was administered to hematology patients 18 years of age and older seen at the Gundersen Lutheran Center for Cancer and Blood Disorders. Additional demographics, diagnoses, and disease-related information were gathered by chart review. Patients reporting a high score were contacted by a social worker to assess the etiology of distress.

Results: At initial visit during the study period, 572 patients reported a distress score (mean = 3.4 ± 2.7). Patients with malignant (3.2 ± 2.7) or non-malignant (3.6 ± 2.6) disease reported similar distress (p = 0.125). Females (4.0 ± 2.8) were more distressed than males (2.9 ± 2.4; p < 0.001). Other factors significantly associated with distress were history of anxiety (p < 0.001), depression (p < 0.001), younger age (p = 0.001), and time from diagnosis (visit day 3.6 ± 2.4; 1–90 days 4.2 ± 2.9; 3mo-1 year 2.9 ± 2.6; 1+ years 3.4 ± 2.7; p = 0.028). Distress based on disease status was (relapse 5.0 ± 3.1, remission/stable 3.3 ± 2.6, treatment 3.7 ± 2.7) also significantly different (p = 0.009).

Conclusion: We found no significant difference in levels of reported distress between patients with malignant and non-malignant hematologic disorders. Significantly higher distress levels were reported in women, younger patients, those with previous psychiatric disease, date of visit, and those at relapse. Patients with either malignant or non-malignant hematologic disorders should be considered for distress screening and management.
Anti-Idiotype Antibody Response after Vaccination Correlates with Better Overall Survival in Follicular Lymphoma.
Authors: Ai Wei Z. , Tibshirani Robert , Taidi Behnaz , Czerwinski Debra , Levy Ronald
Published: October 2019

Purpose Immunoglobulin idiotype (Id) is a clonal marker expressed by B cells. Lymphoma B cells express a unique Id which serves as an ideal target for immunotherapy. It has been shown that induction of anti-Id immune response through vaccination is associated with many clinical benefits, including tumor regression, molecular remission, and prolonged progression free survival (PFS). However, it is unknown if induced immune response is an independent predictor for overall survival (OS). Patients and Methods We analyzed 91 patients who were uniformly treated with CVP followed by idiotype vaccination as the first-line therapy. The induction of anti-Id immune response was evaluated and correlated with OS. Univariate and multivariate analyses were applied to select independent factors that predicted OS.

Results: Those patients who achieved a CR/CRu to CVP chemotherapy or produced anti-Id antibody after vaccination had longer survival than those who did not. At 10 years, OS was 90% and 68% for patients with or without a CR/CRu after CVP (p = 0.024); 90% and 69% for patients with or without tumor-specific antibody production, respectively (p = 0.027). In contrast, generation of anti-Id cellular response did not correlate with OS. Furthermore, CR/CRu to CVP and generation of anti-Id antibody response were the only two independent factors among those tested that were correlated with longer survival.

Conclusion: Generation of anti-Id antibody after active immunotherapy is an independent predictor for better OS in follicular lymphoma.
TNF-alpha Plays a Crucial Role in the JAK2-V617F Induced Myeloproliferative Disorder.
Authors: Bumm Thomas G.P. , VanDyke Jonathan , Loriaux Marc , Gendron Carolyn , Wood Lisa G. , Druker Brian J. , Deininger Michael W.N.
Published: October 2019

Background: The JAK2 V617F mutation is present in almost all patients with polycythemia vera (PV) and to a large proportion in patients with essential thrombocythemia and idiopathic myelofibrosis. Mice transplanted with JAK2 V617F infected bone marrow develop a myeloproliferative disorder (MPD) resembling PV with secondary myelofibrosis. We have shown that mice with JAK2 V617F induced MPD have significantly higher plasma TNF-alpha levels compared to controls (1). We therefore hypothesized that TNF-alpha may be involved in the pathogenesis of murine JAK2 V617F-induced MPD.

Methods: In a side by side experiment 5-FU stimulated murine bone marrow cells from TNF-alpha knock out mice (B6;129S6-Tnftm1Gkl/J, referred to as TNF-alpha null) and wild type F1-hybrid control mice (B6;129S6F1/J, referred to as TNF-alpha WT) were infected with MIGR1-IRES-GFP retroviral vector carrying murine wild type JAK2 (JAK2-WT) or JAK2-V617F or empty vector control (MIGR1). Cells were transplanted into lethally irradiated TNF-alpha null or WT recipients. The mice were monitored by observation and blood counts. At day 90 mice were euthanized for detailed pathologic analysis.

Results: TNF-alpha WT mice transplanted with TNF-alpha WT cells infected with JAK2-V617F (but not JAK2 WT or MIGR1) developed a PV-like MPD characterized by plethora and elevated hematocrit (HCT) levels (Figure 1). Autopsy and histology revealed splenomegaly with extramedullary hematopoiesis, while the bone marrow showed trilineage hyperplasia and severe reticulin fibrosis. In contrast TNF-alpha null or WT mice transplanted with JAK2-V617F infected TNF-alpha null marrow retained normal blood counts and their spleen weights were similar to controls (Figure 1). Spleen histology showed only mild to moderate infiltration with myeloid cells, with preservation of splenic architecture. Bone marrow cellularity was only slightly increased, and reticulin fibrosis was absent except in one mouse with focally moderate fibrosis. As expected, recipients of JAK2 WT or MIGR1 infected marrow showed no signs of MPD.

Conclusion: TNF-alpha is required for induction of murine JAK2-V617F-positive MPD, at least in the B6;129S6 background. The underlying mechanism is under study. One possibility is that suppression of residual normal hematopoiesis by JAK2-V617F-induced TNF-alpha is required for complete penetrance of the MPD phenotype. This would be in analogy to human PV, where erythroblasts from JAK2 V617F-positive PV patients show increased resistance to CD95-induced apoptosis compared to normal erythroblasts, which may give them a proliferative advantage over normal cells (2).

Figure 1. Figure 1.
Addition of Rituximab to CHOP Chemotherapy Significantly Improves Survival of Patients with Transformed Lymphoma.
Authors: Al-Tourah Abdulwahab J. , Savage Kerry J. , Gill Karamjit K. , Klasa Richard J. , Sehn Laurie H. , Shenkier Tamara N. , Gascoyne Randy D. , Connors Joseph M.
Published: October 2019

Background: Transformation of indolent lymphoma into diffuse large B-cell lymphoma (DLBCL) has been associated with poor survival and the optimal therapy is unknown. The addition of rituximab to CHOP chemotherapy (CHOP-R) has been shown to significantly improve the survival of patients (pts) with de novo DLBCL. However, the impact of rituximab on the survival of patients with transformed DLBCL remains unknown.

Methods: As part of a large retrospective analysis examining the long term natural history of pts initially diagnosed with indolent lymphoma in British Columbia (B.C.) (n=690; follicular 600, small lymphocytic 49, lymphoplasmacytic 41), we have identified those pts who developed transformed lymphoma. The definition of transformation was based on either histological confirmation or clinical features, defined as one or more of the following: rapid discordant nodal or extranodal growth; sudden rise in LDH to > 2 x previous baseline; involvement of unusual extranodal sites; or hypercalcemia. Pts were excluded from this analysis if they received high-dose chemotherapy and stem cell transplant as part of the treatment for their transformed lymphoma (n= 9). Pts who were too frail to be treated with multi-agent chemotherapy were also excluded. The extent of disease at transformation was considered limited in pts with single nodal region involvement, no B symptoms, non-bulky disease (<10 cm) and with limited extranodal involvement. Pts with more extensive disease were considered to have advanced transformation.

Results: 108 pts with transformed lymphoma were identified in the B.C. lymphoid database, the majority of whom had a prior history of follicular lymphoma (n=103, 95%). Median age at transformation was 60 y (22–71). Transformation was histologically confirmed in 74 (69%) pts. The outcome of 23 pts who were treated with CHOP-R was compared to 85 pts who were treated with CHOP-like chemotherapy. None of the 108 pts had received rituximab as part the management of their indolent lymphoma. The median follow-up for all living pts in the CHOP-like cohort was 7 y (3–14) and 3 y (0.5–5) in the CHOP-R cohort. The post-transformation 5 y overall survival following CHOP-like chemotherapy was 33% vs 61% after CHOP-R (P= 0.01). This difference in survival remained significant when pts with clinically diagnosed transformation were excluded from the analysis (P= 0.02). For the 78 pts with advanced disease extent (59, CHOP-like; 19, CHOP-R) there was a significant difference in PT-5yOS (21% vs 57%, respectively; P= 0.005). For pts with limited disease extent (CHOP-like, 26; CHOP-R, 4) no significant difference in PT-5yOS was observed (P= 0.2).

Conclusions: In rituximab-naive patients with indolent lymphoma, the addition of rituximab to CHOP chemotherapy significantly improves overall survival after transformation.
Hydroxyurea Utilization in Nigeria, a Lesson in Public Health.
Authors: Aliyu Zakari Y. , Babadoko Aliyu , Mamman Aisha
Published: October 2019

Hydroxyurea is a successful and cost effective drug therapy for sickle cell disease. Treatment with hydroxyurea is associated with a significant decrease in sickle cell complications, hospitalizations and transfusion requirements by about 50% and mortality reduction by 40% in clinical studies. The drug is unfortunately underutilized in sickle cell disease in the United States despite clear efficacy data and management experience. There is no data on the utilization of hydroxyurea in Africa, a part of the world with the highest global burden of sickle cell disease. We prospectively interviewed 206 consecutive adults and pediatric sickle cell patients as part of the Nigerian pulmonary hypertension screening study and reviewed over 1000 patients followed longitudinally at Ahmadu Bello university teaching hospital in Zaria, Nigeria. We also interviewed 10 hematologists (3 specialists and 7 hematologists in training) at the same university hospital. 65% of the 206 prospectively evaluated patients met the Multicenter Study of Hydroxyurea clinical indications for hydroxyurea treatment. No patient (zero percent) was on hydroxyurea therapy. All hematologists (100%) reported their discomfort with instituting hudroxyurea. Barriers to hydroxyurea utilization identified by practitioners included safety and toxicity profile (100%), patient compliance (100%), effective follow up (100%), drug availability (100%), affordability (100%) and specifically concern for reactivation of latent tuberculosis (50%) and carcinogenesis (100%) and teratogenicity (100%). Only 5% of patients had been informed of or were aware of hydroxyurea as a treatment option in sickle cell disease. Patient related barriers to hydroxyurea identified include lack of awareness (95%), cost (100%), availability (100%), need for frequent follow up (90%), pregnancy restrictions and need for concomitant contraceptive use (98%) and risk of infections (98%). Our study indicates the absolute lack of hydroxyurea utilization in a major health care center in Nigeria. Nigeria has the highest incidence of sickle cell disease in the world with about 150,000 children born with the disease annually. Sickle cell disease accounts for about 9 –16% of under-five mortality rates in the country. The sickle cell disease related morbidity, mortality and health systems financial burden remains very high in Nigeria and most of Africa. Local health care provider education and support and patient counseling and education are needed for the successful introduction of hydroxyurea in Nigeria. Clinical studies designed to assess the safety and efficacy of hydroxyurea in unique African settings is needed to facilitate the introduction and utilization of hydroxyurea in Nigeria and other parts of Africa.
Identification of Candidate Genes Associated with Sensitivity and Resistance to the Histone Deacetylase Inhibitor Valproic Acid in Patients with Acute Myeloid Leukemia.
Authors: Craddock Charles F. , Khanim Farhat , Arrazi Julie , Turner Bryan , Bunce Christopher
Published: October 2019

Histone deacetylases inhibitors (HDIs), such as valproic acid (VPA), demonstrate significant clinical activity in a proportion of patients with high risk acute myeloid leukemia (AML). However the mechanism by which HDIs selectively induce apoptosis in leukemic blasts remains unknown. We have therefore correlated the impact of VPA exposure on gene expression in leukemic cell lines and primary AML blasts with its ability to induce cell death in vitro and induce clinical responses in vivo. 14 hemato-lymphoid cell lines were tested for their sensitivity (IC50) to apoptotic cell killing by VPA. Gene expression array analyses using HGMP chip 6500 gene arrays were performed on the same cell lines prior to HDI exposure. A bioinformatics approach combined the array and IC50 data to generate a score for each gene identifying those whose elevated expression correlated with sensitivity or resistance to VPA in vitro. In a concurrent Phase I/II clinical trial 24 patients with high risk AML (relapsed n=11, newly diagnosed n=6, primary refractory n=3) with a median age of 64 yrs (41–83 yrs) received combination treatment with VPA, all trans-retinoic acid (ATRA) and theophylline. Changes in histone acetylation and expression of HDI responsive genes was measured in leukaemic blasts before and after commencement of VPA therapy. Expression of genes associated with VPA sensitivity in vitro were re-analysed with respect to their expression in pre-treatment blasts from non-responding and responding trial patients. By combining LC50 values to VPA and microarray data generated from pre-treatment RNA in 14 hematolymphoid cell lines we were able to identify candidate genes and signalling networks mediating sensitivity and resistance to VPA in vitro. Genes whose higher expression conferred sensitivity included IL-1β and those associated with VPA resistance included PLOD2, cyclin B1 (CCNB1) and ACVR2A. In the clinical trial 5 patients, all with relapsed AML, achieved clinical responses by Cheson criteria (complete remission n=1, partial remission n=4). Comparison of gene expression, as defined by microarray studies, in responding and non-responding patients with the previously identified in vitro VPA signalling networks identified that similar networks to those defined in vitro appeared to be correlated with clinical response. This study demonstrates induction of pro-apoptotic gene expression by VPA in a clinical context and identifies potential mechanisms through which its anti-leukaemic effect may be mediated in vivo. Furthermore we have identified potential VPA-signalling networks containing novel sensitivity and resistance genes whose expression correlates with VPA-mediated tumour cell killing in vitro and may predict clinical activity.
Hematin Promotes Complement Alternative Pathway-Mediated Deposition of C3 Activation Fragments on Human Erythrocytes: Potential Implications for the Pathogenesis of Anemia in Malaria.
Authors: Taylor Ronald P. , Pawluczkowycz Andrew W. , Lindorfer Margaret A. , Waitumbi John W.
Published: October 2019

Childhood malaria caused by Plasmodium falciparum (pf) is often characterized by severe anemia at low parasite burdens; the mechanism(s) responsible for this pathology remain to be defined. We have reported that erythrocyte (E) CR1, the immune adherence receptor specific for C3b, is reduced during anemia in childhood malaria, suggesting a possible role for complement in E destruction. Intravascular lysis of infected E by pf leads to release of E breakdown products hemoglobin and hematin, which have inflammatory properties. Free hematin can bind to E, and we find that in serum and in whole blood anti-coagulated with lepirudin, moderate concentrations of hematin activate the alternative pathway of complement and promote deposition of C3 activation and breakdown products on E. We documented C3 deposition by flow cytometry, and additional fluorescence microscopy studies revealed that most of the deposited C3 fragments are located in close juxtaposition to CR1. Western blots confirmed that the C3 fragments are indeed covalently bound to the E, and immunoprecipitation experiments indicated that a fraction of the deposited C3 is covalently bound to CR1. The degree of C3 fragment deposition is directly correlated with E CR1 levels, both within a given donor’s E population and when E from different donors are compared. E opsonized with complement in the presence of hematin form rosettes with Raji cells, through interaction with CR2, the C3dg receptor expressed on several types of B cells including splenic marginal zone B cells. Thus, hematin-mediated complement activation and C3 fragment deposition on E may promote accelerated splenic (or liver) clearance of the youngest E, which have the most CR1, leading to sudden onset of anemia along with reduction of mean CR1 on surviving E. A monoclonal antibody specific for C3b, mAb 3E7, previously demonstrated to inhibit the alternative pathway of complement, completely blocks the C3 fragment deposition reaction. Use of this monoclonal antibody in non-human primate models of malaria may provide insight into mechanisms of erythrocyte destruction and thus aid in the development of therapies based on inhibiting the alternative pathway of complement.
Depletion and Restoration of the Intravascular Nitrite (NO2-) Reserve Modulate Susceptibility to Liver Vaso-Occlusive Infarction in Sickle Cell Transgenic Mice.
Authors: Hsu Lewis L. , Shiva Sruti , Noguchi Audrey , Curtis Erin , MacArthur Peter , Raat Nicolaas J.H. , Diwan Bhalchandra A. , Schechter Alan N. , Noguchi Constance Tom , Gladwin Mark T.
Published: October 2019

Multiple studies link low bioavailability of nitric oxide (NO) and major complications of sickle cell disease, especially pulmonary hypertension and acute vaso-occlusive pain episodes. A gender dimorphism has been suggested in human studies, with lower levels of NO availability observed in males. These studies support efforts to develop NO-based therapeutics and the newly characterized small molecule nitrite represents an ideal candidate. Nitrite is a circulating anion that is reduced to NO during physiological and pathological hypoxia and mediates potent cellular cytoprotection at physiologic concentrations in the setting of ischemia and reperfusion injury. Supporting a role for nitrite in sickle cell pathogenesis, we found that nitrite levels are depleted in transgenic mice expressing exclusively sickle hemoglobin (sickle mice), with whole blood nitrite levels 50% lower compared to control mice, and are further reduced by 19% in male sickle mice compared with females. Thus, the murine model demonstrates a sexual dimorphism in nitrite levels, consistent with data on human NO bioavailability. We then examined hepatic vaso-occlusion by challenging sickle mice with 2 hours of moderate normobaric hypoxia (10% oxygen), restoring them to normoxia, and then 18 hours later drawing blood to assay plasma alanine aminotransferase (ALT) levels as a quantitative measure of tissue injury. Sickle mice showed a 4- fold rise in ALT with this hypoxia-reoxygenation (HR) challenge; wild-type controls or non-sickling colony control mice with the same HR challenge had no change in ALT. Sexual dimorphism was again evident such that female mice were less susceptible to HR challenge than males. Therapeutic nitrite supplementation, administered either as 2.4 nanomole/g intraperitoneal injection during HR or oral supplementation (estimated 4 micromole/g/day) for 7 days before HR challenge, abrogated the ALT rise in sickle mice. Pharmacokinetic data indicated that nitrite supplementation at these injected and oral doses raise the nitrite levels in blood and tissue, without affecting methemoglobin levels. These studies provide additional mechanistic evidence for a gender contribution to sickle cell endothelial dysfunction and vasculopathy, and suggest that reduced NO bioavailabity in SCD results in dysregulated nitrite homeostasis. Depleted nitrite reserves are associated with enhanced injury with hypoxia-induced vaso-occlusion and therapeutic restoration of nitrite levels, both by intraperitoneal delivery and in the diet, reduces this injury.
MKL1 Promotes Megakaryocytic Differentiation Via Stimulation of Serum Response Factor Target Genes.
Authors: Cheng Ee-chun , Renda Matthew J. , Wang Lin , Krause Diane S.
Published: October 2019

Our studies demonstrate a critical role for MKL1 (megakaryoblastic leukemia 1) in the molecular regulation of megakaryocytopoiesis. MKL1 is part of the fusion protein formed by the t (1; 22) translocation, which is found uniquely in Acute Megakaryoblastic Leukemia (AMKL). The translocation fuses the RBM15 (also known as OTT) gene on chromosome 1 with the MKL1 (also known as MAL) gene on chromosome 22. Previous studies in muscle cells show that MKL1 is a positive cofactor for the transcription factor serum response factor (SRF), and works via the Rho-A pathway to turn on immediate early genes and muscle specific genes. Using qRT-PCR we show that MKL1 mRNA is markedly up-regulated during megakaryocyte (MK) differentiation of primary murine bone marrow and fetal liver cells. When we overexpress MKL1 in the human erythroleukemia (HEL) cell line and differentiate the cells to become MK by phorbol ester (TPA), there is far greater MK differentiation than in control HEL cells. Via analysis of Wright-Geimsa stained cytospins, MKL1 overexpression increases the average percentage of mature MK from 26% to 48%. Using flow cytometry, platelet glycoprotein V positive cells increase from 14% to 43% on average. The percentage of cells with greater than 4N ploidy increases from 13% to 34%. In order to assess the mechanisms by which MKL1 promotes MK differentiation, we tested whether SRF expression was required for the effects of MKL1. The stimulatory effects of MKL1 are strongly abrogated when cells are transfected with siRNA against SRF, proving that MKL1 acts via SRF to stimulate MK differentiation. Interestingly, SRF siRNA also causes a statistically significant decrease in ploidy in control cells stimulated with TPA. By microarray analyses using both Affymetrix and Illumina platforms, enforced MKL1 expression upregulates many cytoskeletal genes and adhesion molecules, enhances the expression of platelet specific genes such as glycoprotein V (consistent with the FACS data), and accelerates the loss of expression of genes associated with erythropoiesis, such as erythrocyte membrane protein band 4.2. These data indicate that MKL1 enhances MK differentiation by promoting endoduplication as well as increasing expression of platelet-specific genes and of multiple cytoskeletal proteins required for proplatelet formation.
Homeostatic Role of the Krüppel-Like Factor 4 (KLF4) in Proliferation and Differentiation of Primitive Hematopoietic Progenitor Cells.
Authors: Park Chun Shik , Yamada Takeshi , Lacorazza H. Daniel
Published: October 2019

Abstract 1497

Poster Board I-520

KLF4 is a tumor suppressor in the gastrointestinal tract known to induce cell cycle arrest in a cell context dependent manner. We recently reported that KLF4 maintains quiescence of T lymphocytes downstream of T-cell receptor signaling (Yamada et al., Nature Immunology, 2009). The role of KLF4 in reprogramming adult somatic cells into pluripotent stem cells along with Oct3/4, c-Myc and Sox2 suggests that KLF4 restricts proliferation of undifferentiated cells. In spite of a redundant role of KLF4 in fetal liver hematopoietic stem cells (HSC), its role in the maintenance of adult bone marrow HSCs has not been studied yet. To study the role of KLF4 in the hematopoietic system we used gain- and loss-of-function mouse models. Retroviral transfer of KLF4 into wild type bone marrow (BM) cells led to significant reduction of colony forming units (CFU) in methylcellulose cultures due to increased apoptosis and lower proliferation. Then, Mx1-Cre was used to induce deletion of Klf4-floxed mice by polyI:C administration. Analysis of peripheral blood cells up to 6-9 months post polyI:C administration showed significant reduction of monocytes, as previously reported, and expansion of CD8+CD44+ T cells due to their increased proliferative potential. BM cells from Klf4-deficient mice exhibited increased number of myeloid progenitor cells measured by flow cytometry (Lin-Sca-1-c-kit+FcRII/III+CD34+ cells), CFU and CFU-S8. Cytoablation with 5-fluorouracil (5-FU) showed lower nadir of peripheral white blood cells in Klf4-deficient mice compared to control mice. In spite of normal multilineage reconstitution in BM transplants experiments, competitive reconstitution with Klf4-deficient and normal BM cells resulted in reduced contribution of Klf4-deficient cells to peripheral blood, likely due to homing and proliferative differences. Collectively, our data shows that KLF4 has an important role in function of hematopoietic stem and progenitor cells.

Disclosures:

No relevant conflicts of interest to declare.
Acute Myeloid Leukemia Cells Depend on VEGF, PDGFR and SCF Receptor Signaling: Leukemia Regression with Pazopanib.
Authors: Gars Eric , McGee Christie S , Bosse Raphael C , Wise Elizabeth , Meacham Amy , Cogle Christopher R.
Published: October 2019

Abstract 1057

Acute myeloid leukemia (AML) cells depend on endothelial cells for survival and proliferation. By targeting endothelial cells with a novel vascular disrupting agent, we recently demonstrated regression of AML (Madlambayan, et al., Blood 2010). In an effort to discover a more selective, anti-vascular therapy for leukemia, we hypothesized that targeting endothelial cell-derived paracrine and leukemia cell-derived autocrine growth factors would result in regression of disease. In particular, our strategy focused on vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF) and stem cell factor (SCF), which are known to promote leukemia cell proliferation. Receptors for each of these growth factors are potently inhibited by pazopanib, which is an orally available tyrosine kinase inhibitor. The aim of our study was to determine the dependence of leukemia cell survival and proliferation on the combined receptor signaling of VEGF, PDGF and SCF. Leukemia cell lines (KG-1, HL60 and K562) were incubated at various durations with varying concentrations of pazopanib. Leukemia cell proliferation was quantified using XTT assay. Apoptosis induction was analysed by flow cytometry (Annexin V and PI staining). Pazopanib effectively impaired proliferation in leukemia cells in vitro in a dose and time dependent fashion. During a 16-hour incubation of leukemia cells, pazopanib showed a 50% lethal concentration (LC50) of 22.57 μM (r = .986), 41.6 μM (r = .991), and 81.97 μM (r = .996) for HL60, K562, and KG-1 cells, respectively. Staining with Annexin V and PI identified apoptosis as the main cause of cell death after exposure to pazopanib. The IC50 of apoptosis for HL60 cells was 132.5 ± 2.7 μM (p = 0.0002). These results indicate that leukemia cells depend on the combined signaling of VEGF, PDGF, and SCF, and suggest that selective inhibition by pazopanib may be a promising therapeutic for AML. Furthermore, pazopanib is orally available and associated with minimal side effects, thus representing an attractive candidate for further testing in AML.

Disclosures:

No relevant conflicts of interest to declare.
Liposomal Targeted Delivery Overcomes Immunostimulatory Effects of Oligonucleotide Based Therapy In Chronic Lymphocytic Leukemia.
Authors: Yu Bo , Mao Yicheng , Bai Li-Yuan , Herman Sarah E.M. , Ramanunni Asha , Jin Yan , Mo Xiaokui , Cheney Carolyn , Chan Kenneth K. , Jarjoura David , Marcucci Guido , Lee Robert , Lee L.James , Byrd John C. , Muthusamy Natarajan
Published: October 2019

Abstract 1475

Therapeutic application of several CpG-containing anti-sense oligodeoxyribonucleotides (ODN) and siRNAs are limited due to their potent immune stimulatory properties. To resurrect these promising agents for clinical application, we have developed an antibody based liposomal delivery strategy that overcomes the immunostimulatory properties without compromising the target down modulation. We show here G3139, an archetypical anti-sense ODN for Bcl-2, induced activation of NF-kB, which results in the up-regulation of its downstream anti-apoptotic targets such as Bcl-2 (22.5%, n=10, p=0.04) and Mcl-1 (35.0%, n=4, p<0.005) and co-stimulatory markers such as CD86 (47.5%, n=10, p<0.05) and HLA-DR (175%, n=10, p<0.005) as well as cytokine flare in B chronic lymphocytic leukemia (CLL) cells. These adverse immunostimulatory responses are abrogated by Rituximab mediated immunoliposomal delivery of G3139, resulting in significant Bcl-2 down-regulation and ∼60% enhanced sensitivity to fludarabine induced cytotoxicity (n=4, p<0.001). Consistent with the in-vitro observations, significant in-vivo therapeutic efficacy was demonstrated in a SCID mouse xenograft leukemia/lymphoma model. A total of 74 female 6- to 8-week-old SCID mice were engrafted with 2×106 Raji cells through tail vein injection on Day 0. The 10-day treatment was initiated via i.p. injection on Day 4. Fludarabine and G3139 were injected into the mice at the dose of 100mg/kg/day and 5mg/kg on alternative days respectively. Hind limb paralysis was considered as the key endpoint while other early removal criteria were also considered. The median survival time for placebo controls was 15 to 18 days without liposomal G3139 treatment. Although the in-vivo experiment is still in progress, CD20-targeting ILP-G3139 has already shown significant therapeutic efficacy with >80% survival rate (n=14) at day 58 of the study as a single agent or in combination with fludarabine treatment, compared to the mice treated with the control Her-ILP-G3139 that showed hind-limb paralysis before Day 45 in all mice. These results indicate potential use of targeted delivery vehicles to resurrect and improve the clinical efficacy of immunostimulatory oligonucleotide therapeutics for B-cell malignancies.

Disclosures:

No relevant conflicts of interest to declare.
F-627, a G-CSF Dimer, Stimulated a More Rapid Neutrophil Recovery In Cyclophosphamide-Treated Monkeys Compared to Monomer Rhg-CSFs.
Authors: Hu Zheng Tao , Huang Zhi Hua , Cen Xiao Bo , Wang Tao , Zhuang Kun , Yang Hong Zhou , Zhang Dan Ping , Sun Nai Chau , Sun Rou Yun , Wang Li , Huang Yu Liang , Yan Xiao Qiang
Published: October 2019

Abstract 1485

Recombinant human granulocyte colony-stimulating factors (rhG-CSF) including filgrastim, lenograstim and pegfilgrastim are widely used to treat chemotherapy-induced neutropenia. However, it remains a challenge to manage severe neutropenia in cancer patients after high dose chemotherapy. The activation of G-CSFR by the G-CSF requires dimerization of two receptor chains bound to two G-CSF ligands. We hypothesized that a G-CSF dimer might generate a faster in vivo response, thus to benefit patients with severe neutropenia. F-627 is a recombinant human G-CSF dimer expressed in mammalian cells. In vitro, F-627 was able to activate STAT3 and to stimulate the proliferation of 32D-GCSFR and M-NSF60 cell lines. To evaluate the efficacy of F-627 in chemotherapy-induced neutropenia, 36 adult cynomolgus monkeys (3/sex) were injected with cyclophoshamide (CY, i.v., 60 mg/kg on day 0 and 65 mg/kg on day 1). Animals were randomized to receive (s.c.) either carrier, or F-627 on day 5 and day 10, at 25, 60 and 150 μg/kg, or rhG-CSF at 10 μg/kg /day (daily injection), or pegfilgrastim at 60 μg/kg on day 5 and day 10. Pharmacokinetics and pharmacodynamics (PK/PD) were evaluated. Significant absolute neutrophil count (ANC) increase was observed in animals treated with F-627 at 25 μg/kg compared to carrier-treated monkeys. At nadir, F-627 at 60 μg/kg generated optimal ANC response with 6.9- and 3.0-fold higher ANC compared to pegfilgrastim- (0.17 × 109/L) and rhG-CSF- (0.39 × 109/L) treated monkeys, respectively. The PK parameters of F627 at 60 ug/kg including MRT, Cmax, Tmax, AUC and CL were comparable to that of pegfilgrastim-treated monkeys, despite the relative G-CSF molar dose in F-627-treated animals was 2.5x lower than the pegfilgrastim-treated animals. The results demonstrate that the G-CSF dimer generated a faster response in CY-treated monkeys compared to the G-CSF monomer-treated animals, suggesting that F-627 could benefit cancer patients with severe neutropenia after high-dose chemotherapy.

Disclosures:

No relevant conflicts of interest to declare.
The Immune-Inhibitory Receptor Osteoactivin Is up-Regulated In Monocyte-Derived Dendritic Cells Upon Exposure to Tyrosine Kinase Inhibitors
Authors: Gutknecht Michael , Schwarzbich Mark-Alexander , Salih Julia , Kanz Lothar , Salih Helmut R. , Schmidt Susanne M. , Grünebach Frank
Published: October 2019

Abstract 1733

Targeted therapies with tyrosine kinase inhibitors (TKI) have significantly improved the treatment of cancer patients. Ex vivo generated dendritic cells (DC) are commonly used in immunotherapeutic strategies due to their unique ability to initiate adaptive immune responses, and multiple approaches presently aim to combine targeted therapies with immunotherapy. However, as many kinases targeted by TKI are, besides governing tumor cell growth, also involved in the activation of DC, TKI therapy may cause immunoinhibitory side effects. Osteoactivin (GPNMB, DC-HIL) is a type I transmembrane glycoprotein that is detected abundantly in DC but not in monocytes. Its expression on antigen-presenting cells can inhibit T cell activation by binding syndecan-4 (SD-4) on T cells. Here we investigated the effect of the BCR/ABL TKI imatinib, dasatinib and nilotinib, which are approved for the treatment of CML, on the expression of osteoactivin and DC functions. DC were generated from blood monocytes by plastic adherence and exposure to GM-CSF and IL-4. Imatinib, nilotinib or dasatinib were added to the culture medium every second day starting from the first day of culture. In some experiments, toll-like receptor (TLR) ligands (L) (LPS (TLR4L), pam3Cys (TLR2L), poly I:C (TLR3L) or R848 (TLR7/8L) were added on day 6 of culture for maturation of DC. We found that DC generated in the presence of therapeutic concentrations of all three TKI displayed an altered phenotype. Imatinib caused significantly reduced expression of the typical DC markers CD1a, CD83 and the co-stimulatory molecule CD86. Nilotinib reduced the expression of CD1a, CD83, CD86 and the DC-specific C-type lectin receptor DC-SIGN (CD209). Dasatinib impaired expression of CD1a, CD83, CD86, CD80 and DC-SIGN. Most notably, we observed excessive up-regulation of osteoactivin on DC upon treatment with all three TKI. Interestingly, incubation with the immunosuppressive and anti-inflammatory cytokine IL-10 also resulted in osteoactivin over-expression. In line with osteoactivin up-regulation, exposure to TKI resulted in reduced stimulatory capacity of DC in MLR with allogenic T cells that could be restored by addition of blocking anti-osteoactivin antibody. In summary, our data demonstrate that up-regulation of osteoactivin is critically involved in the inhibition of DC function upon TKI exposure. These findings are of great importance for future combinatory approaches using TKI and DC-based immunotherapy and indicate that inhibition of osteoactivin expression or function may serve as a novel strategy to enhance the efficacy of immunotherapeutic interventions in cancer patients.

Disclosures:

No relevant conflicts of interest to declare.