bluuebunny/crossref_metadata_2025 huggingface dataset:
Overlapping but Distinct Role of p21WAF1/CDKN1 and Survivin in Hematopoietic Progenitor Cell Proliferation.
Authors: Fukuda Seiji , Abe Mariko , Yamaguchi Seiji , Pelus Louis M.
Published: April 2020

Survivin is a member of the inhibitor of apoptosis protein family that has been implicated in cell cycle control, anti-apoptosis and cell division. Our previous studies and others have shown that Survivin and the cyclin dependent kinase inhibitor p21WAF1/CDKN1 (p21) are functionally associated and are involved in cell cycle, anti-apoptosis and cytokinesis in cancer cells and in normal hematopoietic progenitor cells (HPC). P21 is highly expressed in quiescent hematopoietic stem cells (HSC) in steady state, but the proportion of quiescent HSCs in G0 phase is reduced in p21−/− mice. In contrast, p21 has been shown as positive regulator on cell cycle of normal HPC since p21 deficiency results in fewer total CFU in mouse bone marrow (BM) cells with fewer CFU in S-phase and retrovirus transduction of p21 in p21 deficient bone marrow cells restores total and cycling CFU. We have previously reported that Survivin increases the proliferation of mouse primary HPC and that this enhancing effect is on HPC proliferation is absent when p21 is functionally deleted, suggesting that p21 is required for Survivin to enhance HPC proliferation. In addition, ITD-Flt3 mutations that are normally expressed in patients with acute myeloid leukemia and associate poor prognosis increase expression of both Survivin and p21, implicating their involvement in aberrant proliferation of HPC expressing ITD-Flt3. Herein we have characterized the functional association between p21 and Survivin in normal and transformed cell proliferation. Antagonizing wild-type Survivin in mouse BaF3 cells by retrovirus transduction of a T34A dominant negative mutant Survivin or anti-sense increased p21 expression, even though Survivin requires p21 to enhance HPC proliferation. Ectopic p21 in Survivin+/+ primary mouse bone marrow cells increased the number of immunophenotypically defined c-kit+, lin− (KL) cells, which is consistent with a positive role of p21 in HPC proliferation, however; ectopic expression of p21 failed to increase HPC proliferation in Survivin deficient primary bone marrow cells, suggesting that p21 alone is not sufficient to substitute for Survivin’s enhancing function on normal HPC proliferation. Over-expression of ITD-Flt3 enhanced growth factor independent proliferation of primary mouse marrow c-kit+, Sca-1+, lin− (KSL) cell number; however, co-expression of p21 with ITD-Flt3 dramatically decreased the number of growth factor independent KSL cells (80±6% reduction: P<0.01). Furthermore, the inhibitory effect of p21 on KLS proliferation was further enhanced by Survivin knockout bone marrow cells (64±5% reduction compared with presence of Survivin: P<0.05). These findings indicate that Survivin and p21 have a overlapping but distinct roles in regulating normal HPC proliferation and that manipulating p21 and Survivin may represent a potential therapeutic target for acute leukemia cells expressing ITD-Flt3.
The Bone Morphogenetic Protein (BMP)-4 Is Involved in the Regulation of Human Megakaryocytic Differentiation during Thrombopoietin Signaling.
Authors: Nicolini Franck E , Jeanpierre Sandrine , Kaniewski Bastien , Dumontet Charles , Rimokh Ruth , Puisieux Alain , Maguer-Satta Véronique
Published: April 2020

It has been shown in the past that Activin A, BMP-2 and BMP-4, three members of the TGF-β family, are involved in the regulation of hematopoiesis and particularly erythropoiesis, in humans. In this study, we explored the role of these molecules in human megakaryopoiesis using an in vitro serum-free assay initiated with purified normal CD34+ human bone marrow (BM) cells (from allogeneic BM donors), that allows the analysis of the impact of such molecules on all stages of megakaryocytic differentiation. We could demonstrate for the first time, that in the absence of thrombopoietin (TPO), BMP-4 is able to induce CD34+ progenitor commitment and differentiation into megakaryocytes throughout all stages through means of cytology, flow cytometry, CFU and LTC-IC and ploidy assays, as well as in vitro platelet production. We analyzed as well the expression of megakaryocytic specific factors such as FOG-2, Fli-1 and PF4 by RQ-PCR, and PF4, BMP-4 secretion in culture supernatants. While we have previously shown that Activin A and BMP-2 are involved in the erythropoietic commitment even in the absence of erythropoietin, we were not able to demonstrate any effect of these molecules on megakaryopoietic commitment and differentiation. Using signaling pathways specific inhibitors such as AG490 (JAK-2 pathway inhibitor), PD98059 (ERK pathway inhibitor), LY294002 (PI3-K inhibitor) and Rapamycin (mTOR pathway inhibitor), we could show that BMP-4, as TPO, exerts its effects on human megakaryopoiesis involving specifically the JAK/STAT and mTOR signaling pathways. In addition, the specific inhibition of the BMP signaling pathway with blocking antibodies (CD34+ BM cells cultured in the presence of anti-TPO-R and mouse anti-BMP-4 Antibody), natural soluble inhibitors [such as FLRG (Follistatin related gene) protein or Follistatin], or soluble BMP-receptors (sBMPR-Ia, sBMPR-Ib) has revealed that TPO uses the BMP-4 pathway to induce the megakaryopoietic commitment of human BM CD34+ progenitors. Finally, we could demonstrate that TPO up-regulates a BMP-4 autocrine loop in megakaryocytic progenitors, by inducing their own production of BMP-4 associated to an up-regulation of BMP-receptor expression. In conclusion, this study illustrates that BMP-4 represents an important actor in the regulation of human megakaryopoiesis.
Transformation of E2A-Deficient Pluripotent Progenitors by BCR-ABL Generates Imatinib-Resistant Leukemic Stem Cells.
Authors: Minami Yosuke , Stuart Scott A. , Ikawa Tomokatsu , Abe Akihiro , Naoe Tomoki , Jamieson Catriona H.M. , Wang Jean Y.J.
Published: April 2020

Chronic myeloid leukemia (CML) is effectively treated with imatinib (imatinib mesylate, IM), a small molecule inhibitor of the BCR-ABL tyrosine kinase that is expressed in the entire hematopoietic compartment including stem cells (HSC) and progenitors. However, it is still unclear whether IM-therapy is able to eradicate BCR-ABL-positive HSC and progenitors. By transforming murine E2A-deficient pluripotent hematopoietic cells (Ikawa, et al., Immunity 04) with p210BCR-ABL, we determined that as few as 50 granulocyte macrophage progenitors (GMP)-like cells were sufficient to induce a transplantable CML-like disease in congenic mice, and that the leukemogenic GMP displayed higher levels of β-catenin activity than either the non-transformed GMP or the transformed nonGMP (most of which were myeloid differentiated cells), both in culture and in transplanted mouse bone marrow (Proc Natl Acad Sci USA 08, in press). The initiation of transformation required BCR-ABL kinase activity; however, whereas transformed nonGMP were sensitive to IM-treatment, expansion and survival of the leukemogenic progenitors in ex vivo-culture or in mice were not completely inhibited by IM-treatment. The drug resistance did not correlate with higher levels of BCR-ABL, mutations at ABL-kinase domain, induction of quiescence, stromal support or drug efflux. Additionally, the differential responses between the transformed progenitors and differentiated cells to IM-treatment were not affected by the restoration of E2A-function. These results imply that leukemic progenitors possess innate resistance to IM and that eradication of these cells with other drugs is required to cure CML. We are also investigating BCR-ABL-positive residual disease in HSC and progenitors of chronic phase CML patients on IM-therapy.
Activation of AKT1 in Hematopoietic Stem Cells Causes a Myeloproliferative Disease in a Tet-Inducible Mouse Model.
Authors: Brandts Christian , Rode Miriam , Lindtner Beate , Koehler Gabriele , Koschmieder Steffen , Mueller-Tidow Carsten , Berdel Wolfgang E. , Serve Hubert
Published: April 2020

Activating mutations in Flt3, N- and K-Ras have been reported in all AML subtypes and represent common molecular defects in de novo AML. We have previously shown that these mutations lead to constitutive AKT phosphorylation and activation. As a consequence, Akt phosphorylation is found in myeloid blasts of the majority of AML patients. We reasoned that constitutively active AKT may contribute to leukemia development, and therefore we assessed the contribution of AKT in oncogenic transformation in vivo. For this purpose, we established an inducible mouse model expressing myristylated AKT1 under the control of the scl-3′ enhancer (MyrAKT1). This system restricts activated AKT1 to endothelium, hematopoietic stem cells and myeloid lineage cells at a low but detectable level. About 40% of induced mice developed a myeloproliferative disorder after latencies of 7 to 22 months. Onset of disease was frequently associated with hemangioma formation, due to endothelial MyrAKT1 expression. The myeloproliferative disorder was associated with splenomegaly with increased extramedullary hematopoiesis, while the peripheral blood contained mature granulocytes. Furthermore, the stem cell and progenitor cell compartment in spleens and bone marrow of these mice was altered compared to control mice. Colony formation assays with MyrAKT1-expressing bone marrow suggested that overactivation of AKT1 enhanced proliferation. The AKT1-induced disease was transplantable by both bone marrow and spleen cells. These findings highlight the oncogenic capacity of constitutively activated AKT1 in vivo and indicate that AKT is an attractive target for therapeutic intervention in AML.
HTLV-1 Infection of Human Hematopoietic Stem Cells Induces a Novel T Cell Lymphoma in Humanized SCID Mice
Authors: Banerjee Prabal , Crawford Lindsey , Sieburg Michelle , Green Patrick , Beilke Mark A , Harrington William J , Lairmore Michael D , Feuer Gerold
Published: April 2020

Human T-lymphotropic virus type-1 (HTLV-1) is a human retrovirus linked to cancer and is the etiologic agent of Adult T-cell leukemia/lymphoma (ATLL), an aggressive CD4+/CD25+ T cell malignancy. The early molecular events induced by HTLV-1 infection as well as the role of various viral genes in the induction of leukemia remain unclear, predominantly due to the lack of an animal model that recapitulates ATLL development. HTLV-1 infection of humanized NOD/SCID mice (HTLV-1- HU-SCID) was achieved by inoculation of NOD/SCID mice with CD34+ hematopoietic progenitor cells and stem cells (CD34+ HP/HSCs) infected ex vivo with HTLV-1. HTLV-1-HU-NOD/SCIDmice consistently developed CD4+CD25+ T cell lymphomas with clinical characteristics associated with ATLL and infected mice showed hyperproliferation of infected human stem cells (CD34+CD38−) in the bone marrow. Inoculation of NOD/SCID mice withCD34+ HP/HSCs transduced with a lentivirus vector (LV) expressing the HTLV-1oncoprotein (Tax1) also developed CD4+CD25+ lymphomas. The HTLV-1 bZIP protein(HBZ), encoded by the minus strand of the HTLV-1 genome, is expressed in all ATLL cells and has been implicated in the maintenance of leukemogenesis. HBZ has previously been previously shown to interact with numerous cellular factors and can modulate Tax1 activity in vitro. To establish the role of HBZ in HTLV-1 replication and leukemogenesis in vivo, HU-SCID mice were infected with an infectious proviral clone lacking functional HBZ (HTLV-1ΔHBZ). HTLV-1ΔHBZ-infected HU-SCID mice developed lymphoproliferations with an immature preleukemic CD4−CD8−CD90+ phenotype starting at ~10 weeks post-reconstitution. In contrast wild type HTLV-1 infection reproducibly induces a mature CD4+CD25+ CD90− lymphoma. Lymphoma cells successfully engrafted naïve NOD/SCID mice when injected into the peritoneal cavity and these cells maintain the expression of viral proteins, gp46env and p19gag. HTLV-1 infection of CD34+ HP/HSCs and the recapitulation of a lymphoma similar to ATLL in HU-NOD/SCID mice suggest that hematopoietic stem cells provide a relevant cellular target and viral reservoir in vivo and that infection of these cells contribute to viral lymphomagenesis in humans. The HTLV-1-HU-SCID mouse model presents a compelling in vivo model to characterize molecular initiation and progression of events in the generation of ATL and to establish the role of HTLV-1 auxiliary proteins in viral pathogenesis.
The GATA-1 Cofactor FOG-1 Recruits NuRD to Promote Normal Erythroid and Megakaryocyte Development and Maintain Lineage Fidelity by Restricting Mast Cell Gene Expression.
Authors: Gregory Gregory D , Wang Yuhuan , Hong Wei , Miccio Annarita , Bersenev Alexey , Yu Xiang , Wang Hongxin , Choi John , Shelat Suresh , Tong Wei , Poncz Mortimer , Blobel Gerd
Published: April 2020

Tissue-specific nuclear factors can establish gene expression patterns in one cell lineage and suppress that of another. GATA-1 and its cofactor FOG-1 (Zfpm1) regulate erythroid and megakaryocyte development by activating and repressing gene transcription. We previously showed that a conserved motif within the N-terminus of FOG-1 binds the Nucleosome Remodeling and Deacetylase (NuRD) co-repressor complex. Here we report that mice bearing FOG-1 point mutations that disrupt the NuRD interaction display mild anemia with splenomegaly and macrothrombocytopenia, a phenotype reminiscent of that observed in animals bearing germline mutations that disrupt the GATA-1/FOG-1 interaction. Microarray studies revealed relatively few changes in gene expression pattern sin mutant erythroid cells and megakaryocytes. Among the most prominent findings was a marked increase in the levels of Gata2, which is normally silenced in mature erythroid cells. Strikingly, mutant erythroid cells also displayed activation of several genes of the mast cell lineage where FOG-1 is normally extinguished. Furthermore, mutant megakaryocytes misexpressed the same set of mast cell genes, suggesting that NuRD binding by FOG-1 is required to suppress mast cell fate throughout the erythro-megakaryocytic ontogeny. In agreement, prospectively isolated megakaryocytic-erythroid progenitors (MEP) not only exhibited elevated Gata2 and mast cell gene expression, but maintained a multilineage capacity, generating both mast cells and other myeloid lineage cells in culture. Upregulation of mast cell-specific genes is likely the combined consequence of the failure of mutant FOG-1 to function as a repressor and the high levels of GATA-2. Together, these results underscore the importance of the FOG-1-NuRD interaction as an effector of GATA-1 activity. In particular, recruitment of NuRD to GATA-1/FOG-1 regulated genes is required to optimize erythroid and megakaryocytic maturation and restrict a mast cell program in those lineages. More generally, recruitment of NuRD by lineage-specific transcription factors may be a common mechanism to narrow and focus gene expression during tissue maturation.
Accelerated Repair of Vascular Injury in Diabetes by TGF-β1 Modified Hematopoietic Stem Cells (HSC).
Authors: Bartelmez Stephen , Ruscetti Francis W , Iversen Patrick , Bhatwadekar Ashay , Grant Maria
Published: April 2020

Previously, we demonstrated that a transient blockade of endogenous transforming growth factor-beta type 1 (TGF-β1) in murine and human HSC accelerates bone marrow engraftment while dramatically reducing the number of HSC needed for long-term reconstitution. CD34+ cells give rise to endothelial progenitor cells (EPC) and have been shown to participate in the repair of damaged vasculature. CXCR4 is the receptor for stromal derived factor (SDF-1), a chemoattractant released by ischemic tissue that guides EPCs to damaged sites. In this study, we examined levels of TGF- β1 mRNA in CD34+ blood and bone marrow (BM) cells of diabetic and non diabetic individuals. We also treated CD34+ cells of diabetic and nondiabetic origin with antisense phosphorodiamidate morpholino oligomers (PMOs) to TGF-β1 and examined surface expression of CXCR-4, migratory response to SDF-1 as well as in vivo reparative function in a model of retinal ischemic injury. Our results show that CD34+ EPC from the blood of diabetic patients are markedly defective in their ability to repair damaged retinal vessels compared to control cells in contrast to CD34+ cells from the diabetic BM. Diabetic CD34+EPC from blood express elevated levels of TGF-β1 mRNA and have a blunted migratory response to SDF-1 compared to controls (p<0.05 and p<0.01 respectively). Transient (2–4 days) blockade of endogenous TGF-β1 using PMOs to TGF- β1 in diabetic CD34+/EPC increases CXCR-4 expression in these cells, enhances their migratory prowess and restores their ability to repair damaged retinal vessels. This approach is an enhanced autologous stem cell therapy based on a well studied, rapid and reversible modification of bone marrow CD34+EPCs derived from the diabetic patient.
Bone Morphogenetic Protein 4 Regulates Hematopoietic Stem Cell Maintenance in Vivo.
Authors: Goldman Devorah C. , Bailey Alexis S. , Pfaffle Dana L. , Christian Jan L. , Fleming William H.
Published: April 2020

The secreted signaling molecule Bone Morphogenetic Protein 4 (BMP4) is expressed by osteoblasts and other cell types that comprise the hematopoietic microenvironment. Therefore, we hypothesized that BMP4 may play an important regulatory role in the maintenance and function of hematopoietic stem cells (HSCs). As the deletion of BMP4 is lethal during early embryogenesis, we exploited a viable BMP4 hypomorph in which a point mutation in BMP4 reduces, but does not abolish the amount of active BMP4 ligand. In these mutants, peripheral blood cell lineages and bone marrow cellularity are normal during steady state conditions. However, consistent with our hypothesis, 40% fewer c-kit+, Sca-1+, lineage- (KSL) cells were present in the femurs of mutants (7.4 x103 ± 0.4 x103 SEM) than in age-and sex-matched wild-type (WT) controls (12.4 x103 ± 1.3 x103, p<0.005). Transplantation of mutant KSL cells produced levels of hematopoietic engraftment indistinguishable from transplanted WT KSL cells, consistent with a stem cell extrinsic effect of BMP4. To functionally assess the mutant hematopoietic microenvironment, unfractionated WT bone marrow cells (BM) were transplanted into lethally irradiated mutants or WT controls. Although WT cells could engraft mutant primary recipients to the same degree as WT recipients, serial transplantation of these WT cells into secondary WT hosts revealed a marked depletion of hematopoietic reconstitution activity. Specifically, nearly a 4-fold reduction of donor cells was found in the peripheral blood of secondary recipients that received BM from reconstituted mutant hosts (p<0.0005). To determine whether such defects in the mutant microenvironment exist in the absence of myeloablative conditioning and transplantation, a parabiotic mouse model was employed. CD45.1 WT mice were joined to CD45.2 BMP4 hypomorphs for 8 weeks and then separated. As early as one month following separation, a greater than 2- fold reduction in circulating WT donor cells was detected in mutant hosts relative to the number of mutant donor cells detected in the WT hosts (p<0.005). Analysis of BM at 24 weeks following separation revealed a striking 23- fold reduction in WT donor hematopoietic cells in the mutant host BM compared to the number of mutant hematopoietic cells detected in WT host BM. Together, our findings reveal a novel, critical role for BMP4 in maintaining HSCs in vivo.
Urine Concentrating Ability in Infants with Sickle Cell Anemia: Baseline Data from the BABY HUG Trial.
Authors: Miller Scott T. , Wang Winfred C. , Iyer Rathi V. , Rana Sohail R , Lane Peter A. , Ware Russell E. , Rees Renee C ,   The BABY HUG Investigators
Published: April 2020

A urine concentrating defect is quite common in sickle cell anemia (SCA), has its onset in early childhood, and may be reversible (with transfusion) until age 10 years. BABY HUG is an NHLBI-NICHD sponsored double-blind, placebo-controlled Phase III Clinical Trial (NCT00006400) designed to assess efficacy of hydroxyurea in preventing organ damage in young children with SCA (Hb SS or SβO thalassemia); primary endpoints are spleen function and glomerular filtration rate (GFR). Two hundred thirty-three infants, recruited without regard to disease severity, underwent eligibility screening. To assess urine concentrating ability as a secondary endpoint, parents were instructed to collect and save timed urine specimens from subjects after 4 to 10 hours of fluid deprivation (NPO) overnight for osmolality (OSM) determination. More prolonged deprivation was avoided due to safety concerns. A paired serum sample for OSM, urea nitrogen (BUN), and creatinine was obtained the next morning. All specimens were analyzed in a central laboratory. The analyses included 184 infants with a urine specimen and a reported period of fluid deprivation of at least four hours; 178 had concurrent sera. Mean age was 13.0±2.7 mo (range 7.5 – 17.9 mo) and mean duration of fluid deprivation was 7.4±2.4 hr (4–13 hr). Mean serum OSM was 286±6 mOsm/kg H2O and independent of age, height, weight, or duration NPO. Urine OSM (mean 410±152, median 433, range 58–794 mOsm/kg H2O) was significantly greater than serum osmolality (p<0.0001) and correlated with duration NPO (p=0.001). One hundred forty-two infants (77.2%) concentrated urine (urine OSM > [mean serum OSM + 1 SD]); twenty-two (12.0%) had urine/serum OSM ratio > 2 and 54 (29.4%) had urine OSM > 500 mOsm/L. In addition, five infants (2.7%) were isosthenuric (urine OSM within mean serum OSM ± 1 SD) and 37 (20.1%) hyposthenuric (> 1 SD below mean serum OSM) despite instructions to withhold fluid. Urine OSM was associated with increasing 99mTc-DTPA GFR (p=0.024) and BUN (p<0.0001), but not with serum OSM, age, height, weight or serum creatinine. We conclude that even with a variable, often limited, fluid deprivation challenge, 75 percent of young infants with SCD were able to concentrate urine. We anticipate that at the end of each infant’s two-year study drug treatment period parents will be more successful in achieving the recommended fluid deprivation and urine collection and that differences in concentrating ability between those taking hydroxyurea and those taking placebo will be discernable.
Oxygen Desaturation at Rest and after Exercise in Pediatric Sickle Cell Disease Patients: Correlations with Hemolysis and Elevated Tricuspid Regurgitant Jet Velocity.
Authors: Campbell Andrew D. , Minniti Caterina , Rana Sohail R , Onyekwere Onyinye C. , Nouraie Mehdi , Arteta Manuel , Sable Craig , Ensing Gregory , Dham Niti , Darbari Deepika S. , Luchtman-Jones Lori , Kato Gregory J. , Gladwin Mark , Castro Oswaldo , Gordeuk Victor R.
Published: April 2020

Background. As a part of a multicenter, observational study in determining the prevalence and risk factors of elevated tricuspid regurgitant jet velocity (TRV) in children, oxygen desaturation correlated with TRV. We further investigated the risk factors and clinical associations of oxygen desaturation at rest and after execrcise in children at steady state. Methods. 310 children and adolescents with sickle cell disease were studied under basal conditions. Pulse oximetry was determined at rest and after a six minute walk test. The relationships of oxygen saturation at rest and desaturation during exercise to the available clinical and laboratory variables were investigated. Results. Among 300 patients with available baseline oxygen saturations, 30 (10%) had saturation <95 percent, 129 (43%) had saturation of 95–98 percent, and 141 (47%) had saturation >98 percent. Twenty-three (9%) of 244 patients had >3 percentage point reduction in oxygen saturation during a six minute walk; the median (interquartile range) baseline saturation was 96 (95– 99) percent among these patients versus 98 (97–100) percent among those with less or no reduction in saturation during the walk (P = 0.0009). Hemoglobin (p<0.0001), creatinine (p=0.014) and total lung capacity (p=0.042) were also lower in patients with declines in saturations >3 percentage points during the walk while a hemolytic index (p<0.0001), tricuspid regurgitant jet velocity (p=0.030), pulmonary insufficiency end diastolic velocity (PIEDV) (p=0.019), left ventricular mass index (LVMI) (p=0.0006) and left ventricular internal diameter z score (LVIDD z score) (p=0.0001) were higher. In 3 separate logistic regression models (clinical variables, echocardiographic parameters, and pulmonary function testing), lower hemoglobin, hemolytic index, PIEDV, LVIDD z score, and low TLC were independent predictors of six-minute-walk desaturation of >3 percentage points. Conclusion. Markers of hemolysis, low hemoglobin, PIEDV, LVMI, LVIDD z score, lower TLC, and elevated TRV velocities are associated with ≥3% reduction in oxygen desaturation during six minute walk in children and adolescents with sickle cell disease. A high degree of oxygen desaturation during the six minute walk in sickle cell disease patients might serve as an early biomarker for pulmonary hypertension. Exercise induced changes in oxygen saturation in sickle cell disease children may provide insight into the development of pulmonary hypertension as adults.
Analysis of FDG PET Diffuse Bone Marrow Uptake in Staging of Hodgkin’s Disease: A Reflect of Disease Infiltration or Just Inflammation?.
Authors: Salaun Pierre Y , Gastine Thomas , Campion Loic , Cambefort Pierre , Bodet-Milin Caroline , Moreau Anne , Le Gouil Steven , Harousseau Jean-Luc , Kraeber-Bodéré Françoise
Published: April 2020

FDG-PET has been successfully evaluated in the management of Hodgkin’s lymphoma (HL) and the most recent international guidelines recommended FDG-PET for initial staging and final therapeutic assessment. However, FDG PET diffuse bone marrow uptake (BMU) is frequently observed and remains difficult to analyse. The aim of this study was to evaluate the correlation between bone marrow involvement and BMU in HD. Methods: 106 HD patients (median age was 31 years range from 9 to 81; male gender in 23 cases) who underwent FDG PET/CT were retrospectively analysis. BMU level was graduated according to liver uptake. In addition, as semi-quantitative reference SUVmax was assessed upon sacral promontory. These data were compared with patient characteristics including age, Ann Arbor staging, bulky disease (Tumor burden > 10 cm), presence of B symptoms, bone foci on PET (n=106), bone marrow biopsy (n=75), leukocyte count (n=74), LDH (n=87) and inflammatory parameters measured in the serum (C-Reactive Protein (CRP) (n=83) and fibrinogen (n=60)). Univariate and multivariate analysis were performed. A Fisher’s exact test and a Kruskal-Wallis equality of population rank test were used to analyse qualitative and quantitative parameters, respectively. Results: No statistical link was found between BMU and stage, bulky disease, bone marrow involvement (BMI), bone foci on PET or LDH. In contrast, univariate analysis showed correlation between BMU and age (p =0.0001), B symptoms (p =0.028 for clinical), CRP (p=0.0001), fibrinogen (p=0.0111) and leukocyte (p=0.0024). Multivariate parameters analysis found independent correlation between BMU and CRP level (p<0.0001). Sacrum SUV was correlated with inflammatory parameters as BMU and with disease extension parameters like stage (p=0.0001), BMI on biopsy (p=0.0127) and bone foci on PET (p=0.0498). No BMI was found in patients presented with SUVmax below 3.5. Conclusion: This study demonstrates that the intensity of FDG-PET bone marrow uptake in HD is not correlated to BMI but related to inflammatory parameters probably reflecting the cellular activation within the BM. Semi-quantitative analysis of SUV could be useful to improve bone uptake interpretation because no infiltration is found under a SUV level.
Prognostic Value of Immunohistochemical Angiogenic and Invasion Markers in Hodgkin Lymphoma.
Authors: Mestre Francisco , Gutierrez Antonio , Ramos Rafael , Casasus M. , Nicolau C. , Martinez Jordi , Besalduch Juan , Rifa Julio , Garcia Juan Fernando , Rodriguez Jose
Published: April 2020

Microenvironment has and important role in sustaining the malignant cells by providing survival signals and promoting angiogenesis and invasion. Vascular endothelial growth factor (VEGF), cyclooxygenase-2 (COX-2), metalloproteinase-2 (MM2) and laminin are involved in angiogenesis, growth and metastatic invasion potential of malignancies. Numerous studies have demonstrated up-regulation of this protein at both mRNA and protein level in various tumours and a correlation with advanced stage and prognosis has been demonstrated in several solid tumors and hematologic malignancies such as multiple myeloma or non-Hodgkin lymphomas. There is evidence that also the neoplastic Hodgkin-Reed-Stenberg cells express VEGF and that VEGF expression both in macrophages and the extracellular matrix might facilitate tumour progression. To analyze the role of these markers in the prognosis we retrospectively examined their immunohistochemical expression using a tissue array in a series of 62 cases of Hodgkin’s disease uniformly treated with the standard ABVD regimen (3–6 cycles, in function of clinical prognostic factors). Median age was 30 years (14–71). Prognostic factors were as follows: 35% Ann Arbor stage III–IV, 23% adjusted-international prognostic index >1 and 11% Hasenclever > 3. Median follow-up was 90 months. Relapses were significantly higher in patients with strong VEGF staining (50% vs 19%; p=0.04) with a significant worst progression-free survival (60% vs 84%; p = 0.01). COX-2, MM2 and laminin showed a non-significant trend towards a lower relapse rate and PFS. Conclusion: The presence of VEGF strong staining in Hodgkin-Reed-Stenberg cells or reactive macrophages and the extracellular matrix might facilitate tumour progression as it was related to a shorter PFS. The trend towards a worst prognosis in higher COX-2, MM2 and laminin staining warrants further investigation of their role in the prognosis of patients with Hodgkin lymphoma.
Mannose-Binding Lectin Deficiency - a Risk Factor for Invasive Aspergillosis in Immunocompromised Patients.
Authors: Lambourne Jonathan , Agranoff Dan , Buchbinder Aby , Troke Peter F , Herbrecht Raoul , Lindsay Jodi , Harrison Thomas
Published: April 2020

Invasive aspergillosis is a devastating infection with 30–40% mortality. Immune mechanisms involved in recognition and elimination of aspergillus are incompletely understood. Mannose-binding lectin (MBL) deficiency occurs in 10–15% of the population and has been identified as a susceptibility factor in animal models of aspergillosis. We hypothesise there is an association between human MBL deficiency and invasive aspergillosis.

In this multi-centre case-control study, serum MBL concentrations were measured in 65 patients with probable or proven invasive aspergillosis (as defined by the 2002 EORTC/NIH-MSG criteria) and in 79 immunocompromised controls with fever not due to aspergillosis. MBL concentrations were measured using a commercially available, technically straightforward solid phase ELISA (Hycult Biotechnology, The Netherlands). Median MBL concentrations and the frequency of MBL deficiency were compared using the Mann-Whitney and Fisher’s exact tests.

Cases and controls were well matched in terms of age, gender, ethnicity and cause of immunocompromise. The assay performed well with a mean coefficient of variation of 7%. Patients with invasive aspergillosis had significantly lower MBL concentrations and a greater frequency of MBL deficiency than controls (65% vs. 33%, p = 0.0002, MBL deficiency cut-off 500ng/ml). MBL deficiency was significantly more frequent in cases than in controls at all MBL concentrations ≤500ng/ml. This is the first study to identify a strong association between MBL deficiency and invasive aspergillosis in immunocompromised patients. Because MBL concentrations do not consistently change during acute infection we conclude that MBL deficiency predisposes to invasive aspergillosis. Routine measurement of MBL levels is within the capability of most diagnostic laboratories. These data suggest that replacement therapy with recombinant human MBL may be an opportunity to prevent or mitigate the poor outcome of invasive aspergillosis.
Long-Term Follow-up of Rituximab and Infusional Cyclophosphamide, Doxorubicin, and Etoposide (cde) in Combination with HAART in HIVRelated Non-Hodgkin’s Lymphomas (NHL).
Authors: Spina Michele , Simonelli Cecilia , Vaccher Emanuela , Jaeger Ulrich , Sparano Joseph , Talamini Renato , Tirelli Umberto
Published: April 2020

Background: The combination of Rituximab plus chemotherapy (CT) is more effective than CT alone in the treatment of high grade NHL.

Objective: To report the long-term follow-up of CDE plus Rituximab in HIV-NHL.

Methods: In June 1998, we started a phase II study using infusional CDE (Cyclophosphamide 187.5 mg/m2/day, Doxorubicin 12.5 mg/m2/day and Etoposide 60 mg/m2/day) administered by continuous intravenous infusion for 4 days every 4 weeks and Rituximab 375 mg/m2 i.v. on day 1. HAART was given concomitantly with CT.

Results: Seventy-four patients (pts) have been enrolled. The median CD4+ cell count was 161 (range 3–691) and the median Performance Status was 1 (range 0–3). Diffuse large B-cell NHL was diagnosed in 72% of pts and Burkitt in 28%. Seventy per cent of pts had advanced stage (III–IV) disease and 57% of pts had an age-adjusted international prognostic index ³2. Fifty-two out of 74 pts (70%) achieved a complete remission (cr), 4/74 (5%) had a partial remission and 18 pts progressed. With a median follow-up of 61 months, only 17% of CRs have relapsed and 41/74 pts are alive. The overall survival, disease free survival and time to treatment failure (TTF) at 5 years were 56%, 81% and 52%, respectively. Only one secondary tumor (acute leukemia) has been observed. No case of late pulmonary or cardiac toxicity has been reported.

Conclusions: The combination of Rituximab and CDE in HIV-NHL treated concomitantly with HAART is very active. CR rate (70%) and TTF at 5 years (52%) are comparable to those observed in high grade NHL of the general population. Our data confirm that in HAART era a high proportion of HIV-NHL can be cured. This study was supported by ISS grants.
In Vivo Genetic Mutations Define Predominant Functions of the Human T-Cell Leukemia/Lymphoma Virus p12I Protein.
Authors: Franchini Genoveffa , Fukumoto Risaku , Andresen Vibeke , Bialuk Izabela , Cecchinato Valentina , Walser Jean Claude , Valeri Valerio William , Nauroth Julie M , Gessain Antoine , Nicot Christophe
Published: April 2020

The human T-cell leukemia/lymphoma virus Type 1 (HTLV-1) ORF-I encodes, a ninety-nine amino acid hydrophobic membrane protein, p12I that affects receptors in different cellular compartments. We report here that proteolytic cleavage dictates different cellular localization and functions of p12I. The removal of a non-canonical endoplasmic reticulum (ER) retention/retrieval signal within the amino terminus of p12I is necessary for trafficking to the Golgi apparatus and generation of a completely cleaved 8 kDa protein. The 8 kDa protein in turn traffics to the cell surface, is recruited to the immunological synapse following T-cell receptor (TCR) ligation and down-regulates TCR proximal signaling. The uncleaved 12 kDa form of p12I resides in the ER and interacts with the β and γc chains of the interleukin-2 receptor (IL-2R), the heavy chain of the major histocompatibility complex (MHC) class I, as well as calreticulin and calnexin. Genetic analysis of ORF-I from ex vivo samples of HTLV-1-infected patients reveals predominant amino acid substitutions within ORF-I that inhibits proteolytic cleavage, suggesting that ER associated functions of p12I may contribute to the survival and proliferation of the infected T-cells in the host.
Acute Lymphoblastic Leukemia (ALL) with t(8;14)(q11.2;q32): B-Lineage Disease with High Proportion of Down Syndrome. A Children’s Oncology Group (COG) Study.
Authors: Messinger Yoav H. , Higgins Rodney , Devidas Meenakshi , Hunger Stephen P. , Carroll Andrew J. , Heerema Nyla A.
Published: April 2020

Recurrent chromosome translocations play a critical role in the pathogenesis of ALL and many translocations have important prognostic significance. The t(8;14)(q11.2;q32) is a recurrent translocation that fuses the chromosome 8 CEBPD (CCAAT enhancer binding protein delta) gene to the IgH (immunoglobulin heavy chain) gene, leading most likely to deregulated CEPD expression. We previously reported the clinical data of 10 such patients (Leukemia, 2000;14:238–240). Using the COG ALL cytogenetics database we expand the report to include 22 patients (13 females and 9 males) with the t(8;14)(q11.2;q32). All patients with available immunophenotyping data (n=20) had B-lineage ALL. The median age at diagnosis was 9.2 years (range: 1.6 months – 17.1 years). Median diagnostic white blood cell count (WBC) was 9,950 (range: 700–172,000). Of the 22 patients, 7 were NCI standard risk and 15 were high risk. The most common additional cytogenetic abnormality was trisomy 21. Of the ten cases with trisomy 21, seven were constitutional (Down Syndrome), thus confirming previous reports that a significant fraction of t(8;14) patients (7/22; 31.8%) have Down Syndrome, which is much higher than the 3% (80/2811) overall rate of children with Down Syndrome in a recent COG ALL trial (p<0.0001). In addition to the Down Syndrome cases, one case with phenotypic Turner syndrome had a mosaic constitutional r(Y)(p11q11.2); thus, eight cases (36%) had constitutional abnormalities. Secondary abnormalities included additional X (n=5); additional 5 (n=3); Philadelphia chromosome (n=1). Two cases had a second der(14)t(8;14) and two had loss of the der(8) t(8;14), consistent with the der(14)t(8;14) as the significant abnormality. Four cases (18%) had 9p deletions compared with 11% in the overall ALL population, and two cases (9%) had abnormalities of 13q compared with 2% in the overall population. Numerically, the cases were pseudodiploid (n=12) and hyperdiploid (n=10). Children with Down Syndrome had superior estimated 5-year event-free survival of 100%, compared to non-Down patients with 50.1±17.7% (p=0.04). Overall survival was also different but did not reach statistical significance: 100% vs. 60.9±17.0% (p=0.088). In summary, the rare t(8;14)(q11.2;q32) is associated with B-lineage phenotype and occurs with greatly increased frequency in children with Down Syndrome, who have an excellent outcome with standard COG therapy as compared to non-Down Syndrome patients with this translocation.
Stimulation of B-Cell Lymphoproliferations with CpG-Oligonucleotide DSP30 Plus IL-2 Is More Effective than with TPA to Detect Clonal Abnormalities.
Authors: Struski Stéphanie , Gervais Carine , Helias Catherine , Herbrecht Raoul , Audhuy Bruno , Mauvieux Laurent
Published: April 2020

Conventional cytogenetics (CC) of B-cell lymphoproliferations remains difficult because of low mitotic in vitro activity of the leukemic cells. Therefore, mitogen stimulation of B-cells is required to analyze an adequate number of metaphases. Chromosome abnormalities using CC with TPA can be detected in up to 50% of chronic lymphocytic leukemia (CLL), but the development of FISH techniques has allowed the detection of selected chromosome abnormalities in more than 80% of CLL. However, FISH is restricted, since information is only available for the genes/loci for which probes are used. So, for a comprehensive genetic analysis, CC is essential because it provides an overview of all microscopically visible chromosome abnormalities important as prognostic factors. The use of the immunostimulatory CpG-oligonucleotide DSP30 to effectively induce cell cycle progression of CLL cells in vitro has been reported. This proliferation is markedly enhanced upon addition of Interleukine-2 to cultures. To our knowledge and to date, there has been no direct comparison of classical TPA versus DSP30+IL-2. DSP30+IL-2 stimulation has been successfully tested in CLL but no data are available for other lymphoproliferations. We cultured 132 B-cell lymphoproliferations (80 CLL and 52 other B-cell lymphoid neoplasms (BCLN)) in parallel, in presence of TPA or DSP30+IL-2. The objective of this study was to evaluate the suitability of DSP30+IL-2 as a routine B-cell mitogen for metaphase cytogenetics. CC successfully analyzed 94.9% of CLL and 98.1% of BCNL with more than 80% abnormal karyotypes. For CLL, failures of karyotypes were more frequent in cultures with DSP30+IL-2 (14%) than in those with TPA (4%). The rate of failures were similar for BCLN (6% versus 4%). For CLL, there were significantly fewer metaphases in DSP30+IL-2 than in TPA spreads (mean of 50 versus 72 metaphases per slide respectively, p=0.0007), as well as for BCLN (mean of 50 versus 71 metaphases per slide respectively, p=0.009). However, the proportion of abnormal metaphases was significantly higher in DSP30+IL-2 (mean of 59%) compared to TPA cultures (mean of 26%, p=0.00265) for CLL and for BCLN (mean of 57% versus 33%, p=0.0065). Stimulation with DSP30+IL-2 allowed to detect more abnormalities, more abnormal subclones and more complex karyotypes in CLL and in the majority of BCLN. Though FISH exploration using a large probe panel has yielded valuable results in lymphoproliferative diseases, it underestimates the heterogeneity of chromosomal aberrations. Complexity of chromosomal changes, recently associated to unfavorable outcomes, can only be assessed with CC. In conclusion, our results in both CLL and BCLN indicate that the immunostimulatory oligonucleotide DSP30 in combination with IL-2 is an easy and efficient stimulus in metaphase generation for routine chromosomal banding.
High-Resolution Genomic Profiling (array-CGH) of Childhood T-ALL Identifies Deletions at 6q15-16.1 as a Predictive Marker for Early Treatment Response.
Authors: Remke Marc , Pfister Stefan , Stanulla Martin , Schrappe Martin , Muckenthaler Martina Ulrike , Lichter Peter , Kulozik Andreas
Published: April 2020

Purpose: Precursor T-cell lymphoblastic leukemia (T-ALL) accounts for approximately 15% of pediatric ALL and represents a particular clinical challenge, because relapses are usually fatal. In T-ALL, monitoring treatment response (prednisone prephase; MRD kinetics) provides a powerful tool for risk assessment. We now aimed to identify DNA-based predictive biomarkers that allow risk assessment and treatment stratification at the time of diagnosis. We thus performed a genome-wide analysis for DNA copy-number aberrations to detect circumscribed gains and losses that are specific for molecular and prognostic subgroups.

Experimental design: Samples of 77 T-ALL patients enrolled in BFM-ALL protocols were analyzed by array-based comparative genomic hybridization (array-CGH) on an 8k BAC array allowing for an average resolution of 0.5 megabases. After using a Hidden Markov Model for bioinformatic analysis, our findings were correlated with minimal residual disease (MRD) data. Frequently affected pathways were identified using Babelomics software. This approach was complemented by genomic sequencing and the analysis of mRNA abundance for interesting candidate genes in a subset of leukemias.

Results: Several circumscribed genomic alterations affecting key regulators and downstream intermediates of TGF-β and PI3K-AKT signalling were identified thus suggesting an important role of these pathways in T-ALL leukemogenesis. Furthermore, deletions at chromosome band 6q15-16.1 haboring the caspase 8-associated protein 2 (CASP8AP2) gene were overrepresented in patients with unfavorable MRD. QRT-PCR revealed a marked gene-dosage effect resulting in reduced expression of CASP8AP2 mRNA in samples with 6q15-16.1 deletions (p=0.01). Fisher’s exact test revealed that deletions at 6q15-16.1 constitute a powerful marker for poor early treatment response (p=0.03), which is likely related to the reduced expression of the pro-apoptotic CASP8AP2 gene.

Conclusions: Array-CGH analyses indicate a novel role of TGF-β and PI3K-AKT signalling in T-ALL leukemogenesis. Moreover, deletions at 6q15-16.1 resulting in the reduced expression of the pro-apoptotic CASP8AP2 candidate gene identify a subgroup of T-ALL patients with poor treatment response. Prospective studies will now have to validate the usefulness of DNA-based molecular markers to clinically stratify the individual risk profile in pediatric T-ALL.
Chromosome 17 Abnormalities Are Associated with Worse Overall and Relapse Free Survival in Patients with Acute Myelogenous Leukemia and Poor-Risk Cytogenetics.
Authors: Borthakur Gautam , Tam Constantine , Kantarjian Hagop , Lin E. , Cortes Jorge , O’Brien Susan , Koller Charles , Pierce Sherry , Keating Michael
Published: April 2020

Purpose: Chromosome 17 abnormalities define a group of patients with acute myelogenous leukemia (AML) (Nahi, H. et al. Leukemia and Lymphoma2008;49:508) with poor outcomes. We analyzed the additional impact of chromosome 17 abnormalities (−17, −17p, −17q, der17) among patients with AML and cytogenetic abnormalities traditionally considered to be of adverse prognosis.

Patients and Methods: 1086 patients with AML [excluding inv 16, t (8;21), t (15;17), Diploid/-y abnormality] were included in this analysis. Based on cytogenetic abnormalities patients were grouped into: −5,−7,−5and −7, complex. The following parameters were included in uni and multi-variate analysis: age, performance status, WBC, hemoglobin, platelets, marrow blast percentage, bilirubin, creatinine, albumin, LDH, chromosome 17 abnormality (yes/no).

Results: Four hundred and fourteen (45%) patients achieved complete remission (CR) or CR with incomplete platelet recovery (CRp) and 267 (64.5%) patients relapsed. Two hundred seventy (24.9%) patients had abnormalities of chromosome 17. Abnormalities of chromosome 17 were associated with lower CR or CRp rate (p=0.02) and higher possibility of having cytogenetic abnormality of −5 or −7 (p<0.0001). Multivariate analysis showed that patients with abnormalities of chromosome 17 had worse overall survival (OS) compared to patients without (p= 0.003)(Fig.1). Multi-variate analysis within cytogenetic subgroups showed that chromosome 17 abnormalities were associated with worse OS in patients with chromosome 5 abnormality(p=.02) (data not shown) and in those with complex cytogenetics (p=.04)(Fig.2) and not in patients with chromosome 7 (p=.17)or combined 5 and 7 abnormalities (p=.33). Similar analysis restricted to patients achieving CR/CRp after induction therapy showed that impact of chromosome 17 abnormalities on relapse free survival (RFS) mirrored their impact on OS.

Conclusion: chromosome 17 abnormalities are associated with worse OS and RFS in patients with AML and adverse cytogenetics and have additional negative impact on the outcomes in certain well-known adverse cytogenetic subgroups.

Figure 1: Kaplan-Meier estimates of overall survival by status of chromosome 17 Figure 1:. Kaplan-Meier estimates of overall survival by status of chromosome 17 Figure 2: Kaplan-Meier estimates of overall survival by status of chromosome 17 in subgroup of patient, complex Figure 2:. Kaplan-Meier estimates of overall survival by status of chromosome 17 in subgroup of patient, complex