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•Population-scale genomics (N = 937 939) allowed assembly of a large FVL/PTGM double-heterozygous cohort while mitigating ascertainment bias.•FVL/PTGM DH appeared to be more common than FVL homozygosity and conferred a similar risk of venous thrombosis.

•Zanubrutinib sustained PFS benefit over ibrutinib; sensitivity analyses suggest this was driven by both antileukemic effect and tolerability.•Zanubrutinib maintained a favorable safety/tolerability profile with prevalence of most adverse events remaining stable or decreasing year-over-year.

Introduction:Pts who receive covalent Bruton tyrosine kinase inhibitors (cBTKis) for CLL can develop drug resistance, leading to disease progression. Often, cBTKi resistance results from the emergence of subclones with mutations at the cBTKi binding site (C481). Less frequent, non-C481 mutations, including gatekeeper residue T474 and kinase-impaired L528 mutations, have been reported in pts with progression on cBTKis. Most characterizations of cBTKi resistance mutations are derived from small or retrospective pt populations. To gain further insight into the genetic mechanisms of cBTKi resistance in a randomized population of pts with CLL, we performed next-generation sequencing (NGS) on samples from pts who had progression on zanubrutinib (zanu) or ibrutinib (ibr) in the phase 3 ALPINE study (NCT03734016; Brown et al. NEJM. 2023).

Introduction:ALPINE, a randomized, multinational phase 3 study (NCT03734016) in patients with R/R CLL/SLL, established the statistical and clinically meaningful superiority of zanubrutinib over ibrutinib on progression-free survival (PFS) and overall response rate (ORR) and confirmed the favorable safety/tolerability profile of zanubrutinib (Brown et al. NEJM; 2022). Now, with 3 years of overall study follow-up, we report the results of an extended follow-up analysis.

Activated B-cell diffuse large B-cell lymphoma (ABC-DLBCL) is an aggressive lymphoproliferative disorder involving chronic NF-κB activation. Several mutations in the BCR and MyD88 signaling pathway components, such as MyD88 L265P, are implicated in this aberrant activation. Among heat shock proteins, HSP110 has recently been identified as a prosurvival and/or proliferation factor in many cancers, but its role in ABC-DLBCL survival mechanisms remained to be established. We observed that short hairpin RNA–mediated HSP110 silencing decreased the survival of several ABC-DLBCL cell lines and decreased immunoglobulin M-MyD88 co-localization and subsequent NF-κB signaling. Conversely, overexpression of HSP110 in ABC-DLBCL or non-DLBCL cell lines increased NF-κB signaling, indicating a tight interplay between HSP110 and the NF-κB pathway. By using immunoprecipitation and proximity ligation assays, we identified an interaction between HSP110 and both wild-type MyD88 and MyD88 L265P. HSP110 stabilized both MyD88 forms with a stronger effect on MyD88 L265P, thus facilitating chronic NF-κB activation. Finally, HSP110 expression was higher in lymph node biopsies from patients with ABC-DLBCL than in normal reactive lymph nodes, and a strong correlation was found between the level of HSP110 and MyD88. In conclusion, we identified HSP110 as a regulator of NF-κB signaling through MyD88 stabilization in ABC-DLBCL. This finding reveals HSP110 as a new potential therapeutic target in ABC-DLBCL.

Activated B-cell diffuse large B-cell lymphoma (ABC-DLBCL) is an aggressive lymphoproliferative disorder involving chronic NF-κB activation. Several mutations in the BCR and MyD88 signaling pathway components, such as MyD88 L265P, are implicated in this aberrant activation. Among heat shock proteins, HSP110 has recently been identified as a prosurvival and/or proliferation factor in many cancers, but its role in ABC-DLBCL survival mechanisms remained to be established. We observed that short hairpin RNA–mediated HSP110 silencing decreased the survival of several ABC-DLBCL cell lines and decreased immunoglobulin M-MyD88 co-localization and subsequent NF-κB signaling. Conversely, overexpression of HSP110 in ABC-DLBCL or non-DLBCL cell lines increased NF-κB signaling, indicating a tight interplay between HSP110 and the NF-κB pathway. By using immunoprecipitation and proximity ligation assays, we identified an interaction between HSP110 and both wild-type MyD88 and MyD88 L265P. HSP110 stabilized both MyD88 forms with a stronger effect on MyD88 L265P, thus facilitating chronic NF-κB activation. Finally, HSP110 expression was higher in lymph node biopsies from patients with ABC-DLBCL than in normal reactive lymph nodes, and a strong correlation was found between the level of HSP110 and MyD88. In conclusion, we identified HSP110 as a regulator of NF-κB signaling through MyD88 stabilization in ABC-DLBCL. This finding reveals HSP110 as a new potential therapeutic target in ABC-DLBCL.

The histone methyltransferase EZH2 is frequently mutated in germinal center–derived diffuse large B-cell lymphoma and follicular lymphoma. To further characterize these EZH2 mutations in lymphomagenesis, we generated a mouse line where EZH2Y641F is expressed from a lymphocyte-specific promoter. Spleen cells isolated from the transgenic mice displayed a global increase in trimethylated H3K27, but the mice did not show an increased tendency to develop lymphoma. As EZH2 mutations often coincide with other mutations in lymphoma, we combined the expression of EZH2Y641F by crossing these transgenic mice with Eµ-Myc transgenic mice. We observed a dramatic acceleration of lymphoma development in this combination model of Myc and EZH2Y641F. The lymphomas show histologic features of high-grade disease with a shift toward a more mature B-cell phenotype, increased cycling and gene expression, and epigenetic changes involving important pathways in B-cell regulation and function. Furthermore, they initiate disease in secondary recipients. In summary, EZH2Y641F can collaborate with Myc to accelerate lymphomagenesis demonstrating a cooperative role of EZH2 mutations in oncogenesis. This murine lymphoma model provides a new tool to study global changes in the epigenome caused by this frequent mutation and a promising model system for testing novel treatments.

The histone methyltransferase EZH2 is frequently mutated in germinal center–derived diffuse large B-cell lymphoma and follicular lymphoma. To further characterize these EZH2 mutations in lymphomagenesis, we generated a mouse line where EZH2Y641Fis expressed from a lymphocyte-specific promoter. Spleen cells isolated from the transgenic mice displayed a global increase in trimethylated H3K27, but the mice did not show an increased tendency to develop lymphoma. As EZH2mutations often coincide with other mutations in lymphoma, we combined the expression of EZH2Y641Fby crossing these transgenic mice with Eµ-Myc transgenic mice. We observed a dramatic acceleration of lymphoma development in this combination model of Mycand EZH2Y641F. The lymphomas show histologic features of high-grade disease with a shift toward a more mature B-cell phenotype, increased cycling and gene expression, and epigenetic changes involving important pathways in B-cell regulation and function. Furthermore, they initiate disease in secondary recipients. In summary, EZH2Y641Fcan collaborate with Mycto accelerate lymphomagenesis demonstrating a cooperative role of EZH2mutations in oncogenesis. This murine lymphoma model provides a new tool to study global changes in the epigenome caused by this frequent mutation and a promising model system for testing novel treatments.

Pathologische anatomie leiden endothelium (PAL-E) antibody has been used for more than 20 years as a prototype marker for vascular endothelium. The elusive target of this antibody has been claimed to be plasmalemma vesicle-associated protein-1 (PV-1) and neuropilin-1 (NRP-1). Using immunofluorescence, we show that PAL-E, anti–PV-1, anti–NRP-1, and anti-CD31 antibodies show largely identical staining patterns in the vasculature of different tissues. However, PV-1–transfected cells only bind PAL-E and anti–PV-1 antibodies, whereas NRP-1 transfectants stain with anti–NRP-1 antibodies in flow cytometry. Using lysates from tissues and transfected cells, we further confirm that the molecule recognized by PAL-E and anti–PV-1 antibodies is not NRP-1 but PV-1. Nevertheless, coimmunoprecipitation studies unambiguously demonstrate that NRP-1 can form complexes with PV-1. This connects, for the first time, 2 molecules involved in leukocyte trafficking and angiogenesis, thereby opening interesting possibilities for future research in this field.

Pathologische anatomie leiden endothelium (PAL-E) antibody has been used for more than 20 years as a prototype marker for vascular endothelium. The elusive target of this antibody has been claimed to be plasmalemma vesicle-associated protein-1 (PV-1) and neuropilin-1 (NRP-1). Using immunofluorescence, we show that PAL-E, anti–PV-1, anti–NRP-1, and anti-CD31 antibodies show largely identical staining patterns in the vasculature of different tissues. However, PV-1–transfected cells only bind PAL-E and anti–PV-1 antibodies, whereas NRP-1 transfectants stain with anti–NRP-1 antibodies in flow cytometry. Using lysates from tissues and transfected cells, we further confirm that the molecule recognized by PAL-E and anti–PV-1 antibodies is not NRP-1 but PV-1. Nevertheless, coimmunoprecipitation studies unambiguously demonstrate that NRP-1 can form complexes with PV-1. This connects, for the first time, 2 molecules involved in leukocyte trafficking and angiogenesis, thereby opening interesting possibilities for future research in this field.

Familial hemophagocytic lymphohistiocytosis (FHL) is an autosomal recessive, often-fatal hyperinflammatory disorder. Mutations in PRF1, UNC13D, STX11, and STXBP2are causative of FHL2, 3, 4, and 5, respectively. In a majority of suspected FHL patients from Northern Europe, sequencing of exons and splice sites of such genes required for lymphocyte cytotoxicity revealed no or only monoallelic UNC13Dmutations. Here, in 21 patients, we describe 2 pathogenic, noncoding aberrations of UNC13D. The first is a point mutation localized in an evolutionarily conserved region of intron 1. This mutation selectively impairs UNC13Dtranscription in lymphocytes, abolishing Munc13-4 expression. The second is a 253-kb inversion straddling UNC13D, affecting the 3′-end of the transcript and likewise abolishing Munc13-4 expression. Carriership of the intron 1 mutation was found in patients across Europe, whereas carriership of the inversion was limited to Northern Europe. Notably, the latter aberration represents the first description of an autosomal recessive human disease caused by an inversion. These findings implicate an intronic sequence in cell-type specific expression of Munc13-4 and signify variations outside exons and splice sites as a common cause of FHL3. Based on these data, we propose a strategy for targeted sequencing of evolutionary conserved noncoding regions for the diagnosis of primary immunodeficiencies.

Familial hemophagocytic lymphohistiocytosis (FHL) is an autosomal recessive, often-fatal hyperinflammatory disorder. Mutations in PRF1, UNC13D, STX11, and STXBP2 are causative of FHL2, 3, 4, and 5, respectively. In a majority of suspected FHL patients from Northern Europe, sequencing of exons and splice sites of such genes required for lymphocyte cytotoxicity revealed no or only monoallelic UNC13D mutations. Here, in 21 patients, we describe 2 pathogenic, noncoding aberrations of UNC13D. The first is a point mutation localized in an evolutionarily conserved region of intron 1. This mutation selectively impairs UNC13D transcription in lymphocytes, abolishing Munc13-4 expression. The second is a 253-kb inversion straddling UNC13D, affecting the 3′-end of the transcript and likewise abolishing Munc13-4 expression. Carriership of the intron 1 mutation was found in patients across Europe, whereas carriership of the inversion was limited to Northern Europe. Notably, the latter aberration represents the first description of an autosomal recessive human disease caused by an inversion. These findings implicate an intronic sequence in cell-type specific expression of Munc13-4 and signify variations outside exons and splice sites as a common cause of FHL3. Based on these data, we propose a strategy for targeted sequencing of evolutionary conserved noncoding regions for the diagnosis of primary immunodeficiencies.

Leukocyte migration to sites of inflammation is regulated by several endothelial adhesion molecules. Vascular adhesion protein-1 (VAP-1) is unique among the homing-associated molecules as it is both an enzyme that oxidizes primary amines and an adhesin. Although granulocytes can bind to endothelium via a VAP-1–dependent manner, the counter-receptor(s) on this leukocyte population is(are) not known. Here we used a phage display approach and identified Siglec-9 as a candidate ligand on granulocytes. The binding between Siglec-9 and VAP-1 was confirmed by in vitro and ex vivo adhesion assays. The interaction sites between VAP-1 and Siglec-9 were identified by molecular modeling and confirmed by further binding assays with mutated proteins. Although the binding takes place in the enzymatic groove of VAP-1, it is only partially dependent on the enzymatic activity of VAP-1. In positron emission tomography, the 68Gallium-labeled peptide of Siglec-9 specifically detected VAP-1 in vasculature at sites of inflammation and cancer. Thus, the peptide binding to the enzymatic groove of VAP-1 can be used for imaging conditions, such as inflammation and cancer.

Leukocyte migration to sites of inflammation is regulated by several endothelial adhesion molecules. Vascular adhesion protein-1 (VAP-1) is unique among the homing-associated molecules as it is both an enzyme that oxidizes primary amines and an adhesin. Although granulocytes can bind to endothelium via a VAP-1–dependent manner, the counter-receptor(s) on this leukocyte population is(are) not known. Here we used a phage display approach and identified Siglec-9 as a candidate ligand on granulocytes. The binding between Siglec-9 and VAP-1 was confirmed by in vitro and ex vivo adhesion assays. The interaction sites between VAP-1 and Siglec-9 were identified by molecular modeling and confirmed by further binding assays with mutated proteins. Although the binding takes place in the enzymatic groove of VAP-1, it is only partially dependent on the enzymatic activity of VAP-1. In positron emission tomography, the 68Gallium-labeled peptide of Siglec-9 specifically detected VAP-1 in vasculature at sites of inflammation and cancer. Thus, the peptide binding to the enzymatic groove of VAP-1 can be used for imaging conditions, such as inflammation and cancer.

CD73 is involved in the extracellular ATP metabolism by dephosphorylating extracellular AMP to adenosine and thus regulating permeability of the blood vessels and leukocyte traffic into the tissues. It is also present on lymphatic vessels where its distribution and function have not been characterized. We found that CD73 is expressed on a subpopulation of afferent lymph vessels but is absent on efferent lymphatics, unlike LYVE-1 and podoplanin, which are expressed on both types of lymphatics. The extracellular nucleotide metabolism on lymphatic endothelium differs from that on blood vessel endothelium as lymphatic endothelium has lower NTPDase and higher ecto-5′-nucleotidase/CD73 activity than blood vascular endothelium. In knockout mice, the lack of CD73 on lymphocytes decreases migration of lymphocytes to the draining lymph nodes more than 50% while CD73-deficient lymph vessels mediate lymphocyte trafficking as efficiently as the wild-type lymphatics. Thus, although endothelial CD73 is important for permeability and leukocyte extravasation in blood vessels, it does not have a role in these functions on lymphatics. Instead, lymphocyte CD73 is intimately involved in lymphocyte migration via afferent lymphatic vessels.

CD73 is involved in the extracellular ATP metabolism by dephosphorylating extracellular AMP to adenosine and thus regulating permeability of the blood vessels and leukocyte traffic into the tissues. It is also present on lymphatic vessels where its distribution and function have not been characterized. We found that CD73 is expressed on a subpopulation of afferent lymph vessels but is absent on efferent lymphatics, unlike LYVE-1 and podoplanin, which are expressed on both types of lymphatics. The extracellular nucleotide metabolism on lymphatic endothelium differs from that on blood vessel endothelium as lymphatic endothelium has lower NTPDase and higher ecto-5′-nucleotidase/CD73 activity than blood vascular endothelium. In knockout mice, the lack of CD73 on lymphocytes decreases migration of lymphocytes to the draining lymph nodes more than 50% while CD73-deficient lymph vessels mediate lymphocyte trafficking as efficiently as the wild-type lymphatics. Thus, although endothelial CD73 is important for permeability and leukocyte extravasation in blood vessels, it does not have a role in these functions on lymphatics. Instead, lymphocyte CD73 is intimately involved in lymphocyte migration via afferent lymphatic vessels.

Leukocytes migrate from the blood into areas of inflammation by interacting with various adhesion molecules on endothelial cells. Vascular adhesion protein-1 (VAP-1) is a glycoprotein expressed on inflamed endothelium where it plays a dual role: it is both an enzyme that oxidizes primary amines and an adhesin that is involved in leukocyte trafficking to sites of inflammation. Although VAP-1 was identified more than 15 years ago, the counterreceptor(s) for VAP-1 on leukocytes has remained unknown. Here we have identified Siglec-10 as a leukocyte ligand for VAP-1 using phage display screenings. The binding between Siglec-10 and VAP-1 was verified by different adhesion assays, and this interaction was also consistent with molecular modeling. Moreover, the interaction between Siglec-10 and VAP-1 led to increased hydrogen peroxide production, indicating that Siglec-10 serves as a substrate for VAP-1. Thus, the Siglec-10–VAP-1 interaction seems to mediate lymphocyte adhesion to endothelium and has the potential to modify the inflammatory microenvironment via the enzymatic end products.

Leukocytes migrate from the blood into areas of inflammation by interacting with various adhesion molecules on endothelial cells. Vascular adhesion protein-1 (VAP-1) is a glycoprotein expressed on inflamed endothelium where it plays a dual role: it is both an enzyme that oxidizes primary amines and an adhesin that is involved in leukocyte trafficking to sites of inflammation. Although VAP-1 was identified more than 15 years ago, the counterreceptor(s) for VAP-1 on leukocytes has remained unknown. Here we have identified Siglec-10 as a leukocyte ligand for VAP-1 using phage display screenings. The binding between Siglec-10 and VAP-1 was verified by different adhesion assays, and this interaction was also consistent with molecular modeling. Moreover, the interaction between Siglec-10 and VAP-1 led to increased hydrogen peroxide production, indicating that Siglec-10 serves as a substrate for VAP-1. Thus, the Siglec-10–VAP-1 interaction seems to mediate lymphocyte adhesion to endothelium and has the potential to modify the inflammatory microenvironment via the enzymatic end products.

Pathologische Anatomie Leiden-endothelium antibody has been used for more than 20 years as a marker for vascular endothelium. Despite its widespread use, the target of this antibody was only recently identified as plasmalemma vesicle–associated protein-1 (PV-1). However, no function has been identified for this molecule. Here we report that activation of human umbilical vein endothelial cells with tumor necrosis factor-α resulted in a remarkable redistribution of PV-1 toward the peripheral areas of the cells. Furthermore, in vitro endpoint transmigration experiments showed that transcellularly migrating lymphocytes are surrounded by rings containing PV-1 and caveolin-1. Moreover, PV-1 associates physically with vimentin. In addition, administration of anti–PV-1 antibody during capillary flow assays resulted in a significant inhibition of lymphocyte transmigration through the endothelial cell layer, whereas rolling and adhesion were unaffected. In vivo blockage of PV-1 by an antibody in acute peritonitis and air pouch model resulted in a significant decrease in the number of migrating leukocytes. Here we thus define leukocyte transendothelial migration as the first known function for PV-1.

Pathologische Anatomie Leiden-endothelium antibody has been used for more than 20 years as a marker for vascular endothelium. Despite its widespread use, the target of this antibody was only recently identified as plasmalemma vesicle–associated protein-1 (PV-1). However, no function has been identified for this molecule. Here we report that activation of human umbilical vein endothelial cells with tumor necrosis factor-α resulted in a remarkable redistribution of PV-1 toward the peripheral areas of the cells. Furthermore, in vitro endpoint transmigration experiments showed that transcellularly migrating lymphocytes are surrounded by rings containing PV-1 and caveolin-1. Moreover, PV-1 associates physically with vimentin. In addition, administration of anti–PV-1 antibody during capillary flow assays resulted in a significant inhibition of lymphocyte transmigration through the endothelial cell layer, whereas rolling and adhesion were unaffected. In vivo blockage of PV-1 by an antibody in acute peritonitis and air pouch model resulted in a significant decrease in the number of migrating leukocytes. Here we thus define leukocyte transendothelial migration as the first known function for PV-1.

Macrophage mannose receptor (MR) participates in pathogen recognition, clearance of endogenous serum glycoproteins, and antigen presentation. MR is also present on lymphatic vessels, where its function is unknown. Here we show that migration of lymphocytes from the skin into the draining lymph nodes through the afferent lymphatics is reduced in MR-deficient mice, while the structure of lymphatic vasculature remains normal in these animals. Moreover, in a tumor model the primary tumors grow significantly bigger in MR−/− mice than in the wild-type (WT) controls, whereas the regional lymph node metastases are markedly smaller. Adhesion of both normal lymphocytes and tumor cells to lymphatic vessels is significantly decreased in MR-deficient mice. The ability of macrophages to present tumor antigens is indistinguishable between the 2 genotypes. Thus, MR on lymphatic endothelial cells is involved in leukocyte trafficking and contributes to the metastatic behavior of cancer cells. Blocking of MR may provide a new approach to controlling inflammation and cancer metastasis by targeting the lymphatic vasculature.

Macrophage mannose receptor (MR) participates in pathogen recognition, clearance of endogenous serum glycoproteins, and antigen presentation. MR is also present on lymphatic vessels, where its function is unknown. Here we show that migration of lymphocytes from the skin into the draining lymph nodes through the afferent lymphatics is reduced in MR-deficient mice, while the structure of lymphatic vasculature remains normal in these animals. Moreover, in a tumor model the primary tumors grow significantly bigger in MR−/−mice than in the wild-type (WT) controls, whereas the regional lymph node metastases are markedly smaller. Adhesion of both normal lymphocytes and tumor cells to lymphatic vessels is significantly decreased in MR-deficient mice. The ability of macrophages to present tumor antigens is indistinguishable between the 2 genotypes. Thus, MR on lymphatic endothelial cells is involved in leukocyte trafficking and contributes to the metastatic behavior of cancer cells. Blocking of MR may provide a new approach to controlling inflammation and cancer metastasis by targeting the lymphatic vasculature.

Leukocyte migration from the blood into tissues is pivotal in immune homeostasis and in inflammation. During the multistep extravasation cascade, endothelial selectins (P- and E-selectin) and vascular adhesion protein-1 (VAP-1), a cell-surface–expressed oxidase, are important in tethering and rolling. Here, we studied the signaling functions of the catalytic activity of VAP-1. Using human endothelial cells transfected with wild-type VAP-1 and an enzymatically inactive VAP-1 point mutant, we show that transcription and translation of E- and P-selectins are induced through the enzymatic activity of VAP-1. Moreover, use of VAP-1–deficient animals and VAP-1–deficient animals carrying the human VAP-1 as a transgene show a VAP-enzyme activity–dependent induction of P-selectin in vivo. Up-regulation of P-selectin was found both in high endothelial venules in lymphoid tissues and in flat-walled vessels in noninflamed tissues. VAP-1 activity in vivo led to increased P-selectin–dependent binding of lymphocytes to endothelial cells. These data show that the oxidase reaction catalyzed by VAP-1 alters the expression of other molecules involved in the leukocyte extravasation cascade. Our findings indicate cross-talk between adhesion molecules involved in the tethering and rolling of leukocytes and show that VAP-1–dependent signaling can prime the vessels for an enhanced inflammatory response.

Leukocyte migration from the blood into tissues is pivotal in immune homeostasis and in inflammation. During the multistep extravasation cascade, endothelial selectins (P- and E-selectin) and vascular adhesion protein-1 (VAP-1), a cell-surface–expressed oxidase, are important in tethering and rolling. Here, we studied the signaling functions of the catalytic activity of VAP-1. Using human endothelial cells transfected with wild-type VAP-1 and an enzymatically inactive VAP-1 point mutant, we show that transcription and translation of E- and P-selectins are induced through the enzymatic activity of VAP-1. Moreover, use of VAP-1–deficient animals and VAP-1–deficient animals carrying the human VAP-1 as a transgene show a VAP-enzyme activity–dependent induction of P-selectin in vivo. Up-regulation of P-selectin was found both in high endothelial venules in lymphoid tissues and in flat-walled vessels in noninflamed tissues. VAP-1 activity in vivo led to increased P-selectin–dependent binding of lymphocytes to endothelial cells. These data show that the oxidase reaction catalyzed by VAP-1 alters the expression of other molecules involved in the leukocyte extravasation cascade. Our findings indicate cross-talk between adhesion molecules involved in the tethering and rolling of leukocytes and show that VAP-1–dependent signaling can prime the vessels for an enhanced inflammatory response.

Vascular adhesion protein-1 (VAP-1) is a homodimeric glycoprotein that belongs to a unique subgroup of cell-surface-expressed oxidases. In adults, endothelial VAP-1 supports leukocyte rolling, firm adhesion, and transmigration in both enzyme activity-dependent and enzyme activity-independent manner. Here we studied the induction and function of VAP-1 during human ontogeny. We show that VAP-1 is already found in the smooth muscle at embryonic week 7. There are marked time-dependent switches in VAP-1 expression in the sinusoids of the liver, in the peritubular capillaries of the kidney, in the capillaries of the heart, and in the venules in the lamina propria of the gut. Fetal VAP-1 is dimerized, and it is enzymatically active. VAP-1 in fetal-type venules is able to bind cord blood lymphocytes. Also, adenovirally transfected VAP-1 on human umbilical vein endothelial cells is involved in rolling and firm adhesion of cord blood lymphocytes under conditions of physiologic shear stress. We conclude that VAP-1 is synthesized from early on in human vessels and it is functionally intact already before birth. Thus, VAP-1 may contribute critically to the oxidase activities in utero, and prove important for lymphocyte trafficking during human ontogeny.

Vascular adhesion protein-1 (VAP-1) is a homodimeric glycoprotein that belongs to a unique subgroup of cell-surface-expressed oxidases. In adults, endothelial VAP-1 supports leukocyte rolling, firm adhesion, and transmigration in both enzyme activity-dependent and enzyme activity-independent manner. Here we studied the induction and function of VAP-1 during human ontogeny. We show that VAP-1 is already found in the smooth muscle at embryonic week 7. There are marked time-dependent switches in VAP-1 expression in the sinusoids of the liver, in the peritubular capillaries of the kidney, in the capillaries of the heart, and in the venules in the lamina propria of the gut. Fetal VAP-1 is dimerized, and it is enzymatically active. VAP-1 in fetal-type venules is able to bind cord blood lymphocytes. Also, adenovirally transfected VAP-1 on human umbilical vein endothelial cells is involved in rolling and firm adhesion of cord blood lymphocytes under conditions of physiologic shear stress. We conclude that VAP-1 is synthesized from early on in human vessels and it is functionally intact already before birth. Thus, VAP-1 may contribute critically to the oxidase activities in utero, and prove important for lymphocyte trafficking during human ontogeny.

The pathologische anatomie Leiden-endothelium (PAL-E) antibody has been used for almost 20 years as a specific marker for vascular endothelial cells. Due to the fact that this antibody works only in very limited applications, the molecular identity of PAL-E has remained unknown. In this work, we demonstrate by double stainings, cross-immunoprecipitations, and transfectants that the PAL-E antigen is identical with a protein designated PV-1 (plasmalemmal vesicle 1) or FELS (fenestrated endothelial-linked structure protein) and is not vimentin, as reported earlier. As the expression of this molecule is by no means restricted to fenestrated endothelium, we suggest the use of the name PLVAP for this protein. Molecular identification of PLVAP should help in the production of new tools for the identification of vascular as opposed to lymphatic endothelium and to elucidate the function of this protein.

The pathologische anatomie Leiden-endothelium (PAL-E) antibody has been used for almost 20 years as a specific marker for vascular endothelial cells. Due to the fact that this antibody works only in very limited applications, the molecular identity of PAL-E has remained unknown. In this work, we demonstrate by double stainings, cross-immunoprecipitations, and transfectants that the PAL-E antigen is identical with a protein designated PV-1 (plasmalemmal vesicle 1) or FELS (fenestrated endothelial-linked structure protein) and is not vimentin, as reported earlier. As the expression of this molecule is by no means restricted to fenestrated endothelium, we suggest the use of the name PLVAP for this protein. Molecular identification of PLVAP should help in the production of new tools for the identification of vascular as opposed to lymphatic endothelium and to elucidate the function of this protein.

Common lymphatic endothelial and vascular endothelial receptor-1 (CLEVER-1; also known as stabilin-1 or FEEL-1) is a large multifunctional glycoprotein implicated in scavenging, angiogenesis, and cell adhesion. Here we studied the function of human CLEVER-1 in leukocyte trafficking. Lymphatic vessels expressed CLEVER-1 constitutively in skin in vivo, whereas on vascular endothelium it appeared only upon inflammation. On isolated vascular endothelial cells, CLEVER-1 supported rolling and transmigration of peripheral blood mononuclear cells (PBMCs) under physiologically relevant laminar shear stress. Intriguingly, CLEVER-1 also mediated transmigration of leukocytes through cultured lymphatic endothelium under static conditions. Thus, synthesis of CLEVER-1 is differentially regulated on the 2 anatomically distinct vascular beds, and CLEVER-1 mediates the transmigration step of the leukocyte traffic in both of them. Notably, CLEVER-1 is the first adhesion molecule shown to be involved in the PBMC transmigration through the lymphatic arm of the immune system. (Blood. 2004;104:3849-3857)

Common lymphatic endothelial and vascular endothelial receptor-1 (CLEVER-1; also known as stabilin-1 or FEEL-1) is a large multifunctional glycoprotein implicated in scavenging, angiogenesis, and cell adhesion. Here we studied the function of human CLEVER-1 in leukocyte trafficking. Lymphatic vessels expressed CLEVER-1 constitutively in skin in vivo, whereas on vascular endothelium it appeared only upon inflammation. On isolated vascular endothelial cells, CLEVER-1 supported rolling and transmigration of peripheral blood mononuclear cells (PBMCs) under physiologically relevant laminar shear stress. Intriguingly, CLEVER-1 also mediated transmigration of leukocytes through cultured lymphatic endothelium under static conditions. Thus, synthesis of CLEVER-1 is differentially regulated on the 2 anatomically distinct vascular beds, and CLEVER-1 mediates the transmigration step of the leukocyte traffic in both of them. Notably, CLEVER-1 is the first adhesion molecule shown to be involved in the PBMC transmigration through the lymphatic arm of the immune system. (Blood. 2004;104:3849-3857)

Polymorphonuclear leukocytes (PMNs) migrate from the blood into areas of inflammation by binding to the endothelial cells of blood vessels via adhesion molecules. Vascular adhesion protein-1 (VAP-1) is one of the molecules mediating leukocyte-endothelial cell interactions. It is also an endothelial cell-surface enzyme (amine oxidase) that produces reactive oxygen species during the catalytic reaction. To study the role of the enzymatic activity of VAP-1 in PMN extravasation, we used an enzymatically inactive VAP-1 mutant, specific amine oxidase inhibitors (including a novel small molecule compound), and anti-VAP-1 antibodies in several flow-dependent models. The enzyme inhibitors diminished PMN rolling on and transmigration through human endothelial cells under conditions of laminar shear stress in vitro. Notably, the enzyme inactivating point mutation abolished the capacity of VAP-1 to mediate transmigration. Moreover, the new VAP-1 inhibitor effectively prevented the extravasation of PMNs in an animal model of inflammation. These data show that the oxidase activity of VAP-1 controls PMN exit from the blood during the relatively poorly understood transmigration step. (Blood. 2004;103:3388-3395)

Polymorphonuclear leukocytes (PMNs) migrate from the blood into areas of inflammation by binding to the endothelial cells of blood vessels via adhesion molecules. Vascular adhesion protein-1 (VAP-1) is one of the molecules mediating leukocyte-endothelial cell interactions. It is also an endothelial cell-surface enzyme (amine oxidase) that produces reactive oxygen species during the catalytic reaction. To study the role of the enzymatic activity of VAP-1 in PMN extravasation, we used an enzymatically inactive VAP-1 mutant, specific amine oxidase inhibitors (including a novel small molecule compound), and anti-VAP-1 antibodies in several flow-dependent models. The enzyme inhibitors diminished PMN rolling on and transmigration through human endothelial cells under conditions of laminar shear stress in vitro. Notably, the enzyme inactivating point mutation abolished the capacity of VAP-1 to mediate transmigration. Moreover, the new VAP-1 inhibitor effectively prevented the extravasation of PMNs in an animal model of inflammation. These data show that the oxidase activity of VAP-1 controls PMN exit from the blood during the relatively poorly understood transmigration step. (Blood. 2004;103:3388-3395)

Background: T-cell large granular lymphocytic leukemia (T-LGLL), a rare lymphoproliferative disorder of mature T cells, is characterized by the accumulation of activated effector T cells leading to a clonally restricted T-cell receptor (TCR) repertoire. Chronic antigen stimulation together with activating somatic STAT3mutations have been proposed to lead to clonal expansion of leukemic cells. However, no holistic research has been done to show how leukemic and non-leukemic cells liaise to sustain abnormal immune reactivity in T-LGLL.

Loughran: Kymera Therapeutics: Membership on an entity's Board of Directors or advisory committees; Bioniz Therapeutics: Membership on an entity's Board of Directors or advisory committees; Keystone Nano: Membership on an entity's Board of Directors or advisory committees; Dren Bio: Membership on an entity's Board of Directors or advisory committees. Maciejewski: Alexion: Consultancy; Novartis: Consultancy; Regeneron: Consultancy; Bristol Myers Squibb/Celgene: Consultancy. Mustjoki: Novartis: Research Funding; BMS: Research Funding; Janpix: Research Funding; Pfizer: Research Funding.

In a single institution, we have used recombinant interferon-? (IFN-?) to treat 116 newly diagnosed Philadelphia-positive (Ph+) chronic myeloid leukemia (CML) patients and analyzed the predictive factors for response and survival. The patients whose median age was 50.3 years (range, 9 to 70) were administered IFN-? (5 million units/m2/d) subcutaneously. The IFN-? dose was subsequently adjusted to maintain the white blood cell and platelet counts between 1.5 and 5 × 109/L, 50 and 100 × 109/L, respectively. At diagnosis, the Sokal score was used to classify the patients into three groups: low (n = 57), intermediate (n = 42), and high risk (n = 16). A complete hematological response (CHR) was achieved in 93 cases (80.2%). Of the 116 patients, 113 were available for cytogenetic evaluation. Fifty patients (43%) achieved a major cytogenetic response (MCR) (=65% marrow Ph- cells), 37 of them having a complete cytogenetic response (CCR). The estimated 5-year survival of the 116 patients was 68% ± 11% (95% confidence interval [CI]) with a median follow-up of 42 months (range, 3 to 114) and 85% ± 11% (95% CI) with a median follow-up of 30.9 (range, 3 to 111) when patients were censored at the time of transplantation. Event-free survival at 5 years (adding death and transplant as event) was 46% ± 11% (95% CI). Using proportional hazards regression to study time-dependent variables, we confirmed that the most significant factor associated with survival was the cytogenetic response (MCR or CCR) (P < .0001). This factor was independent compared with the Sokal score and baseline variables used to calculate the Sokal score. Moreover, using either univariate or multivariate analysis, the achievement of CHR within 3 months was strongly correlated with MCR (P < .0001). Minimum cytogenetic response (mCR, ie, at least 5% of Ph- metaphases) at 3 months was also a significant predictive factor for MCR (P < .0001). These results show that IFN-? can induce a high rate of hematological and cytogenetic response when administered in doses leading to myelosuppression. Factors such as the achievement of CHR and mCR within 3 months could be useful to identify early those patients who will not respond to IFN-? and who need alternative treatments such as stem cell transplantation.

The beta 7-integrin subunit can pair with two alpha-chains, alpha 4 and alpha E, and is expressed mainly on lymphocytes. As an alpha 4- heterodimer it binds to the mucosal addressin MAdCAM-1, thus acting as a mucosal homing receptor. As an alpha E-heterodimer it binds to E- cadherin and is mainly found on intestinal intraepithelial lymphocytes. Consequently, beta 7 is mostly expressed on lymphocytes of the mucosal immune system. To study the compartmentalization of these cells further we compared the distribution of such lymphocytes in two strains of mice (BALB/c and NOD) and found that the distribution of beta 7-positive lymphocytes among various lymphoid tissues in these strains was very different. In NOD mice a conspicuous population of beta 7- integrin(high) lymphocytes expressing either alpha 4, alpha E, or both, was found in nonmucosal lymphoid tissues such as peripheral lymph nodes (PLNs). They mostly expressed the PLN homing receptor L-selectin, and included both naive and memory cells on the basis of their expression of CD44/pgp-1 and CD45RB, as did the few beta 7(high) lymphocytes in BALB/c PLNs. Their homing to Peyer's patches (PPs) and PLNs was equally effective and the cells homing to PPs and PLNs were equal in their level of L-selectin and alpha 4/beta 7 expression. However, functional studies indicated that their homing to PPs mostly depended on alpha 4/beta 7-integrin, whereas they mainly used L-selectin to home to PLNs. beta 7(high) lymphocytes were found also in circulating blood of unmanipulated NOD mice, and their L-selectin expression was higher than in BALB/c mice. These results show that lymphocytes of the mucosal immune system may also express the peripheral node homing receptor L- selectin during their recirculation and that in NOD mice they frequently retain a dual homing specificity, which leads to their accumulation in nonmucosal tissues.

The myc proto-oncogenes encode nuclear phosphoproteins, which are believed to participate in the control of cell proliferation and differentiation. Deregulated expression of c-myc has been implicated in several human hematopoietic malignancies. We have studied the expression and mRNA processing of human L-myc, N-myc, and c-myc genes in a panel of human leukemias, leukemia cell lines, and normal hematopoietic cells. L-myc mRNA was expressed in three acute myeloid leukemias (AML) studied and in several myeloid leukemia cell lines. Only low expression levels were observed in adult bone marrow and in fetal spleen and thymus. The K562 and Dami leukemia cell lines showed a unique pattern of L-myc mRNA processing, with approximately 40% of L- myc mRNA lacking exon III and intron I. N-myc was expressed in five of six AML cases studied, in one of nine acute lymphocytic leukemia (ALL) cases, and in several leukemia cell lines, while c-myc mRNA was detected in all leukemias and leukemia cell lines studied. Coexpression of all three myc genes was observed in Dami and MOLT-4 cell lines and in two AMLs, and either L-myc or N-myc was coexpressed with c-myc in several other cases. These results show that in addition to c-myc, the L-myc and N-myc genes are expressed in some human leukemias and leukemia cell lines, and suggest a lack of mutually exclusive cross- regulation of the myc genes in human leukemia cells.

The mycproto-oncogenes encode nuclear phosphoproteins, which are believed to participate in the control of cell proliferation and differentiation. Deregulated expression of c-mychas been implicated in several human hematopoietic malignancies. We have studied the expression and mRNA processing of human L-myc, N-myc, and c-mycgenes in a panel of human leukemias, leukemia cell lines, and normal hematopoietic cells. L-mycmRNA was expressed in three acute myeloid leukemias (AML) studied and in several myeloid leukemia cell lines. Only low expression levels were observed in adult bone marrow and in fetal spleen and thymus. The K562 and Dami leukemia cell lines showed a unique pattern of L-mycmRNA processing, with approximately 40% of L-mycmRNA lacking exon III and intron I. N-mycwas expressed in five of six AML cases studied, in one of nine acute lymphocytic leukemia (ALL) cases, and in several leukemia cell lines, while c-mycmRNA was detected in all leukemias and leukemia cell lines studied. Coexpression of all three mycgenes was observed in Dami and MOLT-4 cell lines and in two AMLs, and either L-mycor N-mycwas coexpressed with c-mycin several other cases. These results show that in addition to c-myc, the L-mycand N-mycgenes are expressed in some human leukemias and leukemia cell lines, and suggest a lack of mutually exclusive cross-regulation of the mycgenes in human leukemia cells.

Congenital factor XIII (FXIII) deficiency is potentially a severe bleeding disorder, but in some cases, the symptoms may be fairly mild. In this study, we have characterized the molecular mechanism of a mild phenotype of FXIII A-subunit deficiency in a Finnish family with two affected sisters, one of whom has even had two successful pregnancies without regular substitution therapy. In the screening tests for FXIII deficiency, no A-subunit could be detected, but by using more sensitive assays, a minute amount of functional A-subunit was seen. 3H-putrescine incorporation assay showed distinct FXIII activity at the level of 0.35% of controls, and also the fibrin cross-linking pattern in the patients clotted plasma showed partial γ-γ dimerization. In Western blot analysis, a faint band of full-length FXIII A-subunit was detected in the patients' platelets. The patients have previously been identified as heterozygotes for the Arg661 → Stop mutation. Here we report a T → C transition at position +6 of intron C in their other allele. The transition affected splicing of FXIII mRNA resulting in low steady state levels of several variant mRNA transcripts. One transcript contained sequences of intron C, whereas two transcripts resulted from skipping of one or two exons. Additionally, correctly spliced mRNA lacking the Arg661 → Stop mutation of the maternal allele could be detected. These results demonstrate that a mutation in splice donor site of intron C can result in several variant mRNA transcripts and even permit partial correct splicing of FXIII mRNA. Further, even the minute amount of correctly processed mRNA is sufficient for producing protein capable of γ-γ dimerization of fibrin. This is a rare example of an inherited functional human disorder in which a mutation affecting splicing still permits some correct splicing to occur and this has a beneficial effect to the phenotype of the patients.

CD44, a cell surface glycoprotein, is involved in lymphocyte trafficking from the blood to lymphatic tissues, and is of importance in dissemination of lymphoma. A variant form of CD44 that has additional amino acids in the common protein backbone (CD44v6) also seems to play a role in the metastatic dissemination of malignancies. We measured serum CD44 and CD44v6 in 34 patients with lymphoma and in healthy controls by dot blot assay. Small amounts of both CD44 (range, 10 to 80 ng/mL) and CD44v6 could be detected in sera of all controls. Serum CD44 was elevated in all patients with lymphoma before treatment (range, 70 to > 2,000 ng/mL, P < .0001), and CD44v6 was also slightly elevated. Serum CD44 levels correlated with response to treatment. Patients with complete response achieved similar CD44 levels as the controls, whereas those with progressive disease had increased serum CD44 levels. We conclude that both the standard and the variant form of CD44 are detectable in sera of healthy individuals, and that serum CD44 may be useful in monitoring treatment response in patients with lymphoma.

Hillmen: Roche: Research Funding; Gilead: Research Funding; Apellis: Research Funding; Acerta: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Expenses, Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Expenses, Research Funding. Brown:AbbVie: Consultancy; Acerta Pharma: Consultancy; AstraZeneca: Consultancy; BeiGene: Consultancy; Catapult Therapeutics: Consultancy; Dynamo Therapeutics: Consultancy; Genentech/Roche: Consultancy; Gilead: Consultancy, Research Funding; Juno/Celgene: Consultancy; Kite, a Gilead Company: Consultancy, Research Funding; Loxo: Consultancy, Research Funding; Novartis: Consultancy; Pfizer: Consultancy; Pharmacyclics: Consultancy; Sunesis: Consultancy; TG Therapeutics: Consultancy; Verastem: Consultancy, Research Funding; Sun Pharmaceuticals: Research Funding; Janssen: Honoraria; Teva: Honoraria; Morphosys: Other: Data safety monitoring board; Invectys: Other: Data safety monitoring board; Octapharma: Consultancy. Byrd:Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; BeiGene: Research Funding; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; BeiGene: Research Funding; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding; Novartis: Other: Travel Expenses, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; Ohio State University: Patents & Royalties: OSU-2S; Ohio State University: Patents & Royalties: OSU-2S. Eichhorst:BeiGene: Research Funding; Gilead Sciences, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; ArQule: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Lamanna:Celgene: Consultancy; Oncternal: Research Funding; TG Therapeutics: Research Funding; Ming: Research Funding; Infinity/ Verastem: Research Funding. O'Brien:GlaxoSmithKline: Consultancy; Gilead: Consultancy, Research Funding; AbbVie: Consultancy, Honoraria; Acerta: Research Funding; Alexion: Consultancy; Regeneron: Research Funding; Eisai: Consultancy; Celgene: Consultancy; Aptose Biosciences, Inc: Consultancy; TG Therapeutics: Consultancy, Research Funding; Amgen: Consultancy; Kite: Research Funding; Janssen: Consultancy, Honoraria; Pharmacyclics LLC, an AbbVie Company: Consultancy, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Verastem: Consultancy; Sunesis: Consultancy, Research Funding; Astellas: Consultancy; Vaniam Group LLC: Consultancy. Salmi:BeiGene: Employment. Hilger:BeiGene: Employment, Equity Ownership. Huang:BeiGene: Employment, Equity Ownership. Tam:AbbVie: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Pharmacyclics LLC, an AbbVie company: Honoraria; Novartis: Honoraria; BeiGene: Honoraria; Roche: Honoraria.Zanubrutinib is an investigational agent and has not yet been approved in the US

Background:Bruton tyrosine kinase (BTK) plays a critical role in B-cell receptor signaling, which mediates B-cell proliferation, migration, and adhesion. Inhibition of BTK has emerged as a strategy for targeting B-cell malignancies including CLL/SLL. Zanubrutinib (BGB-3111) is an investigational, next-generation BTK inhibitor that was designed to maximize BTK occupancy and minimize off-target inhibition of TEC- and EGFR-family kinases. Increased specificity may minimize toxicities reported with ibrutinib potentially due to off-target inhibition such as diarrhea, thrombocytopenia, bleeding, atrial fibrillation, rash, and fatigue (Coutre et al. Blood Advances2019). In non-clinical studies, zanubrutinib has been shown to be highly potent, selective, bioavailable, and irreversible, with potentially advantageous pharmacokinetic (PK) and pharmacodynamic properties. Complete and sustained BTK occupancy has been observed with zanubrutinib treatment in both peripheral blood mononuclear cells and in lymph nodes (Tam et al. Blood2019). Based on drug-drug interaction studies and population PK analyses (internal data), zanubrutinib may also be co-administered with strong or moderate CYP3A inhibitors at a reduced dose, proton pump inhibitors, vitamin K antagonists, as well as direct oral anticoagulants. Zanubrutinib does not prolong the QT interval. Pooled clinical data from 6 zanubrutinib monotherapy trials including 682 patients (pts) with either non-Hodgkin lymphoma, Waldenström macroglobulinemia, or CLL/SLL suggests that zanubrutinib has been generally well tolerated amongst pts with B-cell malignancies (Tam et al. EHA 2019). This data further showed that some toxicities often associated with BTK inhibitors were infrequent with zanubrutinib, including 1.9% atrial fibrillation/flutter (0.6% grade ≥3), 2.5% major hemorrhage (2.1% grade ≥3), 10.9% fatigue (0.7% grade ≥3), 18.0% rash (0.1% grade ≥3), 18.3% thrombocytopenia (6.6% grade ≥3), and 19.4% diarrhea (0.9% grade ≥3). Early clinical data in pts with treatment-naïve (TN; n=22) or relapsed/refractory (R/R; n=56) CLL/SLL showed that zanubrutinib was highly active: 96.2% overall response rate (ORR), including 4.5% and 1.8% with complete response in TN and R/R CLL/SLL, respectively (Tam et al. Blood2019).

No relevant conflicts of interest to declare.

Introduction

Background: The treatment of Acute Myeloid Leukemia (AML) in elderly patients remains unsatisfactory, with an expected survival time of about 1 year post diagnosis. In an attempt to improve outcome for these patients, the prospective open-label phase 3 LAMSA-2007 trial (Clinicaltrial.gov ID, NCT00590837) repeated, at decreasing doses in consolidation and reinduction courses, a standard induction regimen with cytarabine and idarubicin (IC), with or without the randomized addition of lomustine (ICL). This alkylating agent with significant anti-leukemic activity is widely used in France for AML therapy. This study was performed as a confirmatory trial, following our previous report of the French experience in which this compound stood out as a favorable factor of improved outcome for patients with non-unfavorable cytogenetics (Pigneux, JCO 2010).

Vey: Janssen: Honoraria; Roche: Honoraria; Celgene: Honoraria.

No relevant conflicts of interest to declare.

The histone methyl-transferase EZH2 is frequently affected by gain-of-function mutations in germinal-center lymphoma. To further test if these EZH2 mutations can be driver mutations in lymphomagenesis, we have previously generated and characterized a mouse line transgenic for EZH2Y641F. This mouse model leads to an acceleration of lymphoma development in combination with Myc. We have now further investigated this model system and have used RNAseq and ChIPseq to characterize the effects of mutated EZH2 on gene expression and the epigenome.