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The phase 2 CAVALLI (NCT02055820) study assessed efficacy and safety of venetoclax, a selective B-cell lymphoma-2 (Bcl-2) inhibitor, with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) in first-line (1L) diffuse large B-cell lymphoma (DLBCL), including patients demonstrating Bcl-2 protein overexpression by immunohistochemistry (Bcl-2 IHC+). Eligible patients were ≥18 years of age and had previously untreated DLBCL, Eastern Cooperative Oncology Group performance status ≤2, and International Prognostic Index 2 to 5. Venetoclax 800 mg (days 4-10, cycle 1; days 1-10, cycles 2-8) was administered with rituximab (8 cycles) and cyclophosphamide, doxorubicin, vincristine, and prednisone (6-8 cycles) in 21-day cycles. Primary end points were safety, tolerability, and complete response (CR) at end of treatment (EOT). Secondary end points were progression-free survival (PFS) and overall survival. Comparative analyses used covariate-adjusted R-CHOP controls from the GOYA/BO21005 study, an appropriate contemporary benchmark for safety and efficacy. Safety and efficacy analyses included 206 patients. CR rate at EOT was 69% in the overall population and was maintained across Bcl-2 IHC+ subgroups. With a median follow-up of 32.2 months, trends were observed for improved investigator-assessed PFS for venetoclax plus R-CHOP in the overall population (hazard ratio [HR], 0.61; 95% confidence interval [CI], 0.43-0.87) and Bcl-2 IHC+ subgroups (HR, 0.55; 95% CI, 0.34-0.89) vs R-CHOP. Despite a higher incidence of grade 3/4 hematologic adverse events (86%), related mortality was not increased (2%). Chemotherapy dose intensity was similar in CAVALLI vs GOYA. The addition of venetoclax to R-CHOP in 1L DLBCL demonstrates increased, but manageable, myelosuppression and the potential of improved efficacy, particularly in high-risk Bcl-2 IHC+ patient subgroups.

Vaccinations are effective in preventing infections; however, it is unknown if patients with chronic lymphocytic leukemia (CLL) who are treatment naïve (TN) or receiving Bruton tyrosine kinase inhibitors (BTKi's) respond to novel adjuvanted vaccines. Understanding the effect of BTKi's on humoral immunity is timely because BTKi's are widely used and vaccination against coronavirus disease 2019 is urgently needed. In 2 open-label, single-arm clinical trials, we measured the effect of BTKi's on de novo immune response against recombinant hepatitis B vaccine (HepB-CpG) and recall response against recombinant zoster vaccine (RZV) in CLL patients who were TN or on BTKi. The primary end point was serologic response to HepB-CpG (anti-hepatitis B surface antibodies ≥10 mIU/mL) and RZV (≥fourfold increase in anti-glycoprotein E). The response rate to HepB-CpG was lower in patients on BTKi (3.8%; 95% confidence interval [CI], 0.7-18.9) than patients who were TN (28.1%; 95% CI, 15.6-45.4; P= .017). In contrast, the response rate to RZV did not differ significantly between the BTKi (41.5%; 95% CI, 27.8-56.6) and TN cohorts (59.1%; 95% CI, 38.7-76.7; P= .2). BTKi's were associated with a decreased de novo immune response following HepB-CpG, whereas recall immune response following RZV was not significantly affected by BTKi therapy. These trials were registered at www.clinicaltrials.govas #NCT03685708 (Hep-CpG) and #NCT03702231 (RZV).

A minority of patients with follicular lymphoma (FL) undergo histological transformation (HT). This retrospective analysis of 549 patients from the phase 3 GALLIUM study (NCT01332968) assessed the relationship between maximum standardized uptake value (SUVmax) at baseline on positron emission tomography (PET) and HT risk. Previously untreated patients with high tumor burden grade 1-3a FL received obinutuzumab- or rituximab-based chemotherapy induction. The relationship between baseline SUVmax (bSUVmax) and HT risk was assessed using cutoff values for SUVmax >10 and >20. Overall, 15 of 549 (2.7%) patients with baseline PET scans experienced biopsy-confirmed HT (median follow-up, 59 months). More than 65% of patients had bSUVmax > 10, with 3.3% of these experiencing HT. Only 1 of 74 (1.4%) patients with bSUVmax > 20 underwent HT. Median bSUVmax in patients with HT vs without HT was 12.4 (range, 8.1-28.0) vs 11.8 (range, 3.1-64.4), respectively; median bSUVrange (the difference between bSUVmax of the most and least 18F-fluorodeoxyglucose–avid lymphoma sites) was 8.0 (range, 1.1-23.9) vs 7.1 (range, 0.0-59.8), respectively. There was no temporal relationship between bSUVmax and HT. Neither bSUVmax nor bSUVrange predicted HT in GALLIUM, suggesting that there may be little benefit in rebiopsy of lesions to exclude HT based on SUVmax alone before initiating therapy in patients with high tumor burden FL.

Vladimer: Allcyte GmbH: Current Employment, Current equity holder in private company, Other: Founder. Jaeger:Karyopharm: Honoraria; Amgen: Honoraria; Gilead: Honoraria, Research Funding; BMS/Celgene: Consultancy, Honoraria, Research Funding; True North: Honoraria, Research Funding; Miltenyi: Consultancy, Honoraria; CDR Life AG: Consultancy, Research Funding; F. Hoffmann-La Roche: Honoraria, Research Funding; Infinity: Honoraria; Takeda: Honoraria; Novartis: Consultancy, Honoraria, Research Funding; AbbVie: Honoraria. Krall:Allcyte GmbH: Current Employment, Current equity holder in private company, Other: Founder. Valent:Allcyte GmbH: Research Funding; Cellgene: Honoraria, Research Funding; Pfizer: Honoraria. Wolf:Celgene: Honoraria, Research Funding. Zielinski:MSD: Consultancy, Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; Imugene: Consultancy, Honoraria, Speakers Bureau; Ariad: Consultancy, Honoraria, Speakers Bureau; Pfizer: Consultancy, Honoraria, Speakers Bureau; Merrimack: Consultancy, Honoraria, Speakers Bureau; Merck KGaA: Consultancy, Honoraria, Speakers Bureau; Fibrogen: Consultancy, Honoraria, Speakers Bureau; AstraZeneca: Consultancy, Honoraria, Speakers Bureau; Tesaro: Consultancy, Honoraria, Speakers Bureau; Gilead: Consultancy, Honoraria, Speakers Bureau; Servier: Consultancy, Honoraria, Speakers Bureau; Shire: Consultancy, Honoraria, Speakers Bureau; Eli Lilly: Consultancy, Honoraria, Speakers Bureau; Athenex: Consultancy, Honoraria, Speakers Bureau. Superti-Furga:Allcyte GmbH: Current equity holder in private company, Other: Founder. Snijder:Allcyte GmbH: Current equity holder in private company, Other: Founder. Staber:Roche: Consultancy, Honoraria, Research Funding; Astra Zeneca: Consultancy, Honoraria; Celgene/ BMS: Consultancy, Honoraria; msd: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria.

Common lymphoid progenitors (CLPs) differentiate to T and B lymphocytes, dendritic cells, natural killer cells, and innate lymphoid cells. Here, we describe culture conditions that, for the first time, allow the establishment of lymphoid-restricted, but uncommitted, long-term proliferating CLP cell lines and clones from a small pool of these cells from normal mouse bone marrow, without any genetic manipulation. Cells from more than half of the cultured CLP clones could be induced to differentiate to T, B, natural killer, dendritic, and myeloid cells in vitro. Cultured, transplanted CLPs transiently populate the host and differentiate to all lymphoid subsets, and to myeloid cells in vivo. This simple method to obtain robust numbers of cultured noncommitted CLPs will allow studies of cell-intrinsic and environmentally controlled lymphoid differentiation programs. If this method can be applied to human CLPs, it will provide new opportunities for cell therapy of patients in need of myeloid-lymphoid reconstitution.

Several gene editing strategies for autosomal dominant ELANE-associated severe congenital neutropenia (CN) have been proposed. All strategies efficiently correct CN with different degrees of the investigated comprehensiveness of toxicity and off-target activity. The next step now is to select the best approach for clinical translation. The first-line awareness should consider not only the universal applicability of gene editing but, most importantly, its safety. Since CN patients have a high cumulative incidence of leukemogenic transformation and pre-leukemia cells in the patient's bone marrow are detected at an early age, safety considerations are of the utmost importance.

Introduction:

Background

Severe congenital neutropenia (CN) is an inherited bone marrow failure syndrome characterized by impaired maturation of neutrophil granulocytes. Due to the lack or very low levels of neutrophils in the peripheral blood, patients experience severe, life-threatening bacterial infections as early as birth. Autosomal-dominant ELANEmutations are the most common cause of CN. Although most patients respond to daily treatment with recombinant human granulocyte colony-stimulating factor, about 15% of patients do not respond to this cytokine, and approximately 20% of patients develop myelodysplasia or acute myeloid leukemia. The only curative treatment available thus far is allogenic hematopoietic stem cell transplantation, which is associated with serious side effects. Autologous transplantation of gene-edited patients' HSPCs offers a novel curative therapy. Considering the wide range of mutations distributed throughout the ELANEgene, A universal, high-safety-profile approach could be advantageous for all ELANE-CN patients. Our group has already published a universal CRISPR/Cas9-mediated knockout approach for the ELANEgene; however, targeting the coding sequence region (CDS) of the ELANEgene could lead to the introduction of new disease-causing variants, so it may be harmful for patients. In order to address this concern, we reformatted our previously published knockout concept into a clinical-grade gene therapy strategy.

Although the role of classic driver mutations (i.e., JAK2, MPL, and CALR) in myeloproliferative neoplasm (MPN) development is well characterized, additional somatic mutations likely modulate the phenotype. Recurrent somatic mutations, such as those in TET2, DNMT3A, ASXL1genes are commonly identified in MPN using targeted myeloid panels, however identification of somatic mutations using an unbiased approach may unveil novel pathways involved in MPN pathogenesis.

A single subcutaneous (SC) injection of plerixafor results in rapid mobilization of hematopoietic progenitors, but fails to mobilize 33% of normal allogeneic sibling donors in 1 apheresis. We hypothesized that changing the route of administration of plerixafor from SC to IV may overcome the low stem cell yields and allow collection in 1 day. A phase 1 trial followed by a phase 2 efficacy trial was conducted in allogeneic sibling donors. The optimal dose of IV plerixafor was determined to be 0.32 mg/kg. The primary outcome of reducing the failure to collect ≥2 × 106CD34+/kg recipient weight in 1 apheresis collection to ≤10% was not reached. The failure rate was 34%. Studies evaluating the stem cell phenotype and gene expression revealed a novel plasmacytoid dendritic cell precursor preferentially mobilized by plerixafor with high interferon-α producing ability. The observed cytomegalovirus (CMV) viremia rate for patients at risk was low (15%), as were the rates of acute grade 2-4 graft-versus-host disease (GVHD) (21%). Day 100 treatment related mortality was low (3%). In conclusion, plerixafor results in rapid stem cell mobilization regardless of route of administration and resulted in novel cellular composition of the graft and favorable recipient outcomes. These trials were registered at clinicaltrials.govas #NCT00241358 and #NCT00914849.

Introduction: The treatment landscape for hematologic malignancies is evolving rapidly, and a range of therapeutic options with differing routes of administration is now available. The shifting dynamics of these novel therapies and increasing total treatment costs highlight the importance of value-based healthcare decisions that take patient, payer, and societal perspectives into account. It is therefore increasingly important to consider both direct and indirect costs when evaluating therapeutic options. Reducing healthcare visits for administration of non-oral therapies (injectable and/or mixed therapies) results in indirect cost savings and is of particular relevance during the current coronavirus disease (COVID-19) pandemic where there are distinct challenges with respect to visiting hospital settings. However, the indirect cost savings of utilizing oral versus non-oral treatments have yet to be fully assessed from a patient and societal perspective. The objective of this review was to assess the differences in indirect non-treatment-related costs between oral and non-oral therapies for hematologic malignancies.

Venous thromboembolism (VTE) is common in patients with brain tumors, and underlying mechanisms are unclear. We hypothesized that podoplanin, a sialomucin-like glycoprotein, increases the risk of VTE in primary brain tumors via its ability to induce platelet aggregation. Immunohistochemical staining against podoplanin and intratumoral platelet aggregates was performed in brain tumor specimens of 213 patients (mostly high-grade gliomas [89%]) included in the Vienna Cancer and Thrombosis Study, a prospective observational cohort study of patients with newly diagnosed cancer or progressive disease aimed at identifying patients at risk of VTE. Platelet aggregation in response to primary human glioblastoma cells was investigated in vitro. During 2-year follow-up, 29 (13.6%) patients developed VTE. One-hundred fifty-one tumor specimens stained positive for podoplanin (33 high expression, 47 medium expression, 71 low expression). Patients with podoplanin-positive tumors had lower peripheral blood platelet counts (P< .001) and higher D-dimer levels (P< .001). Podoplanin staining intensity was associated with increasing levels of intravascular platelet aggregates in tumor specimens (P< .001). High podoplanin expression was associated with an increased risk of VTE (hazard ratio for high vs no podoplanin expression: 5.71; 95% confidence interval, 1.52-21.26; P =.010), independent of age, sex, and tumor type. Podoplanin-positive primary glioblastoma cells induced aggregation of human platelets in vitro, which could be abrogated by an antipodoplanin antibody. In conclusion, high podoplanin expression in primary brain tumors induces platelet aggregation, correlates with hypercoagulability, and is associated with increased risk of VTE. Our data indicate novel insights into the pathogenesis of VTE in primary brain tumors.

Defibrotide (DF) has received European Medicines Agency authorization to treat sinusoidal obstruction syndrome, an early complication after hematopoietic cell transplantation. DF has a recognized role as an endothelial protective agent, although its precise mechanism of action remains to be elucidated. The aim of the present study was to investigate the interaction of DF with endothelial cells (ECs). A human hepatic EC line was exposed to different DF concentrations, previously labeled. Using inhibitory assays and flow cytometry techniques along with confocal microscopy, we explored: DF-EC interaction, endocytic pathways, and internalization kinetics. Moreover, we evaluated the potential role of adenosine receptors in DF-EC interaction and if DF effects on endothelium were dependent of its internalization. Confocal microscopy showed interaction of DF with EC membranes followed by internalization, though DF did not reach the cell nucleus even after 24 hours. Flow cytometry revealed concentration, temperature, and time dependent uptake of DF in 2 EC models but not in other cell types. Moreover, inhibitory assays indicated that entrance of DF into ECs occurs primarily through macropinocytosis. Our experimental approach did not show any evidence of the involvement of adenosine receptors in DF-EC interaction. The antiinflammatory and antioxidant properties of DF seem to be caused by the interaction of the drug with the cell membrane. Our findings contribute to a better understanding of the precise mechanisms of action of DF as a therapeutic and potential preventive agent on the endothelial damage underlying different pathologic situations.

Interstrand crosslinks (ICLs) are toxic DNA lesions that cause severe genomic damage during replication, especially in Fanconi anemia pathway-deficient cells. This results in progressive bone marrow failure and predisposes to acute myeloid leukemia (AML). The molecular mechanisms responsible for these defects are largely unknown. Using Ercc1-deficient mice, we show that Trp53 is responsible for ICL-induced bone marrow failure and that loss of Trp53 is leukemogenic in this model. In addition, Ercc1-deficient myeloid progenitors gain elevated levels of miR-139-3p and miR-199a-3p with age. These microRNAs exert opposite effects on hematopoiesis. Ectopic expression of miR-139-3p strongly inhibited proliferation of myeloid progenitors, whereas inhibition of miR-139-3p activity restored defective proliferation of Ercc1-deficient progenitors. Conversely, the inhibition of miR-199a-3p functions aggravated the myeloid proliferation defect in the Ercc1-deficient model, whereas its enforced expression enhanced proliferation of progenitors. Importantly, miR-199a-3p caused AML in a pre-leukemic mouse model, supporting its role as an onco-microRNA. Target genes include HuR for miR-139-3p and Prdx6, Runx1, and Suz12 for miR-199a-3p. The latter genes have previously been implicated as tumor suppressors in de novo and secondary AML. These findings show that, in addition to TRP53-controlled mechanisms, miR-139-3p and miR-199a-3p are involved in the defective hematopoietic function of ICL-repair deficient myeloid progenitors.

Interstrand crosslinks (ICLs) are toxic DNA lesions that cause severe genomic damage during replication, especially in Fanconi anemia pathway-deficient cells. This results in progressive bone marrow failure and predisposes to acute myeloid leukemia (AML). The molecular mechanisms responsible for these defects are largely unknown. Using Ercc1-deficient mice, we show that Trp53is responsible for ICL-induced bone marrow failure and that loss of Trp53is leukemogenic in this model. In addition, Ercc1-deficient myeloid progenitors gain elevated levels of miR-139-3pand miR-199a-3pwith age. These microRNAs exert opposite effects on hematopoiesis. Ectopic expression of miR-139-3pstrongly inhibited proliferation of myeloid progenitors, whereas inhibition of miR-139-3pactivity restored defective proliferation of Ercc1-deficient progenitors. Conversely, the inhibition of miR-199a-3pfunctions aggravated the myeloid proliferation defect in the Ercc1-deficient model, whereas its enforced expression enhanced proliferation of progenitors. Importantly, miR-199a-3pcaused AML in a pre-leukemic mouse model, supporting its role as an onco-microRNA. Target genes include HuRfor miR-139-3pand Prdx6, Runx1, and Suz12for miR-199a-3p.The latter genes have previously been implicated as tumor suppressors in de novo and secondary AML. These findings show that, in addition to TRP53-controlled mechanisms, miR-139-3pand miR-199a-3pare involved in the defective hematopoietic function of ICL-repair deficient myeloid progenitors.

NKR-P1B is a homodimeric type II transmembrane C-type lectinlike receptor that inhibits natural killer (NK) cell function upon interaction with its cognate C-type lectin-related ligand, Clr-b. The NKR-P1B:Clr-b interaction represents a major histocompatibility complex class I (MHC-I)-independent missing-self recognition system that monitors cellular Clr-b levels. We have generated NKR-P1BB6-deficient (Nkrp1b−/−) mice to study the role of NKR-P1B in NK cell development and function in vivo. NK cell inhibition by Clr-b is abolished in Nkrp1b−/− mice, confirming the inhibitory nature of NKR-P1BB6. Inhibitory receptors also promote NK cell tolerance and responsiveness to stimulation; hence, NK cells expressing NKR-P1BB6 and Ly49C/I display augmented responsiveness to activating signals vs NK cells expressing either or none of the receptors. In addition, Nkrp1b−/− mice are defective in rejecting cells lacking Clr-b, supporting a role for NKR-P1BB6 in MHC-I-independent missing-self recognition of Clr-b in vivo. In contrast, MHC-I-dependent missing-self recognition is preserved in Nkrp1b−/− mice. Interestingly, spontaneous myc-induced B lymphoma cells may selectively use NKR-P1B:Clr-b interactions to escape immune surveillance by wild-type, but not Nkrp1b−/−, NK cells. These data provide direct genetic evidence of a role for NKR-P1B in NK cell tolerance and MHC-I-independent missing-self recognition.

Serum cytokines and chemokines may reflect tumor biology and host response in follicular lymphoma (FL). To determine whether the addition of these biological factors may further refine prognostication, 30 cytokines and chemokines were measured in pretreatment serum specimens from newly diagnosed FL patients (n = 209) and from 400 matched controls. Cytokine levels were correlated with clinical outcome in patients who were observed or received single agent rituximab, or those who received chemotherapy. Correlations with outcome in chemotherapy treated patients were further examined in a separate cohort of 183 South West Oncology Group (SWOG) patients and all patients were then included in a meta-analysis. Six cytokines were associated with outcome in the Molecular Epidemiology Resource (MER) after adjusting for the FL international prognostic index. In patients who were observed or treated with rituximab alone, increased serum IL-12 and interleukin 1 receptor antagonist (IL-1RA) (P = .005 and .02) were associated with a shorter event-free survival. In patients receiving chemotherapy, hepatocyte growth factor, IL-8, IL-1RA, and CXCL9 (P = .015, .048, .004, and .0005) predicted a shorter EFS. When the MER chemotherapy treated patients and SWOG patients were combined in a meta-analysis, IL-2R, IL-1RA, and CXCL9 (P = .013, .042, and .0012) were associated with a poor EFS.

Ly49-mediated recognition of MHC-I molecules on host cells is considered vital for natural killer (NK)–cell regulation and education; however, gene-deficient animal models are lacking because of the difficulty in deleting this large multigene family. Here, we describe NK gene complex knockdown (NKCKD) mice that lack expression of Ly49 and related MHC-I receptors on most NK cells. NKCKDNK cells exhibit defective killing of MHC-I–deficient, but otherwise normal, target cells, resulting in defective rejection by NKCKDmice of transplants from various types of MHC-I–deficient mice. Self–MHC-I immunosurveillance by NK cells in NKCKDmice can be rescued by self–MHC-I–specific Ly49 transgenes. Although NKCKDmice display defective recognition of MHC-I–deficient tumor cells, resulting in decreased in vivo tumor cell clearance, NKG2D- or antibody-dependent cell-mediated cytotoxicity–induced tumor cell cytotoxicity and cytokine production induced by activation receptors was efficient in Ly49-deficient NK cells, suggesting MHC-I education of NK cells is a single facet regulating their total potential. These results provide direct genetic evidence that Ly49 expression is necessary for NK-cell education to self–MHC-I molecules and that the absence of these receptors leads to loss of MHC-I–dependent “missing-self” immunosurveillance by NK cells.

MicroRNAs (miRNAs) have the potential to regulate cellular differentiation programs; however, miRNA deficiency in primary hematopoietic stem cells (HSCs) results in HSC depletion in mice, leaving the question of whether miRNAs play a role in early-lineage decisions un-answered. To address this issue, we deleted Dicer1, which encodes an essential RNase III enzyme for miRNA biogenesis, in murine CCAAT/enhancer-binding protein α (C/EBPA)–positive myeloid-committed progenitors in vivo. In contrast to the results in HSCs, we found that miRNA depletion affected neither the number of myeloid progenitors nor the percentage of C/EBPA–positive progenitor cells. Analysis of gene-expression profiles from wild-type and Dicer1-deficient granulocyte-macrophage progenitors (GMPs) revealed that 20 miRNA families were active in GMPs. Of the derepressed miRNA targets in Dicer1-null GMPs, 27% are normally exclusively expressed in HSCs or are specific for multipotent progenitors and erythropoiesis, indicating an altered gene-expression landscape. Dicer1-deficient GMPs were defective in myeloid development in vitro and exhibited an increased replating capacity, indicating the regained self-renewal potential of these cells. In mice, Dicer1deletion blocked monocytic differentiation, depleted macrophages, and caused myeloid dysplasia with morphologic features of Pelger-Huët anomaly. These results provide evidence for a miRNA-controlled switch for a cellular program of self-renewal and expansion toward myeloid differentiation in GMPs.

MicroRNAs (miRNAs) have the potential to regulate cellular differentiation programs; however, miRNA deficiency in primary hematopoietic stem cells (HSCs) results in HSC depletion in mice, leaving the question of whether miRNAs play a role in early-lineage decisions un-answered. To address this issue, we deleted Dicer1, which encodes an essential RNase III enzyme for miRNA biogenesis, in murine CCAAT/enhancer-binding protein α (C/EBPA)–positive myeloid-committed progenitors in vivo. In contrast to the results in HSCs, we found that miRNA depletion affected neither the number of myeloid progenitors nor the percentage of C/EBPA–positive progenitor cells. Analysis of gene-expression profiles from wild-type and Dicer1-deficient granulocyte-macrophage progenitors (GMPs) revealed that 20 miRNA families were active in GMPs. Of the derepressed miRNA targets in Dicer1-null GMPs, 27% are normally exclusively expressed in HSCs or are specific for multipotent progenitors and erythropoiesis, indicating an altered gene-expression landscape. Dicer1-deficient GMPs were defective in myeloid development in vitro and exhibited an increased replating capacity, indicating the regained self-renewal potential of these cells. In mice, Dicer1 deletion blocked monocytic differentiation, depleted macrophages, and caused myeloid dysplasia with morphologic features of Pelger-Huët anomaly. These results provide evidence for a miRNA-controlled switch for a cellular program of self-renewal and expansion toward myeloid differentiation in GMPs.

Acute myelogenous leukemias (AMLs) and endothelial cells depend on each other for survival and proliferation. Monotherapy antivascular strategies such as targeting vascular endothelial growth factor (VEGF) has limited efficacy in treating AML. Thus, in search of a multitarget antivascular treatment strategy for AML, we tested a novel vascular disrupting agent, OXi4503, alone and in combination with the anti-VEGF antibody, bevacizumab. Using xenotransplant animal models, OXi4503 treatment of human AML chloromas led to vascular disruption in leukemia cores that displayed increased leukemia cell apoptosis. However, viable rims of leukemia cells remained and were richly vascular with increased VEGF-A expression. To target this peripheral reactive angiogenesis, bevacizumab was combined with OXi4503 and abrogated viable vascular rims, thereby leading to enhanced leukemia regression. In a systemic model of primary human AML, OXi4503 regressed leukemia engraftment alone and in combination with bevacizumab. Differences in blood vessel density alone could not account for the observed regression, suggesting that OXi4503 also exhibited direct cytotoxic effects on leukemia cells. In vitro analyses confirmed this targeted effect, which was mediated by the production of reactive oxygen species and resulted in apoptosis. Together, these data show that OXi4503 alone is capable of regressing AML by a multitargeted mechanism and that the addition of bevacizumab mitigates reactive angiogenesis.

Endothelial sialomucin CD34 functions as an L-selectin ligand mediating lymphocyte extravasation only when properly glycosylated to express a sulfated carbohydrate epitope, 6-sulfo sialyl Lewis x (6-sulfo SLex). It is thought that multivalent 6-sulfo SLex expression promotes high-affinity binding to L-selectin by enhancing avidity. However, the reported low amount of 6-sulfo SLex in total human CD34 is inconsistent with this model and prompted us to re-evaluate CD34 glycosylation. We separated CD34 into 2 glycoforms, the L-selectin–binding and nonbinding glycoforms, L-B-CD34 and L-NB-CD34, respectively, and analyzed released O- and N-glycans from both forms. L-B-CD34 is relatively minor compared with L-NB-CD34 and represented less than 10% of total tonsillar CD34. MECA-79, a mAb to sulfated core-1 O-glycans, bound exclusively to L-B-CD34 and this form contained all sulfated and fucosylated O-glycans. 6-Sulfo SLex epitopes occur on core-2 and extended core-1 O-glycans with approximately 20% of total L-B-CD34 O-glycans expressing 6-sulfo SLex. N-glycans containing potential 6-sulfo SLex epitopes were also present in L-B-CD34, but their removal did not abolish binding to L-selectin. Thus, a minor glycoform of CD34 carries relatively abundant 6-sulfo SLex epitopes on O-glycans that are important for its recognition by L-selectin.

Chronic active Epstein-Barr virus infection (CAEBV) is a rare disease in which previously healthy persons develop severe, life-threatening illness. Mutations in the perforin gene have been found in familial hemophagocytic lymphohistiocytosis, which shares some features with CAEBV. We studied a patient who died at age 18, 10 years after the onset of CAEBV. The patient had high titers of antibodies to EBV, EBV RNA in lymph nodes, T-cell lymphoproliferative disease, and hemophagocytic lymphohistiocytosis. DNA sequencing showed novel mutations in both alleles of the perforin gene that resulted in amino acid changes in the protein. The quantity of the native form of perforin from the patient’s stimulated peripheral blood mononuclear cells (PBMCs) was extremely low and immunoblotting showed accumulation of an uncleaved precursor form of perforin. Stimulated PBMCs from the patient were defective for Fas-independent cytotoxicity. These data imply that mutations in this patient resulted in reduced perforin-mediated cytotoxicity by his lymphocytes. This is the first case in which perforin mutations have been shown to result in accumulation of the uncleaved, immature form of perforin. Mutations in the perforin gene are associated with some cases of CAEBV with hemophagocytic lymphohistiocytosis.

Following stress signals, the p53 tumor suppressor protein plays a critical role in regulation of cell proliferation, mainly through induction of growth arrest or apoptosis. Therefore, this protein needs to be strictly regulated and numerous studies have shown that the MDM2 protein is an essential element for p53 regulation in normal cells and, most importantly, that overexpression of MDM2 is responsible for p53 inactivation in various types of tumors. A previous study showed that this is the case in some Burkitt lymphoma (BL) cell lines, where enhanced translation of mdm2 messenger RNA results in overexpression of the protein that complexes and inactivates wild-type p53. To further investigate the role of the p53/MDM2 complex in these BL cells, as well as in other lymphoid cells that do not overexpress MDM2, this study used antisense oligodeoxynucleotides directed either against mdm2 or against p53. Results show that the mdm2 antisense oligodeoxynucleotide induces apoptosis of cells that express a high or low level of MDM2 protein, only if they contain wild-type p53. Moreover, apoptosis is independent of the accumulation of p53 following mdm2 antisense treatment. Finally, the p53 antisense oligodeoxynucleotide, which inhibits the expression of wild-type p53, also induces a decrease of the MDM2 level in cells, whether or not they overexpress this protein, and causes apoptosis of these cells. These results indicate that decreasing the MDM2 protein level by directly or indirectly targeting its biosynthesis is a potent tool for the induction of apoptosis.

CD30 is a member of the tumor necrosis factor (TNF) receptor superfamily that is expressed on activated lymphocytes, as well as on neoplastic cells of Hodgkin disease (HD) and anaplastic large cell lymphoma (ALCL). A number of reports have shown that, depending on cellular context, CD30 signaling can exert a variety of effects, ranging from cell death to cellular proliferation. In the present study this disparity was examined, using a number of ALCL- and HD-derived cell lines. Activation of CD30 led to the induction of apoptotic death of ALCL cells, along with the selective reduction of TNF receptor-associated factor 2 and impairment in the ability of these cells to activate the pro-survival transcription factor nuclear factor ?B (NF-?B). In contrast, HD cells, which constitutively express NF-?B, were not susceptible to CD30-induced apoptosis but could be sensitized following ectopic overexpression of a superdominant I?B. These studies suggest that NF-?B plays a determining role in the sensitivity or resistance of lymphoma cells to CD30-induced apoptosis, which may have important consequences in the clinical treatment of CD30-positive neoplasia.

Introduction

Purpose: To provide evidence-based recommendations for health care professionals on diagnosis and management of diffuse large B cell lymphoma (DLBCL) in resource constraint settings with variable and often limited access to standard of care and advanced diagnostic and therapeutic facilities.

Several members of a black family from Southern Illinois were found to be heterozygous for HB JBangkok (αA2β56 gly→asp2), a hemoglobin abnormality previously described only in individuals of Thai, Chinese, or Indonesian ancestry. In two children (ages 3 and 8) Hb JBangkok was present in combination with sickle hemoglobin. Neither of these children demonstrated evidence of hemolytic disease, enlargement of the liver or spleen, or symptomatic sickle crises. The Hb J comprised 54%-59% of the total in all family members having this variant, in common with previous reports of this hemoglobin. Hb A2 and alkali-resistant hemoglobin were present in normal concentrations in all of the family members studied. Deoxygenated mixtures of Hbs S and JBangkok exhibited minimum gelation concentrations similar to those of equivalent mixtures of Hbs S and A.

Circulating IgG autoantibodies that produce cytoplasmic immunofluorescence staining of ethanol-fixed normal neutrophils have recently been found in a large percentage of patients with active Wegener's granulomatosis. Such autoantibodies are rarely found in other diseases and are therefore virtually diagnostic of Wegener's granulomatosis. The nature of the neutrophil antigen defined by these autoantibodies is controversial and the roles of the antigen and/or autoantibodies in the pathogenesis of Wegener's granulomatosis are unknown. We studied serum samples that produce the cytoplasmic pattern of staining from 10 patients with a diagnosis of Wegener's granulomatosis. By Western blot analysis, all 10 sera reacted with a 29-Kd neutrophil protein (p29). We generated a mouse monoclonal antibody directed against this antigen. The monoclonal antibody produced the same immunofluorescence staining pattern as the serum autoantibodies and was used to affinity-purify p29. The purified antigen had a novel N-terminal sequence homologous to members of the serine proteinase family and bound to radiolabled diisopropyl fluorophosphate (DFP). We conclude that the neutrophil antigen responsible for the cytoplasmic staining pattern produced by autoantibodies in patients with active Wegener's granulomatosis is a distinctive serine proteinase.

Globin chain synthesis was studied in vitro with reticulocytes from a patient heterozygous for Hb Abraham Lincoln, an unstable beta chain variant. Synthesis of α-chains by the reticulocytes exceeded total β-chain synthesis, and a substantial fraction (about 16%) of radioactivity incorporated into globin was recovered from the cells as uncombined α subunits. In a time-course study, the ratio of α: β-chain specific activity was found to increase progressively in a nearly linear manner, suggesting that a fraction of newly synthesized β-chains had undergone rapid destruction. The specific activity of the abnormal β-chain was nearly three times that of ßA. The rate of synthesis of the β-chain of Hb Abraham Lincoln appeared to be approximately half that of the β-chain of Hb A.

Hemoglobin Petah Tikva, a new unstable α-Chain structural variant, was identified in two unrelated children whose families are from the Iraqi Jewish community of Baghdad. Both children have α-thalassemia. with hematologic features of mild HbH disease. The abnormal hemoglobin from the two patients did not separate from hemoglobin A by electrophoresis or chromatography, but the mutant α-chain emerged ahead of αAin carboxymethylcellulose column chromatography, allowing it to be isolated in pure form. Hemoglobin Petah Tikva was unstable in solution and could also be isolated by isopropanol precipitation. Structural analyses indicate that the variant α-Chains contain a normal number of amino acids and have a substitution of Ala → Asp at α110. Each of the patients with HbH disease has one parent with hematologic findings and globin chain synthesis ratios that are characteristic of α-thalassemia trait, and a hematologically normal parent who is a carrier of the variant α-chain of Hb Petah Tikva. The patients each appear to be doubly heterozygous for α-thal-1 and for the structurally abnormal hemoglobin. The mutant α-chains of Hb Petah Tikva were synthesized by reticulocytes of the heterozygous carriers, but only traces of αPTglobin could be detected in their blood, suggesting that the abnormal α-chains underwent postsynthetic degradation or precipitation in these individuals. In the patients with Hb Petah Tikva in combination with α-thalassemia, the normal and mutant α-chains were synthesized by bone marrow erythroid cells at rates proportional to their concentrations in the blood, but there was a substantially decreased rate of synthesis of the variant α-chain by their blood reticulocytes. The α-thalassemia-like expression of Hb Petah Tikva appears to result from instability of the abnormal α-chain as well as from the premature termination of its synthesis during erythroid cell maturation.

Globin chain synthesis was studied in vitro with reticulocytes from a patient heterozygous for Hb Abraham Lincoln, an unstable beta chain variant. Synthesis of α-chains by the reticulocytes exceeded total β-chain synthesis, and a substantial fraction (about 16%) of radioactivity incorporated into globin was recovered from the cells as uncombined α subunits. In a time-course study, the ratio of α : β-chain specific activity was found to increase progressively in a nearly linear manner, suggesting that a fraction of newly synthesized β-chains had undergone rapid destruction. The specific activity of the abnormal β-chain was nearly three times that of βA. The rate of synthesis of the β-chain of Hb Abraham Lincoln appeared to be approximately half that of the β-chain of Hb A.

An electrophoretically fast-moving hemoglobin variant was found in a 2-yr-old boy who was referred for evaluation with findings of iron deficiency anemia. The anemia was corrected, and no hematologic abnormality remained after treatment with iron. Oxygen affinity of the blood was normal, and no evidence was found of instability of the variant hemoglobin. Structural studies demonstrated a substitution of aspartic acid for alanine at β76 (E20). This change did not appear to cause any functional disruption of the hemoglobin in this patient, as would be predicted by the position of the affected amino acid residue on the surface of the molecule.

Hemoglobin variants having electrophoretic mobility more rapid than that of HbA were identified in combination with sickle hemoglobin in two patients at the Cook County Hospital. Neither individual had symptomatic hematologic disease. In one patient, the rapidly migrating hemoglobin had the amino acid substitution characteristic of Hb Tacoma (β-40arg → ser), a mildly unstable variant. In the other patient, Hb Camden (β-131 gln → glu) was identified, and the hematologic findings also indicated that he has α-thalassemia trait. In the patient with HbS-Camden-α-thalassemia, globin synthesis was unbalanced (αlβ0.66), and HbS represented only 19.5% of the total hemoglobin. The latter finding suggests that under conditions of limited α-chain availability βCamden may combine with αsubunits at least as efficiently as does βA.HbS represented 56% of the hemoglobin of the patient with HbS Tacoma, although the rate of synthesis of βTacoma by her reticulocytes was consistently greater than that of βs.A time-course synthesis study demonstrated a progressive increase in the specific activity of βTacoma in relation to that of βs.suggesting that the unstable β-chains of Hb Tacoma underwent selective intracellular degradation. This process appears to explain the disparity between the rates of synthesis of the two βchains and the relative representation of HbS and Hb Tacoma in the patient's erythrocytes.

Hematologic evaluation of a Nigerian obstetrical patient disclosed the presence of sickle-cell trait as well as evidence of a hemoglobin α-chain abnormality. Hemoglobins containing the variant α-chain were isolated by DEAE-cellulose column chromatography, and analysis of the purified α-chain demonstrated a ser→cys substitution at α-81. The abnormal α-chain represented approximately 45% of the total, and hemoglobins containing this α-chain appeared to have normal stability and functional properties. In addition to the abnormal hemoglobins that were identified in this patient, she also was found to have persistent microcytosis in the absence of iron deficiency, and the percentage of HbS in her erythrocytes was less than that usually present in individuals with sickle cell trait. These findings, together with a reduced α/βglobin synthesis ratio from her peripheral blood reticulocytes, indicated the presence of α-thalassemia trait. Hematologic findings from members of the patient’s family suggest that an α-thalassemia gene may be linked to that of the structurally abnormal α-chain.

Bauer: Pfizer: Consultancy; Syros Pharmaceuticals: Consultancy; Fulcrum Therapeutics: Consultancy; Sanofi: Research Funding.

Introduction

Introduction