Search

Lymphatic dysfunctions are associated with several human diseases, including lymphedema and metastatic spread of cancer. Although it is well recognized that lymphatic capillaries attach directly to interstitial matrix mainly composed of fibrillar type I collagen, the interactions occurring between lymphatics and their surrounding matrix have been overlooked. In this study, we demonstrate how matrix metalloproteinase (MMP)–2 drives lymphatic morphogenesis through Mmp2-gene ablation in mice, mmp2 knockdown in zebrafish and in 3D-culture systems, and through MMP2 inhibition. In all models used in vivo (3 murine models and thoracic duct development in zebrafish) and in vitro (lymphatic ring and spheroid assays), MMP2 blockage or down-regulation leads to reduced lymphangiogenesis or altered vessel branching. Our data show that lymphatic endothelial cell (LEC) migration through collagen fibers is affected by physical matrix constraints (matrix composition, density, and cross-linking). Transmission electron microscopy and confocal reflection microscopy using DQ-collagen highlight the contribution of MMP2 to mesenchymal-like migration of LECs associated with collagen fiber remodeling. Our findings provide new mechanistic insight into how LECs negotiate an interstitial type I collagen barrier and reveal an unexpected MMP2-driven collagenolytic pathway for lymphatic vessel formation and morphogenesis.

Lymphatic dysfunctions are associated with several human diseases, including lymphedema and metastatic spread of cancer. Although it is well recognized that lymphatic capillaries attach directly to interstitial matrix mainly composed of fibrillar type I collagen, the interactions occurring between lymphatics and their surrounding matrix have been overlooked. In this study, we demonstrate how matrix metalloproteinase (MMP)–2 drives lymphatic morphogenesis through Mmp2-gene ablation in mice, mmp2knockdown in zebrafish and in 3D-culture systems, and through MMP2 inhibition. In all models used in vivo (3 murine models and thoracic duct development in zebrafish) and in vitro (lymphatic ring and spheroid assays), MMP2 blockage or down-regulation leads to reduced lymphangiogenesis or altered vessel branching. Our data show that lymphatic endothelial cell (LEC) migration through collagen fibers is affected by physical matrix constraints (matrix composition, density, and cross-linking). Transmission electron microscopy and confocal reflection microscopy using DQ-collagen highlight the contribution of MMP2 to mesenchymal-like migration of LECs associated with collagen fiber remodeling. Our findings provide new mechanistic insight into how LECs negotiate an interstitial type I collagen barrier and reveal an unexpected MMP2-driven collagenolytic pathway for lymphatic vessel formation and morphogenesis.

The objective of these studies was to characterize the macrophage mannose receptor binding and pharmacological properties of carbohydrate remodeled human placental-derived and recombinant β-glucocerebrosidase (pGCR and rGCR, respectively). These are similar but not identical molecules that were developed as enzyme replacement therapies for Gaucher disease. Both undergo oligosaccharide remodeling during purification to expose terminal mannose sugar residues. Competitive binding data indicated carbohydrate remodeling improved targeting to mannose receptors over native enzyme by two orders of magnitude. Mannose receptor dissociation constants (Kd) for pGCR and rGCR were each 13 nmol/L. At 37°C, 95% of the total macrophage binding was mannose receptor specific. In vivo, pGCR and rGCR were cleared from circulation by a saturable pathway. The serum half-life (t1/2) was 3 minutes when less than saturable amounts were injected intravenously (IV) into mice. Twenty minutes postdose, β-glucocerebrosidase activity increased over endogenous levels in all tissues examined. Fifty percent of the injected activity was recovered. Ninety-five percent of recovered activity was in the liver. Parenchymal cells (PC), Kupffer cells (KC), and liver endothelium cells (LEC) were responsible for 75%, 22%, and 3%, respectively, of the hepatocellular uptake of rGCR and for 76%, 11%, and 12%, respectively, of the hepatocellular uptake of pGCR. Both molecules had poor stability in LEC and relatively long terminal half-lives in PC (t1/2= 2 days) and KC (t1/2= 3 days).

The objective of these studies was to characterize the macrophage mannose receptor binding and pharmacological properties of carbohydrate remodeled human placental-derived and recombinant ß-glucocerebrosidase (pGCR and rGCR, respectively). These are similar but not identical molecules that were developed as enzyme replacement therapies for Gaucher disease. Both undergo oligosaccharide remodeling during purification to expose terminal mannose sugar residues. Competitive binding data indicated carbohydrate remodeling improved targeting to mannose receptors over native enzyme by two orders of magnitude. Mannose receptor dissociation constants (Kd) for pGCR and rGCR were each 13 nmol/L. At 37°C, 95% of the total macrophage binding was mannose receptor specific. In vivo, pGCR and rGCR were cleared from circulation by a saturable pathway. The serum half-life (t1/2) was 3 minutes when less than saturable amounts were injected intravenously (IV) into mice. Twenty minutes postdose, ß-glucocerebrosidase activity increased over endogenous levels in all tissues examined. Fifty percent of the injected activity was recovered. Ninety-five percent of recovered activity was in the liver. Parenchymal cells (PC), Kupffer cells (KC), and liver endothelium cells (LEC) were responsible for 75%, 22%, and 3%, respectively, of the hepatocellular uptake of rGCR and for 76%, 11%, and 12%, respectively, of the hepatocellular uptake of pGCR. Both molecules had poor stability in LEC and relatively long terminal half-lives in PC (t1/2 = 2 days) and KC (t1/2 = 3 days).

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

Purpose:

Background.Acute Myeloid Leukemia is a heterogeneous disease, characterized by the uncontrolled proliferation of hematopoietic precursor cells. AML accounts for 15% of acute leukemias in children, with a cure rate of 55-60% as overall survival (Pession A Blood 2013). Complete remission (CR) is achieved in 80-85% of children with AML. Current protocol defines CR by morphological evaluation (blasts <5%) and cytofluorimetric analyses (blasts <0,1%), after two cycles of high-dose chemotherapy. Around 10-15% of children and 30% of adults do not achieve remission and are defined as Primary Induction Failure (PIF). The majority of these cases are addressed to other form of high-dose chemotherapy blocks, experimental therapies or directly to hematopoietic stem cell transplantation (HSCT) as salvage treatment, showing a very poor prognosis (Quarello P et al. British Journal of Haematology 2015). Strong efforts to generate biological data in order to define this subtype of AML are needed for early identification and for target therapy design.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

Background. Aberrations in the DNA repair pathway among children with acute lymphoblastic leukemia (ALL) are largely unknown. Knowledge of such mutations may improve the understanding of tumorigenesis and direct patient care. Germline aberrations of the DNA-repair machinery are crucial for generating chromosomal instability and occurrence of acute leukemia. In order to better understand this mechanism, we addressed our research to NBS1 (Nijmegen Breakage Syndrome) gene and Fanconi Anemia/BRCA1 and BRCA2 pathway, in the attempt to identify mutations and aberrant expression, predisposing and/or cooperating with the leukemogenic process, among biological subgroups of childhood ALL.

Background. Although the prognosis of acute lymphoblastic leukemia (ALL) has improved considerably in recent years, some cases still exhibit therapy-resistance and late relapse. CD9-CXCL12-CXCR4 pathway has been implicated in hematopoietic and leukemic stem cell homing, promoting cell adhesion and survival in bone marrow stromal niches and mediating cell dissemination to secondary lymphoid organs. We characterized the CD9-CXCL12-CXCR4 expression in specific biological subgroups of childhood ALL, comparing, in some cases, diagnosis vs relapse, in order to investigate the role of these genes, in the process of leukemia relapse and to preliminarily evaluate their impact as prognostic markers, even for those subtypes with well-established good outcome.

Background. Acute lymphoblastic leukemia (ALL) is the most common malignancy of childhood and a major cause of childhood cancer-related mortality. Although the cure rate now approaches 90%, certain pediatric ALL subgroups present subsequent relapse. For this reason, analyses of cell signaling pathways will help to identify new markers and/or targets for tailored therapy. PI3K/AKT/mTOR activation is frequently found in both B-ALL and T-ALL. Protein kinase Ck2 (CSNK2) activity in pediatric ALL was increased and its inhibition restored PTEN phosphatase activity with subsequent inactivation of AKT. Moreover, Ck2 may serve the activity of oncogenes such as BCR-ABLand c-MYC, control the activation of other critical signaling cascades (JAK-STAT), and sustain multiple cellular stress-elicited pathway such as the proteotoxic stress, unfolded protein and DNA-damage responses. Ck2 has also been shown to have an essential role in tuning signals derived from the stromal tumor microenvironment (Piazza F et al, Oncogene2016).

Belcher: CSL Behring: Research Funding; Seattle Genetics: Research Funding; Biogen Idec: Research Funding. Vercellotti:Biogen Idec: Research Funding; CSL Behring: Research Funding; Cydan: Research Funding; Seattle Genetics: Research Funding.

Off Label Use: We used Imatinib Mesylate and PEG-L-Asparaginase in two children with AML: an off-label use for indication and age. .

BackgroundAML is an aggressive disease. Current pediatric protocols ensure a 60% survival rate (Pession A et al, Blood 2013), reaching a plateau of intensiveness. Nevertheless, patients with primary induction failure (PIF) (10%) or relapse (30%) after stem cell transplantation (SCT) still have a very poor outcome. Novel therapeutic strategies are needed.

Background: Sickle cell disease (SCD) is one of the world's most common inherited diseases, due to a point mutation in hemoglobin that leads to the polymerization of hemoglobin S, giving red blood cells (RBCs) their sickle shape. The hallmarks of SCD are hemolysis and vaso-occlusion. Vaso-occlusive crises (VOC) frequently occur in response to inflammatory and hypoxic conditions such as infections, surgery, etc. Pain is a major defining characteristic of SCD starting early in life and continuing throughout adulthood. VOC-induced pain can be chronic or episodic and unpredictable, requiring frequent hospitalizations and analgesics. Opioids, predominantly morphine, have been the mainstay treatment for pain management of SCD but pose a serious challenge attributed to the many adverse side effects, including tolerance, respiratory depression, sedation, nausea, constipation, pruritus, and dependence. Given that analgesics characteristically used to treat pain appear to be relatively ineffective in alleviating pain associated with SCD as well as display their own dose-limiting adverse effects, there exists a requisite need for superior pharmacological agents in its treatment. Here we reveal that the bivalent ligand MCC22, is highly effective as an analgesic in a murine model of SCD. MCC22 (MW=1255) contains both mu agonist and chemokine CCR5 antagonist pharmacophores that are linked through a 22-atom-spacer. It was designed specifically to target the mu opiate receptor (MOR)-CCR5 heteromer, as there is evidence for crosstalk between MOR and CCR5 in cultured cells that reduces the efficacy of opioid analgesics employed in SCD. Intrathecal (i.t.) MCC22 was potent in reducing the mechanical and heat hyperalgesia in Complete Freund's Adjuvant (CFA) and lipopolysaccharide (LPS) inflammatory pain assays. ED50s for the i.t. administration were 0.019 and 0.015 pmol/mouse, respectively. MCC22 when given intraperitoneally (i.p.) to LPS pretreated mice had an ED50of 5.6 µmol/kg. We next investigated whether MCC22 would be effective in alleviating pain in SCD mice.

All newly diagnosed lymphoma patients seen at our institution from 2005 – 2009 were evaluated for protocol eligibility. Exclusion criteria included bone fractures, BMD T-scores worse than –2.0, creatinine clearance less than 60 mL/min, dental problems, and recent steroid or bisphosphonate use. Patients on study were stratified as male, pre-menopausal female, or post-menopausal female. Accrued patients were randomized to receive either: 1) oral calcium and vitamin D (Ca+D) or 2) Ca+D and 4 mg zoledronic acid IV at baseline and at 6 months. Urine NTx and bone specific AP levels were measured at baseline, 3, 6, 9, and 12 months.To date, 33 patients have completed the study and have evaluable data. Patient characteristics include: 17 male, 4 pre-menopausal female, 12 post-menopausal female, median age 62 (range 33 – 80). Seventeen patients had osteopenia upon enrollment. Patients that received zoledronic acid had stable median T-scores at all locations during the 12 month observation, whereas the T-scores of the control group decreased at every location evaluated (Location: L1-4, p=0.004; L Neck, p= 0.001; L Hip, p=0.118; R Neck, p=0.009; R Hip, p=0.040). Each group had a similar baseline level of urine NTx and bone specific AP. At 6 months, the urine NTx decreased significantly in the zoledronic acid-treated group compared to urine NTx levels in the control group which increased (p<0.001). The separation between groups remained constant throughout the evaluation period (12 months, p<0.001). The bone specific AP level decreased in the zoledronic acid group by 3 months, and remained below baseline levels at 12 months. The control group had increases in bone specific AP levels throughout the 12 month period (6 months, p=0.001; 12 months, p<0.001).Treatment with zoledronic acid in newly-diagnosed lymphoma patients prevents the bone mineral density loss commonly seen in this population. Bone mass lost is difficult to restore, thus necessitating effective prevention strategies. Urine NTx and bone specific AP levels demonstrate early response to bisphosphonate therapy. The ability to predict response to bisphosphonates at 6 months may allow for early intervention, potentially preventing further bone loss. Our study demonstrates the bone markers urine NTx and bone specific AP are significantly associated with bone health in lymphoma patients receiving chemotherapy.Thompson: Novartis: Research Funding. Toth:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Hagemeister:Novartis: Research Funding.

Abstract 3922

Treatment of lymphoma with alkylating agents and steroids is known to cause bone loss and increased fracture risk. These effects result from multiple causes including systemic lymphoma-related cytokine activity, steroid-mediated bone loss, and therapy-induced hypogonadism. Furthermore, over half of all untreated lymphoma patients are either osteopenic or osteoporotic at the time of diagnosis.1,2 Pamidronate is known to reduce bone loss and risk of vertebral fractures in lymphoma patients undergoing chemotherapy. However, the effects of the more potent bisphosphonate zoledronic acid in this setting are unknown. Therefore, we report on an ongoing phase III trial designed to evaluate the effect of zoledronic acid on the change in bone mineral density (BMD) in patients with newly-diagnosed lymphoma undergoing chemotherapy. In total, 72 patients will be randomized to either the control arm consisting of therapy with calcium carbonate (1200 mg orally per day) plus vitamin D (400 mg orally per day), or the bisphosphonate arm consisting of therapy with calcium carbonate and vitamin D as in the control arm plus zoledronic acid (4 mg IV given at baseline and at 6 months). The primary endpoint of the study is to compare the absolute change between the baseline and 12-month measures of BMD at the lumbar spine and femoral necks. Thus far, 97 patients have been screened for enrollment. Twenty-four patients (24.7%) failed screening due to periodontal disease, a predetermined exclusion criterion of the study. Twenty-three patients have been randomized to the control arm, and 18 patients have been randomized to the bisphosphonate arm. To date, 11 patients in the control arm and 7 patients in the bisphosphonate arm have completed the one-year follow up period including baseline and one-year BMD evaluations. In comparing the change in BMD at one year of patients in the control arm to that of patients in the bisphosphonate arm, the average change in T scores at the lumber spine was −0.482 vs. 0, at the left femoral neck was −0.218 vs. 0.057, and at the right femoral neck was −0.309 vs. 0.243, respectively. There have been no therapy-related serious adverse events in either arm of the study. In addition, no occurrences of osteonecrosis of the jaw have been noted. In conclusion, treatment with the bisphosphonate zoledronic acid in combination with calcium carbonate and vitamin D supplementation appears to provide prevention of bone loss or improvement in the BMD of patients with lymphoma undergoing chemotherapy. Given the large number of patients with below-average BMD prior to therapy and the known deleterious effects of lymphoma therapy on bone density, baseline BMD evaluation may be warranted in all lymphoma patients. In addition, the unexpectedly high rate of periodontal disease in this patient population further supports the need for careful dental evaluation prior to the consideration of bisphosphonate therapy given the well-described, albeit rare, risk of osteonecrosis of the jaw from zoledronic acid.

Treatment of lymphoma with alkylating agents and steroids is known to cause bone loss and increased fracture risk. These effects result from multiple causes including systemic lymphoma-related cytokine activity, steroid-mediated bone loss, and therapy-induced hypogonadism. Furthermore, over half of all untreated lymphoma patients are either osteopenic or osteoporotic at the time of diagnosis.1,2Pamidronate is known to reduce bone loss and risk of vertebral fractures in lymphoma patients undergoing chemotherapy. However, the effects of the more potent bisphosphonate zoledronic acid in this setting are unknown. Therefore, we report on an ongoing phase III trial designed to evaluate the effect of zoledronic acid on the change in bone mineral density (BMD) in patients with newly-diagnosed lymphoma undergoing chemotherapy. In total, 72 patients will be randomized to either the control arm consisting of therapy with calcium carbonate (1200 mg orally per day) plus vitamin D (400 mg orally per day), or the bisphosphonate arm consisting of therapy with calcium carbonate and vitamin D as in the control arm plus zoledronic acid (4 mg IV given at baseline and at 6 months). The primary endpoint of the study is to compare the absolute change between the baseline and 12-month measures of BMD at the lumbar spine and femoral necks. Thus far, 97 patients have been screened for enrollment. Twenty-four patients (24.7%) failed screening due to periodontal disease, a predetermined exclusion criterion of the study. Twenty-three patients have been randomized to the control arm, and 18 patients have been randomized to the bisphosphonate arm. To date, 11 patients in the control arm and 7 patients in the bisphosphonate arm have completed the one-year follow up period including baseline and one-year BMD evaluations. In comparing the change in BMD at one year of patients in the control arm to that of patients in the bisphosphonate arm, the average change in T scores at the lumber spine was −0.482 vs. 0, at the left femoral neck was −0.218 vs. 0.057, and at the right femoral neck was −0.309 vs. 0.243, respectively. There have been no therapy-related serious adverse events in either arm of the study. In addition, no occurrences of osteonecrosis of the jaw have been noted. In conclusion, treatment with the bisphosphonate zoledronic acid in combination with calcium carbonate and vitamin D supplementation appears to provide prevention of bone loss or improvement in the BMD of patients with lymphoma undergoing chemotherapy. Given the large number of patients with below-average BMD prior to therapy and the known deleterious effects of lymphoma therapy on bone density, baseline BMD evaluation may be warranted in all lymphoma patients. In addition, the unexpectedly high rate of periodontal disease in this patient population further supports the need for careful dental evaluation prior to the consideration of bisphosphonate therapy given the well-described, albeit rare, risk of osteonecrosis of the jaw from zoledronic acid.

Background: Myelomonocytic precursors from acute or chronic leukemias can differentiate to dendritic cells in vitro, but leukemias with a dendritic cell immunophenotype are rare, have been reported mainly in adults, and their molecular pathogenesis is unknown. Dendritic cells are classified as Langherans, myeloid and lymphoid/plasmacytoid cells, but leukemias arising from dendritic cells are unclassified in the FAB system. We identified a new entity of pediatric acute myeloid leukemia (AML) presenting with morphologic and immunophenotypic features of mature dendritic cells, which is characterized by MLL gene translocation. Methods and Results: Standard methods were used to characterize the morphology, immunophenotype, karyotype and MLL translocations in 3 cases of pediatric AML. The patients included two boys and one girl diagnosed with AML between 1–6 years old. Their clinical histories and findings included fever, pallor, abdominal and joint pain, adenopathy, hepatosplenomegaly, normal WBC counts but anemia and thrombocytopenia. and no evidence of CNS disease. The bone marrow aspirates were hypocellular and replaced completely by large blasts with irregular nuclei, slightly basophilic cytoplasm, and prominent cytoplasmic projections. There were no cytoplasmatic granules or phagocytosis. Myeloperoxidase and alpha napthyl esterase reactions were negative, excluding FAB M5 AML, and the morphology was not consistent with any standard FAB morphologic diagnosis. The leukemic blasts in all three cases were CD83+, CD86+, CD116+, consistent with differentiated myeloid dendritic cells, and did not express CD34, CD56 or CD117. MLL translocations were identified in all 3 cases. In the first case FISH analysis showed t(10;11)(p12;q23) and RT-PCR identified and a ‘5-MLL-AF10-3’ fusion transcript. In the second case FISH analysis showed t(9;11)(p22;q23) and RT-PCR identified and a ‘5-MLL-AF9-3’ fusion transcript. In the remaining case, the MLL gene rearrangement was identified by Southern blot analysis and RT-PCR showed an MLL-AF9 fusion transcript. The fusion transcripts in all 3 cases were in-frame. Remission induction was achieved with intensive chemotherapy, and all three patients have remained in durable remission for 30–60 months after hematopoietic stem cell transplantation. Conclusions. We have characterized a new pediatric AML entity with features of mature dendritic cells, MLL translocation and an apparently favorable prognosis. The in-frame MLL fusion transcripts suggest that chimeric MLL oncoproteins underlie its pathogenesis. The partner genes in all 3 cases were known partner genes of MLL that encode transcription factors. This study increases the spectrum of leukemias with MLL translocations. Comprehensive morphological, immunophenotypic, cytogenetic and molecular analyses are critical for this diagnosis, and will reveal its frequency and spectrum as additional cases are uncovered.

Background: Myelomonocytic precursors from acute or chronic leukemias can differentiate to dendritic cells in vitro, but leukemias with a dendritic cell immunophenotype are rare, have been reported mainly in adults, and their molecular pathogenesis is unknown. Dendritic cells are classified as Langherans, myeloid and lymphoid/plasmacytoid cells, but leukemias arising from dendritic cells are unclassified in the FAB system. We identified a new entity of pediatric acute myeloid leukemia (AML) presenting with morphologic and immunophenotypic features of mature dendritic cells, which is characterized by MLLgene translocation.