bluuebunny/crossref_metadata_2025 huggingface dataset:
Human Antagonistic Anti-CD40 Antibody, CHIR-12.12, Inhibits CD40-Mediated Survival of Chronic Lymphocytic Leukemia B Cells Leading to Cellular Apoptosis.
Authors: Cherukuri Anu , Kadel Edward , Lee Sang H. , Goldbeck Cheryl , Heise Carla , Luqman Mohammad , Aukerman Sharon L. , Aziz Natasha , O’Brien Susan , Wierda William G. , Jallal Bahija
Published: April 2020

CD40 and CD40 ligand (CD40L) interaction is a key regulator of B-chronic lymphocytic leukemia (CLL) survival. CD40 activation leads to binding with tumor necrosis factor receptor-associated factors (TRAFs) and the subsequent activation of multiple downstream signaling pathways involved in cellular proliferation and survival. We have generated a novel fully human IgG1 anti-CD40 antagonistic monoclonal antibody, CHIR-12.12, using XenoMouse® mice (Abgenix, Inc). CHIR-12.12 blocks CD40L binding to CD40 and inhibits CD40L-induced proliferation/survival of normal human B cells, primary CLL cells, and primary non-Hodgkin’s lymphoma (NHL) cells. We have also demonstrated that it has highly potent antibody-dependent cellular cytotoxicity (ADCC) against primary CLL and non-Hodgkin’s lymphoma cells. We have now investigated its effects on primary CLL cell survival. Soluble human CD40L prolongs primary CLL cell survival in culture, and treatment with CHIR-12.12 inhibits this survival when measured 48–72 hours after addition of CHIR-12.12. CD40L-mediated survival is associated with activation and phosphorylation of Akt, p38 MAPK, ERK, and IkB kinases a and b. Additionally, the anti-apoptotic proteins Mcl-1, Bcl-xl, and XIAP are induced, and markers of apoptosis (cleaved PARP and Caspase-3) are reduced. In contrast, CHIR-12.12 treatment of CD40L-stimulated primary CLL cells ex vivo inhibited downstream phosphorylation of Akt, p38 MAPK, ERK, and IkB kinases (IKK) a and b. Additionally, CHIR-12.12 treatment resulted in induction of cleaved caspase-3 and PARP, and reduction of XIAP, Mcl-1, and Bcl-xl expression, ultimately leading to CLL cell apoptosis. These results demonstrate that CHIR-12.12 inhibits CD40L-mediated signaling pathways and cell survival and could be a potential therapeutic treatment for CLL. CHIR-12.12 is currently in a Phase I clinical study for CLL.
The Shaving Reaction. Rituximab/CD20 Complexes Are Removed from Mantle Cell Lymphoma Cells (Z138) and Chronic Lymphocytic Leukemia (CLL) Cells by Monocyte/Macrophages: In Vitro Studies and a SCID Mouse Model.
Authors: Taylor Ronald P. , Beum Paul V. , Li Yongli , Lindorfer Margaret A. , Williams Michael E.
Published: April 2020

Clinical investigations have revealed that infusion of immunotherapeutic monoclonal antibodies directed to normal or tumor cells can lead to loss of targeted epitopes, a phenomenon called antigenic modulation. Recently we reported that Rituximab (RTX) treatment of CLL patients induced substantial loss of CD20 on B cells found in the circulation after RTX infusion, when RTX plasma concentrations were high. Antigenic modulation can severely compromise therapeutic efficacy, and we postulated that B cells had been “shaved” of RTX/CD20 complexes by monocytes or macrophages of the mononuclear phagocytic system (MPS) in a reaction mediated by Fcγ receptors. Our in vitro model is based on reacting RTX-opsonized CD20+ cells with acceptor THP-1 monocytes differentiated to a macrophage-like phenotype. After 45 min at 37°C, ~50–80% of both RTX and CD20 are removed from opsonized cells, and both proteins are demonstrable on acceptor THP-1 cells, as ascertained by flow cytometry, fluorescence microscopy, and immunoblotting. Tests with inhibitors and use of F(ab’)2 fragments of RTX indicate shaving is mediated by Fcγ receptors, and shaving occurs equally well in the presence and absence of complement. We developed a pre-clinical xenograft mouse model to replicate shaving: 5 X 106 Z138 cells were infused iv or sc, into SCID/CB17 mice, and ~25 days later animals were treated with varying doses of RTX. Z138 cells recovered from lungs (iv model) or tumors (sc model) of treated and control mice 30 min to 3 days after RTX infusion were analyzed by flow cytometry. Substantial loss of CD20 was demonstrable on RTX-targeted cells, and this loss was more pronounced at longer times after RTX infusion. Moreover, Z138 cells in untreated mice had moderate to high levels of CD20 and few cell-associated C3 fragments, but in RTX-treated mice the situation was reversed: the Z138 cells were CD20 poor (<20% of the CD20 levels compared to untreated mice) but had significant amounts of deposited C3dg. These findings replicate our earlier studies which demonstrated that treatment of CLL patients with RTX promotes loss of CD20 as well as complement activation and C3 fragment deposition on circulating B cells. It is likely that previously described antigenic modulation, usually reported for conditions of high circulating malignant cell burdens targeted by monoclonal antibodies, was also mediated by cells of the MPS via the shaving reaction. Our findings may have profound implications for the use of monoclonal antibodies in the immunotherapy of cancer.
Pilot Study of Thrice-Weekly Low-Dose Rituximab (RTX) in Chronic Lymphocytic Leukemia (CLL): Decreased CD20 Loss Via “Shaving” and a Novel Strategy for Enhanced Therapeutic Targeting.
Authors: Williams Michael E. , Densmore John J. , Pawluczkowycz Andrew W. , Beum Paul V. , Kennedy Adam D. , Lindorfer Margaret A. , Hamil Susan H. , Petroni Gina R. , Eggleton Jane C. , Taylor Ronald P.
Published: April 2020

Treatment of CLL patients with RTX 375 mg/m2 induces 90% loss of CD20 from circulating cells via “shaving” of RTX-CD20 complexes from B cells, presumably by tissue macrophages (Kennedy, et al, J Immunol, 2004). To reduce CD20 loss and potentially enhance leukemic cell clearance, we investigated frequent low-dose RTX for CLL. Following informed consent, patients were treated 3 times/wk with RTX 20 or 60 mg/m2 x 2 wks (1 pt) or 4 wks (11 pts) with frequent blood sampling during and post-RTX. 9/12 were previously treated (1–4 prior therapies); only 2 had received prior RTX. Cytogenetics by FISH analysis: del 17p13 (6 pts), del 13q14 (5 pts) and trisomy 12 (1 pt).

Results: In each initial infusion, the first 30 mg of RTX caused a 3 to >10-fold decrease in leukemic cells in 11/12 patients. 60 mg/m2group (n=6): Two hrs after the 1st RTX infusion, cells expressed <20% of baseline CD20 levels; these levels further decreased with subsequent RTX treatments. 2/6 achieved normal lymphocyte counts. One patient achieved CR, ongoing at 9+ mo.; 5 had stable adenopathy and/or splenomegaly (SD) for 3–8 mo.; 3 required subsequent therapy. 20 mg/m2group (n=6): Two hrs after the 1st RTX infusion, cell surface CD20 levels were at ≥50% of initial values (4/6 pts) and were preserved throughout RTX therapy in 5/6 patients. 3/6 achieved normal lymphocyte counts with subsequent RTX infusions. One patient achieved CR lasting 8 mo. There was 1 PR, 3 SD and 1 with no response; 4 required subsequent therapy. The 2 patients with highest cell surface CD20 expression achieved CR, despite the presence of the del 17p13 in both cases. RTX infusion activated Complement (C); large amounts of the C3 activation product C3dg were deposited on targeted cells. However, 70% of deposited C3dg was demonstrable on circulating cells which had lost CD20 and persisted in the circulation.

Toxicity: Treatments were well tolerated, with grade 1–2 infusion reactions and transient neutropenia and thrombocytopenia during initial infusions.

Conclusions: Thrice-weekly low-dose RTX is clinically active and can induce remissions in patients with CLL. Low-dose RTX may promote enhanced cytotoxic attack and clearance of circulating CLL cells via CD20 target preservation; 20 mg/m2 dosing better preserves CD20 levels via decreased shaving vs 60 mg/m2. Our findings suggest that C3dg can serve as a molecular marker for circulating CLL cells which have lost CD20 due to shaving during RTX therapy. These results support further investigation of thrice-weekly low-dose RTX as a novel strategy for improved therapeutic targeting of RTX in CLL.
A Phase 2 Clinical Trial of AP23573, an mTOR Inhibitor, in Patients with Relapsed or Refractory Hematologic Malignancies.
Authors: Rizzieri David A. , Feldman Eric , Moore Joseph O. , Roboz Gail J. , DiPersio John F. , Gabrail Nashat , Stock Wendy , Rivera Victor M. , Albitar Maher , Bedrosian Camille L. , Giles Francis
Published: April 2020

Background: AP23573 is a novel non-prodrug rapamycin analog that inhibits mTOR, a downstream effector of the PI3K/Akt and nutrient-sensing pathways. In phase I clinical trials, AP23573 completely inhibited in vivo mTOR activity in peripheral blood mononuclear cells (PBMCs) as measured by decreased phosphorylation of the mTOR target protein 4E-BP1. Because the mTOR pathway has been implicated in the pathobiology of hematologic malignancies, this study is undertaken in this patient population to determine the safety and efficacy of AP23573 treatment. In addition, correlative studies in PBMC and bone marrow samples will examine if tumoral mTOR pathway status predicts clinical response.

Methods: Eligible patients with relapsed or refractory hematologic malignancies are being enrolled, stratified into 5 disease specific groups and given AP23573 12.5 mg I.V. daily x 5 days (QDx5) every 2 weeks. Enrollment into each group is proceeding according to a Simon’s two-stage design with a minimum of 21 patients per group and an overall maximum enrollment of 205. Efficacy is being assessed according to standard response criteria at the end of each 4-week cycle (leukemia patients) or every two cycles (lymphoma patients). In addition, pharmacodynamic (PD)/biomarker samples for correlative studies are collected during Cycle 1.

Results: At this time, a total of 51 patients have been enrolled. There are 35 males and 16 females with a median age of 61 years (range 31–83). Best or most recent response to treatment by disease cohort is shown below (5 are too early for response evaluation).

Disease Cohort (N) PR HI SD PD PR: Partial Response, HI: Hematologic Improvement, SD: Stable Disease, PD: Progressive Disease Cohort 1: AML/MDS (26) 2 6 18 Cohort 2: ALL (2) 2 Cohort 3: AMM (7) 2 3 2 Cohort 4: CLL (9) 4 3 Cohort 5: T-cell Lymphoma (2), MCL (5) 2 2

AP23573 is generally well tolerated with the most prevalent mild or moderate treatment-related adverse events (AEs) being nausea, mucositis, hyponatremia, pruritis, rash, and hypokalemia. Serious, possibly treatment-related AEs include diarrhea, mucositis, hypertriglyceridemia, neutropenic sepsis, dyspnea, syncope, pleural effusion and pneumonia. Analysis of correlative PD/biomarker studies is in progress.

Conclusions: QDx5, every other week administration of AP23573 has an acceptable side-effect profile with potential efficacy in some patients with advanced stage hematologic malignancies. Indications of anti-cancer activity have been observed in 4 of the 5 disease cohorts with 19 of the 46 evaluable patients (41%) showing at least stable disease. Patient enrollment, treatment and correlative biomarker analyses continue.
The Novel Anti-Leukemic Agent LC-1, Is Preferentially Cytotoxic in CLL Cells Derived from Poor Prognostic Subsets.
Authors: Pepper Chris , Fegan Chris , Brennan Paul , Mills Ken , Burnett Alan K.
Published: April 2020

Chronic lymphocytic leukemia (CLL) has a variable clinical course but once treatment is required the development of drug resistance often ensues. Therefore, delineation of the biological mechanisms of drug resistance and the development of novel therapies designed to overcome this is an important goal of research into this condition. We have recently shown that the transcription factor NF-κB plays a central role in regulating CLL cell survival and this is mediated, at least in part, through the transactivation of a number of anti-apoptotic genes including Bcl-2. In this pre-clinical study we evaluated the cytotoxic effects of the novel parthenolide analog, LC-1 (Leuchemix Inc.), in primary tumor cells derived from 49 CLL patients (20 treated, 29 untreated). LC-1 induced apoptosis, as assessed by the Annexin V assay, in a concentration-dependent manner (0.1 – 10 μM) in all the CLL samples tested with a mean LD50 value (the concentration of drug required to kill 50% of the cells) of 2.9 μM. The induction of apoptosis was preceded by a marked loss of NF-κB activity, as evidenced by electrophoretic mobility shift assay, and a down regulation in Bcl-2 protein expression. Caspase-3 activation was a consistent feature of LC-1-induced apoptosis pointing to the involvement of the intrinsic apoptotic pathway. Importantly, CD38+ samples (> 30% expression) and those derived from patients with unmutated VH genes were more sensitive to LC-1 (LD50 values = 2.3 μM versus 3.4 μM and 2.4 μM versus 3.2 μM; P = 0.0003 and 0.01 respectively) suggesting that these CLL cells have a disproportionate reliance on NF-κB activity to maintain their survival and proliferative advantage. Taken together our data clearly demonstrates that LC-1 preferentially targets CLL cells from poor prognostic subsets. This unique cytotoxicity profile warrants further investigation and supports the use of this agent in early clinical trials for patients with CLL.
Reconstitution of the T Cell Repertoire Following Treatment with Alemtuzumab in Patients with B-Cell Chronic Lymphocytic Leukemia (B-CLL).
Authors: Rezvany Mohammad R. , Tehrani Mahmood J. , Karlsson Claes , Lundin Jeanette , Rabbani Hodjattallah , Mellstedt Håkan , Österborg Anders
Published: April 2020

Background and Methods: B-cell chronic lymphocytic leukemia (B-CLL) occurs as a result of clonal accumulation of functionally abnormal B cells. Alemtuzumab is a humanized monoclonal antibody specific for the CD52 antigen, which is highly expressed on both B-CLL cells and normal lymphocytes, but not on hematopoietic (CD34) stem cells. Alemtuzumab has been shown to effectively deplete the blood and bone marrow of lymphocytes, including CD4 and CD8 T cells, which may lead to profound immunosuppression and make patients more susceptible to infections. We and others have previously shown that the CD4 T cells in B-CLL patients may be clonally distinct from the normal population in that they present a more clonal pattern of the T-cell receptor (TCR) repertoire (Rezvany et al, Blood2003;101:1063–1070). It is therefore of interest to study the T cell repertoire following alemtuzumab administration as well as factors affecting T cell reconstitution following CD52 targeted therapy. In this study, we evaluated in depth the T-cell receptor-beta-variable sequence (TCR BV) in CD4 and CD8 T cells by real-time PCR, before and repeatedly after/during long term follow-up, in 5 B-CLL patients who had received alemtuzumab as first-line therapy (Lundin et al, Blood2002;100:768–773). Also, an analysis was conducted of CDR3 length polymorphism to describe changes in the clonality pattern.

Results: A decline in most of BV genes either in CD4 or CD8 T cells was observed shortly after alemtuzumab treatment, which was followed by a gradual increase in most of the BV genes during long-term follow up. CDR3 length polymorphism analysis shortly after treatment revealed an even more highly restricted pattern in CD4 T cells compared to baseline with a shift towards a monoclonal/oligoclonal pattern regardless of increased or decreased BV usage. Furthermore, in the analysis of the clonal spectrum that was expressed shortly after alemtuzumab therapy, the number of peaks was significantly reduced in CD4 (P <0.01) but not in CD8 T cells, which was followed by a gradual increase in diversity towards a polyclonal repertoire during long-term follow up.

Conclusions: These results indicate that perturbations in the T cell repertoire following alemtuzumab are complex, and are not reflected by changes in CD4/CD8 T cell numbers only. The restricted CDR3 pattern present prior to therapy became even more restricted after end of treatment, followed by a normalization of CDR3 patterns in CD4 T-cells during long-term follow-up. These results further suggest a regulatory role for T cells in relation to the malignant B cell clone in patients with B-CLL.
Pivotal Role of Survivin in Leukemogenesis by E2A-HLF Chimeric Transcription Factor.
Authors: Okuya Mayuko , Kurosawa Hidemitsu , Matsunaga Takayuki , Eguchi Mitsuoki , Furukawa Yusuke , Inukai Takeshi , Matsui Hirotaka , Look A. Thomas , Inaba Toshiya
Published: April 2020

The E2A-HLF fusion transcription factor generated by the t(17;19)(q22;p13) translocation is found in a small population of pro-B cell ALL. Patients associated with this chimera share distinct clinical features such as hypercalcemia, coagulopathy and very poor prognosis due to resistance to intensive chemotherapy including aggressive conditioning for BMT, all of which are unusual for this type of ALL. We have previously demonstrated that inhibition of the trans-activation potential of the E2A-HLF chimera by the dominant negative mutant results in apoptosis in t(17;19)+ ALL cells but does not affect cell cycle. Moreover, E2A-HLF blocks apoptosis induced by cytokine deprivation in IL-3-dependent cells, suggesting that this fusion protein contributes to leukemogenesis by substituting for the anti-apoptotic function of cytokines. The present study shows that survivin is a downstream target molecule of E2A-HLF. Four t(17;19)+ ALL cell lines expressed survivin at high levels and down-regulation of E2A-HLF function by the dominant negative mutant suppressed survivin expression. In addition, forced expression of E2A-HLF in Nalm-6, a t(17;19)− ALL cell line, up-regulated survivin expression. Survivin is known to be expressed predominantly in the G2/M phase. Indeed, separation of the fractions enriched for in each phase of the cell cycle using a counterflow centrifugal elutriator revealed G2/M phase-dominant survivin expression in t(17;19) − ALL cells including Nalm-6. In t(17;19)+ ALL cells, however, survivin was expressed throughout the cell cycle. Moreover, Nalm-6 cells forced to express E2A-HLF showed cell cycle-independent survivin expression. Reporter assay revealed that E2A-HLF induced luciferase activity by transfecting with each reporter construct containing the survivin promoter at a different length from the initial ATG, suggesting that E2A-HLF induces survivin expression at the transcriptional level, but not by direct binding of E2A-HLF to the survivin promoter. To test whether survivin plays anti-apoptotic roles in t(17:19)+ cells, we used a survivin mutant lacking a phosphorylation site (T34A-survivin) and considered to inhibit survivin function in a dominant negative manner. T34A-survivin induced massive apoptosis throughout the cell cycle in t(17;19)+ cells. In contrast, T34A-survivin in t(17;19) − cells induced cell death in only a small population in G2/M phase. In addition to caspase-dependent pathways, T34A-survivin induced apoptosis in t(17;19)+ ALL cells through caspase-independent pathways, in which apoptosis-inducing factor (AIF) translocated from cytoplasm to the nucleus. These results indicate that cell cycle-independent up-regulation of survivin by the E2A-HLF chimera is indispensable for the survival of t(17;19)+ ALL cells, and that inhibition of survivin may offer an effective therapeutic strategy against this refractory ALL.
Pediatric Acute Myeloid Leukemia with Nucleophosmin Mutations Is Characterized by a Gene Expression Signature with Dysregulated HOX Gene Expression Distinct from MLL-Rearranged Leukemias.
Authors: Mullighan Charles G. , Kennedy Alyssa L. , Zhou Xiaodong , Shurtleff Sheila A. , Downing James R.
Published: April 2020

Recent studies have identified mutations in the nucleophosmin (NPM) gene in up to one-third of cases of acute myeloid leukemia (AML) lacking a recurring cytogenetic abnormality. NPM mutations (NPMm+) result in aberrant cytoplasmic localization of nucleophosmin, which may potentially perturb multiple cellular pathways including cell cycle regulation. However the exact mechanisms by which mutant NPM contributes to leukemogenesis are unclear. Gene expression analysis may identify mediators or pathways in NPMm+ AML that are important in the development of leukemia. We have examined NPM mutation status in a 93 cases of pediatric AML and correlated mutation status with gene expression profile. All major AML subgroups were studied including AML-ETO (n=18), MLL rearranged (n=14) CBFB-MYH11 (n=13), PML-RARA and variants (n=4), acute megakaryoblastic leukemia (n=4), and AML with normal cytogenetics (n=20), or non-recurring cytogenetic abnormalities (n=20). Exon 12 of NPM was PCR amplified, sequenced, and abnormal cases verified by cloning and sequencing. Gene expression profiling was performed using Affymetrix U133A arrays. Six cases with tetranucleotide insertion mutations in NPM exon 12 were identified. Four had normal cytogenetics, and two non-recurring cytogenetic abnormalities. Differential gene expression between NPMm+ and NPMm- AML (either all NPMm- cases, or only those NPMm- cases lacking recurring cytogenetic abnormalities) was assessed by t-test. NPMm+ AML was characterized by upregulation of multiple homeobox genes (e.g. HOXA9, A10, B2, B6) as well as multiple genes with known or potential roles in tumorigenesis, such as MEIS1 and the NPM fusion partner ALK. This pattern of disordered HOX expression is similar to that of MLL-rearranged leukemia; however comparison of the signatures of NPMm+ and MLL-rearranged leukemia identified important differences, such as upregulation of HOXB2, B3, B6 and D4 in NPMm+ AML but not MLL-leukemia. These results confirm a recently published report describing perturbed HOX expression in NPMm+ AML (Alcalay et al. Blood2005;106:899), and provide the first evidence that the NPMm+ signature is similar to but distinct from MLL-rearranged AML. These findings provide important insights into the pathogenesis of NPMm+ leukemia, and support the hypothesis that mutated NPM dysregulates HOX expression via a different mechanism than MLL rearrangement.
Gene Expression Profiles in Acute Myeloid Leukemia (AML): From Diagnosis to Prognosis.
Authors: Lee Sanggyu , Chen Jianjun , Zhou Goulin , Shi Run , Kocherginsky Masha , Karrison Theodore G. , Kim Yeong Cheol , Ge Xijin , Jayathilaka Nimanthi , Neilly Mary Beth , Bouffard Gerard , Young Alice , Kornblau Steven , Bloomfield Clara , Bohlander Stefan , Liu Pu Paul , Sun Miao , Wang San Ming , Rowley Janet D.
Published: April 2020

Chromosome translocations are among the most common genetic abnormalities in human leukemia. The abnormally expressed genes from each translocation can be used to identify specific markers for clinical diagnosis of each translocation. Microarrays have identified genes differentially expressed in different translocations but the results between laboratories are not always compatible. We used SAGE to quantitate gene expression in bone marrow(BM) samples from 22 patients with four types of AML, [de novo AML M2 with t(8;21), AML M3 or M3V with t(15;17), AML M4Eo with inv(16), AML M5 with t(9;11) or secondary t(9;11)].We made SAGE libraries from CD15+ leukemic myeloid progenitor cells, collecting over 106 SAGE tags, of which 209,486 were unique tags; 136,010 were known genes and ESTs, and 73,476 were novel transcripts. SAGE tags for further analysis were selected based on a 5-fold difference between patient’s samples and normal CD15+ BM; they were also statistically significantly different at the 5% level. Using these strict criteria, we identified 2,381 unique tags, of which 2,053 were known genes and ESTs, and 328 were novel transcripts that were either specific for each translocation or were common(55) SAGE tags for all 4 translocations. The major change in all translocations was a decrease in expression in leukemia cells compared with normal cells; the decrease was least in the t(8;21) cells. Changes in expression of these known genes, which fall into different gene ontology functional categories, varied by translocation. Those associated with macromolecular biosynthesis, transport and transcription were most altered in the t(8;21); those related to defense response and apoptosis were altered in the t(15;17); cell proliferation genes were most affected by the t(9;11). From this analysis, we identified the functional molecular signature of each translocation. We designed a custom microarray to validate our SAGE data analysis. Our initial microarray contained 349 probes including 212 known genes, 61 ESTs, 28 novel sequences based on our data and 48 genes reported by others. We have now included 65 additional probes that appeared to be correlated with survival. Using 63 samples with the four translocations [16 inv(16), 4 t(9;11), 20 t(15;17), 4 t(8;21) and 19 other translocations], we are validating which genes provide a robust, reproducible “fingerprint” for each translocation, for all translocations, and which ones provide reliable information related to prognosis and survival. Our results will provide new insights into genes that collaborate with each translocation to lead to a fully leukemic phenotype as well as which genes appear to provide valid prognostic information.
Initial Results of a Randomized, Prospective Trial of Prophylaxis To Prevent Joint Disease in Young Children with Factor VIII (FVIII) Deficiency.
Authors: Manco-Johnson Marilyn J. , Abshire Thomas C. , Brown Deborah , Buchanan George R. , Cohen Alan R. , DiMichele Donna , Hoots W. Keith , Leissinger Cindy A. , McRedmond Kevin , Nugent Diane J. , Shapiro Amy D. , Thomas Gregory A. , Valentino Leonard A. , Riske Brenda
Published: April 2020

Background: Retrospective reviews of hemophilia therapy correlate normal joint X-ray (XR) and physical exam (PE) with early institution of routine infusions of FVIII dosed to prevent bleeding. Although the National Hemophilia Foundation recommended prophylaxis for all persons with severe hemophilia in 1995, fewer than half of affected persons in the US have adopted prophylaxis due to cost, effort, lack of perceived need and complications.

Objective: The “Joint Outcome Study” was constructed as a multi-center, open-label, two-arm, prospective, randomized clinical trial to compare full prophylaxis with an intensive treatment regimen for joint hemorrhage.

Methods: A regimen of every other day infusions of FVIII at 25 U/kg to prevent hemorrhage (prophylaxis, P) was compared with intensive therapy using ≥ 3 infusions totaling ≥ 80 U/kg FVIII at the time of each joint hemorrhage to minimize joint damage (enhanced episodic, EE). Primary outcome was the proportion of boys in each arm with bone or cartilage damage on XR or magnetic resonance imaging (MR) of index joints (elbows, knees, and ankles). Outcomes were judged independently by two research radiologists who were blinded to treatment arm and bleed history, with a third for discrepant readings. Other outcomes included joint function on a physical exam scale validated for young children (PE), # of joint hemorrhages and factor utilization. Boys were followed from entry between 12 and 30 months until age 6 years. At entry all children had normal joints on XR and MR and had no more than two hemorrhages into any one joint.

Results: Sixty-five boys were randomized to P (32) or EE (33). The study has been completed. While adjudication of XR and MR outcomes is still ongoing, clinical study results show the following: Children on P consumed more FVIII (mean 163 vs 47 infusions/year, P < 0.001) and had fewer joint hemorrhages per year (0.47 vs 4.9, p < 0.001) than boys on EE. PE scores for the six index joints were lower on the P arm compared with EE (mean score 4.7 vs. 8.6, p< 0.01).

Conclusion: This first randomized clinical trial of prophylaxis in young children with FVIII deficiency showed improved joint function by age 6 years in children on early every other day prophylaxis in comparison to an aggressive program of multiple infusions administered promptly at the time of joint hemorrhage. Joint structural outcome confirmation will be based on MR and XR.
MicroRNA Expression Profiling of Diffuse Large B-Cell Lymphoma Reveals Differences between Germinal Center-Like and Activated B Cell-Like Subtypes.
Authors: Lawrie Charles H. , Soneji Shamit , Cooper Christopher D. , Hatton Chris
Published: April 2020

MicroRNAs (miRNA) are a recently discovered class of short non-coding RNA molecules that negatively regulate gene expression. They have been shown to play a critical role in many biological functions. In humans about 320 miRNAs have been identified, some of which are expressed in a cell-specific and developmental stage-specific manner. Recently it has been shown that the expression profile of miRNAs can be used to subtype clinical cases (and cell lines) according to diagnosis with a greater degree of accuracy than traditional gene expression analysis. The identity of miRNAs associated with different lymphoma types however remains poorly defined. Previous expression studies have revealed the presence of at least two subtypes of diffuse large B-cell lymphoma (DLBCL) representing the postulated cell of origin; those that are germinal center B cell derived (GCB-type) and those that are activated B-cell derived (ABC-type). The latter subtype has been linked with poor prognostic outcome. It is not known whether these subtypes are also defined at the miRNA level. Therefore we examined the miRNA expression profile of DLBCL cell lines of defined subtypes as well as sub-populations of B-lymphocytes by microarray analysis. Consistent with recent publications, we found that mir-19a, 19b and 17-5p (part of mir-17-92 cluster) were up-regulated in cell lines but not in normal lymphocyte populations. Furthermore, cluster analysis showed that GCB-type cell lines (SUD-HL4, SUD-HL6 & SUD-HL10) have a distinct miRNA profile from ABC-type cell lines (OCI-Ly3 & OCI-Ly10). Most notably, high levels of expression of mir-155, mir-181b and mir-325 were found in ABC-type cell lines whilst high levels of mir-181a were found in GCB-type cell lines. We looked at expression of mir-155, 181a, 143, 145, 378 and 16 in these cell lines as well as clinical cases of DLBCL by RNase-protection assay. Consistent with the microarray data, we found that mir-155 was expressed in ABC-type cell lines but not GCB-type cell lines whilst the converse was true for mir-181a. Clinical cases showed similar patterns of expression but have still to be sub-typed according to immunohistochemical markers. Although still preliminary, our data suggests that miRNA profiling may be a useful tool in predicting the subtype of DLBCL cases and hence clinical outcome.
The MDS-Associated Protein EVI1 Impairs Erythroid and Megakaryocytic Differentiation by Direct Interaction with GATA-1.
Authors: Laricchia-Robbio Leopoldo , Li Donglan , Fazzina Raffaella , Chakraborty Soumen , Hay Maher Abdul , Nucifora Giuseppina
Published: April 2020

EVI1 is an aggressive nuclear oncoprotein deregulated by recurring chromosomal abnormalities in acute myeloid leukemia and myelodysplastic syndrome. This protein has two Zn finger domains containing 7 motifs at the N-terminus and 3 motifs at the C-terminus. The expression of this gene is a very poor prognostic marker and is associated with diseases characterized by erythroid and megakaryocytic defects. We have recently shown that the forced expression of EVI1 in murine bone marrow results in a fatal disease with features characteristic of MDS, including fatal dyserythropoiesis, dysmegakaryopoiesis, and anemia. These lineages are regulated by the transcription factor GATA-1, a DNA-binding protein that in addition to erythrocytes and megakaryocytes exerts a strict control also on the differentiation of mast cells and eosinophils, on the basis of its expression and association with specific partners.

In the present study, we used biochemical assays and in vitro culture to show that GATA-1 and the N-terminus of EVI1 are involved in the formation of a protein complex that is unable to regulate efficiently GATA-1-dependent promoters in reporter gene assays. EMSA studies with a GATA-1-specific probe indicate that EVI1 does not recognize and bind to the DNA probe but disrupts the DNA-binding of GATA-1. By deletion analysis and point mutations, we mapped the interaction between the proteins to two motifs in the proximal Zn finger domain of EVI1 and to the C-terminus Zn finger of GATA-1. Cys to Ala mutations in the two EVI1 motifs abrogate the interaction and restore the response of a promoter in reporter gene assays. We propose that the association between EVI1 and the DNA-binding motif of GATA-1 impairs efficient promoter binding by GATA-1 and the regulation of erythroid and megakaryocytic lineage. There studies suggest that the interaction surface between the two proteins could be an attractive target for the development of competing small molecules as a treatment in EVI1-associated leukemia.
Outcomes of Unrelated Cord Blood and Haploidentical Stem Cell Transplantation in Adults with Acute Leukaemia.
Authors: Rocha Vanderson , Aversa Franco , Labopin Myriam , Sanz Guillermo , Ciceri Fabio , Arcese William , Bunjes Donald , Rowe Jacob M. , Di Bartolomeo Paolo , Frassoni Francesco , Martelli Massimo F. , Gluckman Eliane
Published: April 2020

Unrelated cord blood transplantation (UCBT) and Haploidentical T-cell depleted peripheral blood stem cell transplantation (Haplo) are alternative options to treat patients with high risk acute leukaemia. With the aim to compare the outcomes of both strategies we have analysed retrospectively adults patients with ALL or AML receiving either UCBT or Haplo performed in EBMT centers from 1998–2002. One hundred forty four patients had ALL (Haplo=75 and UCBT=73) and 220 had de novo AML (Haplo=154, UCBT=66). In the ALL group, UCBT patients were younger and received less TBI than Haplo recipients. The frequency of patients with advanced phase of the disease at transplant was 45% in Haplo and 51% in UCBT (p=0.79). Cytogenetics and interval from diagnosis to transplant were not statistically different between the 2 groups. In the AML group, UCBT recipients were younger than Haplo recipients. Many patients were transplanted in advanced phase of the disease in both groups (58% of Haplo recipient and 59% in UCBT group), other disease-related factors were not significantly different. Unadjusted outcomes in both groups are reported in table 1.

After statistical adjustment for differences between the 2 groups, in both analysis (ALL and AML), UCBT recipients had delayed neutrophil recovery and higher incidence of acute GVHD (II–IV) compared to Haplo recipients. In the AML group, relapse incidence, TRM and LFS were not statistically different after UCBT or Haplo, however in ALL group, LFS were superior and relapse incidence decreased after UCBT compared to Haplo transplants. In conclusion, in this based registry retrospective analysis, UCBT and Haplo showed similar TRM, relapse and LFS for adults with AML, however LFS were superior after UCBT for patients with ALL due to decreased incidence of relapse.

ALL Cumulative incidence of aGvHD Cumulative incidence of 2y-RI Cumulative incidence of 2y-TRM Kaplan Meyer estimate 2y-LFS Haplo (n=75) 8+/−3% 38+/−5% 49+/−6% 13+/−4% UCBT (n=73) 26+/−5% 23+/−4% 41+/−6% 36+/−6% p value 0.004 0.07 0.55 0.01 AML Haplo (n=154) 5+/−5% 18+/−3% 58+/−4% 24+/−4% UCBT (n=66) 23+/−5% 24+/−5% 46+/−2% 30+/−6% p value <0.0001 0.44 0.23 0.39
A microRNA Cluster as a Target of Genomic Amplification in B-Cell Lymphomas.
Authors: Tagawa Hiroyuki , Karbe Kennosuke , Ohshima Koichi , Morishima Yasuo , Nakamura Shigeo , Seto Masao
Published: April 2020

Background: Genomic gain/amplification of 13q31-q32 is frequently observed in malignant lymphomas. C13orf25, recently established as a candidate gene in malignant lymphoma via 13q31-q32 genomic amplification, encodes two variant transcripts by alternative splicing (Ota et al, Cancer Res 2004). Seven microRNA genes (miR-17-5p, miR-17-3p, miR-18, miR-19a, miR-19b, miR-20 and miR-92) are clustered in C13orf25 transcript variant 2 (C13orf25 v2). Because microRNAs display dynamic temporal and spatial expression patterns, disruption of these microRNAs may be associated with tumorigenesis.

Purpose: The purposes of this study are i) to reveal frequencies of the 13q gain/amplification in various lymphoma types, and ii) to examine the expression of C13orf25 v2 and seven microRNAs using various lymphoma cell lines and tumors with and without 13q gain/amplification.

Experimental Design: We analyzed genomic alterations of chromosome 13 for 12 malignant lymphoma cell lines (eight B-cell and four T-cell lymphomas), and 214 cases of B-cell lymphomas (136 cases of diffuse large B-cell lymphoma (DLBCL), 27 cases of sporadic Burkitt’s lymphoma (sBL), 29 of mantle cell lymphoma (MCL), 22 of follicular lymphoma (FCL)) and 20 cases of T-cell lymphoma by using array-based comparative genomic hybridization. The expression levels of seven microRNAs using 12 lymphoma cell lines with (four) and without (eight) 13q gain/amplification were examined by Northern-blot and quantitative real-time PCR (RQ-PCR) analyses. RQ-PCR for C13orf25 v2 (microRNA cluster) was also conducted for 21 cases of DLBCL (eight cases with 13q gain/13 cases without), 10 cases of sBL (four cases with 13q gain/amp/six cases without) and 10 cases of mantle cell lymphoma.

Results: Frequent (> 20%) gain/amplification of 13q were detected in DLBCL (31 cases, 23%) and Burkitt’s lymphoma (8 cases, 30%) but no gain/amplification at 13q was found in MCL, FCL and T-cell lymphomas. Genomic amplification of 13q31-q32 was observed in four cases of DLBCL and two cases of sBL, four of which were c-MYC rearranged (two cases of DLBCL and two cases of sBL). RQ-PCR and Northern blot analyses revealed that five of the seven mature microRNAs displayed overexpression in lymphoma cell lines with 13q31 genomic gain/amplification but not in those without. RQ-PCR analysis for 21 cases of DLBCL demonstrated that the cases with 13q gain/amplification (8 cases) showed significantly higher expression of C13orf25 v2 than those without (13 cases) (Mann Whitney U test, P < 0.05). Significant higher levels of the five microRNAs in sBL with 13q gain/amplification were also confirmed by Northern blot analysis. Lower expression levels of the microRNAs were found in T cell lymphoma cell lines and tumors.

Conclusion: These results suggest that the microRNA cluster (C13orf25 v2) is a target of 13q/13q31 genomic gain/amplification in DLBCL and sBL.
Isolation of Cytomegalovirus (CMV) Specific T-Cells from Sersopositve Donors: A Possible Tool for CMV-Prophylaxis.
Authors: Borchers Sylvia , Topp Max , Ganser Arnold , Einsele Hermann , Weissinger Eva M. , Hertenstein Bernd
Published: April 2020

Reactivation of endogenous viruses, e. g. CMV, is a major cause of morbidity and mortality after allogeneic hemopoietic stem cell transplantation (allo-HSCT). Risk factors for the reactivation of CMV include the conditioning and immunosuppressive regimen, T-cell depletion of the graft, the development, and the treatment of acute or chronic graft versus host disease (GvHD). T-cells play an important role in the control of viral reactivation, lack of T-cells often leads to recurrent viral infections. The cytokine secretion assay provides the possibility to screen for CMV-reactive cells in a simple FACS assay by detection of interferon-gamma (IFN-gamma) secreting T-cells after stimulation of the cells with CMV-antigen. In addition IFN-g secreting cells can be enriched using the same assy. We have recently optimized enrichment to achieve 80-95% purity of IFN-gamma secreting T-cells.

In order to further optimize stimulation, several CMV-antigens were tested: While viral lysate provides a broad activation of CMV-specific T-cells, recombinant proteins like pp65 may offer more reproducible conditions. Comparison of viral lysate to pp65 regarding activation and enrichment of CMV-specific T-cells showed a possible HLA-dependency of pp65-performance: in HLA-A2-positve donor’s stimulation by pp65 was equal to stimulation with lysate, while in other donors pp65 was less effective. Interestingly, stimulation with lysate yielded mainly CD4-positive T-cells, whereas stimulation with pp65 mainly yielded CD8-positive T-cells.

Five patients, who reactivated CMV after allo-HSCT, were monitored closely for CMV-reactive cells to date. Initially all patients had no or reduced numbers of CMV-reactive T-cells. After one year, in 4 patients CMV-responsiveness was restored, none of these reactivated CMV more than twice. One patient had recurrent CMV-reactivations after HSCT, starting as early as day +20 after HSCT despite of GCV-treatment. Thus, CMV-specific enriched donor T-cells were transfused to control the reactivation. In an effort to minimize the risk of GvHD and to maximize protection from CMV-disease after allo-HSCT, we applied immunomagnetic selection for the isolation of CMV-reactive T-cells from the sero-positive donor. The persistence and proliferation of the transfused CMV-specific T-cells was monitored. During the first 4 weeks the percentage of CMV-reactive T-cells raised from undetectable levels to 3.5%, while CMV-antigenemia declined. After 3 weeks a complete clearance of CMV was achieved. No GvHD occurred, despite a mismatch in HLA-DR between donor and recipient. These preliminary data suggest the feasibility and safety of the transfusion of CMV-specific T-cells after immunomagnetic selection for the treatment of CMV-reactivation.
Neutropenic Mice Can Be Protected from Fungus Infection by Ex Vivo Expanded Allogeneic Myeloid Progenitors.
Authors: Domen Jos , Wahedi Mastura , Danenberg Esther , Smith-Berdan Stephanie , Christensen Julie , Fong Tim , Brown Janice (Wes)
Published: April 2020

Neutropenia induced by chemotherapy, hematopoietic cell transplantation, as well as radiation-induced neutropenia is accompanied by a significant morbidity and mortality due to increased susceptibility to a variety of pathogens. Despite clinical advances, such as the availability of growth factors that can stimulate granulopoiesis, like G-CSF, and new generations of antibiotics and antifungal agents, the problem persists. Direct cellular therapy with mature granulocytes has met with limited success, and would be difficult to implement due to the large number of cells needed, their very short lifespan and the inability to store the cells between harvesting and use. Our long-term goal is to develop a non-HLA-restricted cell-based short-term bridging therapy to reduce susceptibility to infection and enhance recovery from infections in the setting of neutropenia. The recent characterization of surface marker profiles of early myeloid progenitors has allowed the prospective testing of these cells. It has been clearly shown that the use of limited numbers of a combination of Common Myeloid Progenitors (CMP) and Granulocyte-Macrophage-Progenitors (GMP), purified from bone marrow, can protect mice from a normally lethal challenge with fungus or bacteria [Blood100:4660-7(2002)]. We have extended these observations and tested whether myeloid progenitors (MP) with similar properties can be obtained in large numbers from hematopoietic stem cells (HSC) in vitro. Here we report that a one week, serum-free culture can result in a 200-fold expansion of cells with myeloid progenitor activity with respect to the starting CD117+, CD90.1low, Linneg/low and Sca-1+ KTLS-HSC. Furthermore, these myeloid progenitor cells are as effective as Myeloid Progenitors sorted directly from bone marrow in preventing death due to invasive aspergillosis, even when the cells are fully allogeneic and have been cryopreserved prior to use. These results indicate a way in which myeloid progenitor cells, which can be stored frozen prior to use, can be obtained in large numbers. This process, once translated to human cells, would also open the way for the stockpiling of MP for use in nuclear disasters.
Outcomes after Unrelated Cord Blood Transplantation in Children with Acute Lymphoblastic Leukemia. An Eurocord-Netcord Survey.
Authors: Rocha Vanderson , Michel Gerard , Kabbara Nabil , Arcese William , Ortega Juan , Iori Anna Paola , Madero Luis , Chan Ka-Wah , Locatelli Franco , Garnier Federico , Ionescu Irina , Wernet Peter , Gluckman Eliane
Published: April 2020

Unrelated cord blood transplantation (UCBT) is an alternative option to treat children with haematological diseases without an HLA-identical donor. We have analyzed a total of 323 children with ALL receiving an UCBT, from 1994 to 2004 in 99 transplant centres in 24 countries, mostly in Europe. Cumulative incidence with competing risk and KM estimates were used to calculate outcomes. Seventy six children were transplanted in CR1, 136 in CR2 and 111 in more advanced phase of the disease. Among those children poor cytogenetics were observed in 89% of children in 1CR, 33% in 2CR and 42% in advanced phase. Twenty percent of children transplanted in advanced phase had been previously autografted. The median age was 6.5 years at UCBT, median cell dose infused was 4.1x107/kg and the median follow time was 22 months (3–96). The cord blood was HLA identical (6/6) in 12% of the cases, 5/6 in 46%, 4/6 in 39% and 3/6 in 3%. All children received myeloablative conditioning regimen (TBI in 66%) and the majority (67%) received CsA+corticoids as GVHD prophylaxis. Cumulative incidence of neutrophil recovery at day 60, platelets recovery (>20.000) at day 180, acute (grade II–IV) and chronic GVHD were 76±5%, 54±5%, 42±3%, 14±2%, respectively. Overall 2 year-LFS was 36±3%. In a multivariate analysis, only CR1 or CR2 were associated with better LFS (HR=1.8; p<0.0001).

Outcomes CR1 (n=76) CR2 (n=136) Advanced (n=111) TRM at day 100 22+/−5% 25+/−4% 34+/−5% Relapse at 2 years 34+/−8% 37+/−5% 48+/−7% LFS at 2 years 42+/−6% 41+/−4% 24+/−4%

For those patients transplanted with poor cytogenetics, LFS at 2 years was 32±6% and it was 37% for CR1, 43% for CR2 and 0% for advanced phase of the disease. In conclusion, in these large series of high risk ALL patients, these results show that UCBT should be proposed as alternative source of allogeneic transplantation for children lacking an HLA identical donor, in earlier status of the disease.
Functional Correction of Lymphocytes and Dendritic Cells of Patients with WAS or XLT, Using Lentiviral Vectors Targeting the Expression of WAS Protein to Hematopoietic Cells.
Authors: Charrier Sabine , Dupre Loic , Blundell Mike , Scaramuzza Samantha , Jeanson Laurence , Vainchenker William , Danos Olivier , Roncarolo Maria-Grazia , Thrasher Adrian , Galy Anne
Published: April 2020

The WAS gene is mutated in Wiskott-Aldrich syndrome (WAS) and in X-linked thrombopenia (XLT). These diseases associate platelet defects with variable immune dysfunction, as a result of abnormal signalling and impaired cytoskeletal regulation in hematopoeitic cells. Conceivably, severe forms of WAS could be treated by gene therapy because retroviral or lentiviral-mediated WAS gene transfer restores protein expression and function in several WAS protein-deficient models. The purpose of the present study was to improve the safety and efficacy of such lentiviral vectors. Endogenous WAS promoter elements were used to restrict transgene expression to the target cell population and to provide the possibility of regulated expression in these cells. Sequences 0.5 or 1.6 kb upstream of transcription start, were operational in the transfer vector and restricted transgene expression to hematopoietic cells. Vectors utilizing either one of the WAS promoter sequences or the ubiquitously-active PGK-1, SFFV or EF1-a promoters, were compared. Equivalent levels of WAS protein were induced in lymphocytes and dendritic cells (DC), although slightly inferior mRNA levels were obtained in B cells using WAS promoters. At the functional level, all vectors restored a similar degree of proliferation and IL-2 production in T cells and equivalent numbers of podosome cytoskeletal structures in DC. The 0.5 kb or 1.6 kb-long WAS promoter sequences functioned similarly in WAS B lymphocytes or in a model of WASP-deficient T cells generated by RNA interference. No toxicity was induced by over-expression of these vectors in CD34+ cells. Altogether, the data show that lentiviral vectors with WAS promoters function efficiently in several lineages of patient cells and support further development of a hematopoietic-restricted approach for the gene therapy of WAS.