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Angiogenesis and MYC expression associate with poor outcome in diffuse large B-cell lymphoma (DLBCL). MYC promotes neo-vasculature development but whether its deregulation in DLBCL contributes to angiogenesis is unclear. Examination of this relationship may uncover novel pathogenic regulatory circuitry as well as anti-angiogenic strategies in DLBCL. Here, we show that MYC expression positively correlates with vascular endothelial growth factor (VEGF) expression and angiogenesis in primary DLBCL biopsies, independently of dual expressor status or cell-of-origin classification. We found that MYC promotes VEGFA expression, a correlation that was validated in large datasets of mature B-cell tumours. Using DLBCL cell lines and patient-derived xenograft models, we identified the second messenger cyclic-AMP (cAMP) as a potent suppressor of MYC expression, VEGFA secretion and angiogenesis in DLBCL in normoxia. In hypoxia, cAMP switched targets and suppressed hypoxia-inducible factor 1α, a master regulator of VEGFA/angiogenesis in low oxygen environments. Lastly, we used the phosphodiesterase 4b (Pde4b) knockout mouse to demonstrate that the cAMP/PDE4 axis exercises additional anti-angiogenesis by directly targeting the lymphoma microenvironment. In conclusion, MYC could play a direct role in DLBCL angiogenesis, and modulation of cAMP levels, which can be achieved with clinical grade PDE4 inhibitors, has cell and non-cell autonomous anti-angiogenic activity in DLBCL.

ObjectivesRBC alloantibodies can lead to ABO grouping discrepancies unrelated to A or B antigens or antibodies posing challenges in the blood bank testing. Routine blood bank testing and flow cytometry were used to immunophenotype reagent cells and elucidate the cause of ABO discrepancies in two patients.MethodsABO discrepancy was identified in two patients after transfusion with several units of RBCs. For both patients, the pretransfusion type and screen demonstrated blood group A. Eight and 16 days later, both patients showed an apparent antibody to reagent group A cells, which prompted additional study with patients’ samples and flow cytometric testing of commercial reagent cells.ResultsIn both patients’ specimens, posttransfusion evaluation demonstrated an emerging antibody to the Kell antigen (K). The RBCs of both patients typed negative for K, and both were transfused with K-positive RBCs. Flow cytometric analysis of reagent RBCs demonstrated that five of seven lot numbers were positive for K.ConclusionsEmerging anti-K antibody led to agglutination of the K-positive reagent A1 cells, highlighting the importance of considering RBC alloantibodies and the composition of reagent cells when interpreting cases with an apparent ABO grouping discrepancy.

In mature B-cell malignancies, chromosomal translocations often juxtapose an oncogenic locus to the regulatory regions of the immunoglobulin genes. These genomic rearrangements can associate with specific clinical/pathological sub-entities and inform diagnosis and treatment decisions. Recently, we characterized the t(14;16)(q32;q24) in diffuse large B-cell lymphoma (DLBCL), and showed that it targets the transcription factor IRF8, which is also somatically mutated in ~10% of DLBCLs. IRF8 regulates innate and adaptive immune responses mediated by myeloid/monocytic and lymphoid cells. While the role of IRF8 in human myeloid/dendritic-cell disorders is well established, less is known of its contribution to the pathogenesis of mature B-cell malignancies. To address this knowledge gap, we generated the Eμ-Irf8 mouse model, which mimics the IRF8 deregulation associated with t(14;16) of DLBCL. Eμ-Irf8 mice develop normally and display peripheral blood cell parameters within normal range. However, Eμ-Irf8 mice accumulate pre-pro-B-cells and transitional B-cells in the bone marrow and spleen, respectively, confirming that this model amplifies the physiologic role of Irf8 in B-cell development. Notably, in Eμ-Irf8 mice, the lymphomagenic Irf8 targets Aicda and Bcl6 are overexpressed in mature B-cells. Yet, the incidence of B-cell lymphomas is not increased in the Eμ-Irf8 model, even though their estimated survival probability is significantly lower than that of WT controls. Together, these observations suggest that the penetrance on the Irf8-driven phenotype may be incomplete and that introduction of second genetic hit, a common strategy in mouse models of lymphoma, may be necessary to uncover the pro-lymphoma phenotype of the Eμ-Irf8 mice.

Angiogenesis associates with poor outcome in diffuse large B-cell lymphoma (DLBCL), but the contribution of the lymphoma cells to this process remains unclear. Addressing this knowledge gap may uncover unsuspecting proangiogenic signaling nodes and highlight alternative antiangiogenic therapies. Here, we identify the second messenger cyclic-AMP (cAMP) and the enzyme that terminates its activity, phosphodiesterase 4B (PDE4B), as regulators of B-cell lymphoma angiogenesis. We first show that cAMP, in a PDE4B-dependent manner, suppresses PI3K/AKT signals to downmodulate vascular endothelial growth factor (VEGF) secretion and vessel formation in vitro. Next, we create a novel mouse model that combines the lymphomagenic Myc transgene with germline deletion of Pde4b. We show that lymphomas developing in a Pde4b-null background display significantly lower microvessel density (MVD) in association with lower VEGF levels and PI3K/AKT activity. We recapitulate these observations by treating lymphoma-bearing mice with the FDA-approved PDE4 inhibitor, Roflumilast. Lastly, we show that primary human DLBCLs with high PDE4B expression display significantly higher MVD. Here, we defined an unsuspected signaling circuitry in which the cAMP generated in lymphoma cells downmodulates PI3K/AKT and VEGF secretion to negatively influence vessel development in the microenvironment. These data identify PDE4 as an actionable antiangiogenic target in DLBCL.

We describe a case of acute promyelocytic leukemia in a 61-yr-old woman with a cryptic insertion of RARA gene into PML gene. Using a combination of cytogenetic and molecular methods, we confirmed the insertion and presence of the PML-RARA transcript and lack of the reciprocal RARA-PML transcript. Although such cryptic insertions leading to a PML-RARA fusion have been previously reported, we show that such variant insertions, based on our case, appear to have the same prognostic significance as the classical t(15;17)(q22;q21).

The characterization of immunoglobulin heavy chain (IGH) translocations provides information on the diagnosis and guides therapeutic decisions in mature B-cell malignancies while enhancing our understanding of normal and malignant B-cell biology. However, existing methodologies for the detection of IGH translocations are labor intensive, often require viable cells, and are biased toward known IGH fusions. To overcome these limitations, we developed a capture sequencing strategy for the identification of IGH rearrangements at nucleotide level resolution and tested its capabilities as a diagnostic and discovery tool in 78 primary diffuse large B-cell lymphomas (DLBCLs). We readily identified IGH-BCL2, IGH-BCL6, IGH-MYC, and IGH-CCND1 fusions and discovered IRF8, EBF1, and TNFSF13 (APRIL) as novel IGH partners in these tumors. IRF8 and TNFSF13 expression was significantly higher in lymphomas with IGH rearrangements targeting these loci. Modeling the deregulation of IRF8 and EBF1 in vitro defined a lymphomagenic profile characterized by up-regulation of AID and/or BCL6, down-regulation of PRMD1, and resistance to apoptosis. Using a capture sequencing strategy, we discovered the B-cell relevant genes IRF8, EBF1, and TNFSF13 as novel targets for IGH deregulation. This methodology is poised to change how IGH translocations are identified in clinical settings while remaining a powerful tool to uncover the pathogenesis of B-cell malignancies.

Diffuse large B cell lymphoma (DLBCL) is a common and heterogeneous tumor. Extensive genetic examinations of these malignancies were performed in the past decade, but this knowledge has yet to be translated into rationally-designed treatment strategies that effectively change its cure rate. Recognizing and acquiring basic biology data in specific priority areas may accelerate clinical translation in DLBCL. One such knowledge gap concerns the interplay between lymphoma cells, the microenvironment and angiogenesis. This is particularly important because high circulating levels of vascular endothelial growth factor (VEGF) and elevated microvessel density (MVD) are associated with poor outcome in DLBCL, while clinical trials that tested classical anti-VEGFA agents in this setting were negative and plagued by serious adverse events. Cyclic-AMP (cAMP) is a pervasive second messenger that in immune cells exerts primarily negative effects, including suppression of proximal B or T cell receptor signaling and induction of apoptosis. In immune cells, cAMP signaling is terminated by phosphodiesterase 4 (PDE4). Earlier, we identified PDE4B in an outcome prediction signature of DLBCL and showed subsequently that its inhibition had anti-lymphoma properties. cAMP activity is also highly contextualized and it was recently suggested to attenuate vessel development in non-neoplastic cell models. Thus, we speculated that high PDE4B expression/activity, by abrogating cAMP signaling, could modulate angiogenesis in DLBCL. To examine this idea, we first used a panel of DLBCL cell lines and found that cAMP suppressed VEGF expression (mRNA) and secretion (protein) in PDE4B-low but not in PDE4B-high DLBCLs. In human umbilical vein endothelial cell (HUVEC) tube formation assays, we noted that conditioned media from PDE4B-high DLBCLs were significantly more angiogenic than those from PDE4B-low models. To isolate the role of PDE4B in this process, we used genetic and pharmacological models. Stable ectopic expression of PDE4B blocked the anti-angiogenic properties of cAMP, whereas a siRNA-mediated PDE4B knockdown, or exposure to the FDA-approved PDE4 inhibitor Roflumilast, suppressed VEGF levels and activity. Mechanistically, we demonstrated that cAMP, in a PDE4B-dependent manner, suppresses PI3K and AKT activities to impose its anti-angiogenic properties. Thus, ectopic expression of a constitutively active AKT gene in PDE4-low DLBCL cell lines abrogated cAMP effects in a manner similar to PDE4B reconstitution, indicating that PI3K/AKT are key mediators of the cAMP/PDE4 effects on angiogenesis. To expand these observations to more elaborate models, we created a composite mouse where c-Myc-driven lymphomas develop in Pde4b null or wild-typebackgrounds. Remarkably, primary lymphomas from Eµ-Myc;Pde4b-/- mice displayed significantly lower MVD (quantified by immunohistochemistry - IHC - with anti-CD34 staining) than the lymphomas that developed in Eµ-Myc;Pde4b+/+ mice (n= 19, p Next, we tested the hypothesis that pharmacological inhibition of PDE4 in vivo could effectively suppress lymphoma angiogenesis. To that end, we used adoptive transfer to generate multiple independent cohorts of Eµ-Myc-driven lymphoma-bearing mice (n=68), which were randomized to receive vehicle or Roflumilast (5mg/kd/day gavage). B cell lymphomas from Roflumilast-treated mice showed a marked suppression of angiogenesis (p=0.01, for MVD of Roflumilast vs. vehicle groups) and significant decrease in PI3K/AKT activity (p=0.003), which were accompanied by lower serum levels of VEGF (p=0.005). In addition, in comparison to their vehicle-treated isogenic controls, mice that received Roflumilast displayed a smaller tumor burden (p Disclosures No relevant conflicts of interest to declare.