bluuebunny/crossref_metadata_2025 huggingface dataset:
Co-Transplantation of Ucbmn with UC-MSC Enhances Engraftment of Ucbmn in Iron Overload NOD/SCID Mice
Authors: Huang Zhi , Xiao Yuhua , Chen Xiaomin , Li Chunfu , Feng Xiaoqin
Published: October 2019

Background: Umbilical cord blood (UCB)-derived hematopoietic stem cells (HSC) are ideal for allogeneic HSCT when a compatible donor is unavailable. However, the insufficient number of HSCs per UCB unit delays engraftment and may lead to graft failure and/or mortality. Besides, transfusion-induced iron overload has been reported to be involved in the delayed engraftment of HSC. Previously mesenchymal stem cells (MSC) have been implicated in playing a significant role in HSC engraftment. This study aimed to determine the effect of co-transplantation of umbilical cord mesenchymal stem cells (UC-MSC) on engraftment and hematopoietic recovery.

Methods: The iron overload NOD/SCID mice were induced by dose-escalation injection of iron dextran. And the sublethally irradiated iron overload NOD/SCID mice were divided into four groups: (1) intravenous injection of normal saline (control group, n=8), (2) intravenous injection of umbilical cord mesenchymal MN) alone (n=10), (3) intravenous injection of UCBMN and UC-MSC (n=10), and (4) intrafemoral injection of UCBMN and UC-MSC (n=12). Six weeks after injection of UC, the bone marrow (BM) was harvested and analyzed. The semi-quantitative analysis of cytokines including SDF-1a, OPN, and VEGF-α were examined by immunohistochemistry.

Results: As a result, iron deposits in bone liver, BM and spleen. In the BM of control group, no human CD45+ cells were found. Via the intra-bone marrow (iBM) injection, the percentage of human CD45+ cells was 9.86% ±1.86% (n=10) in the presence and 3.18% ±0.98% (n=6) in the absence of MSC, respectively, via the tail vein, the percentage of human CD45+ cells was 3.5%±0.94%. In additional, the levels of SDF-1a, OPN, and VEGF-a were all significantly higher in The mice that received MSC showed significantly enhanced engraftment in all three experiments (P=0.000).

Conclusion: Co-transplantation of UC-MSC is effective for improving engraftment in iron overload NOD/SCID mice. The point underlying the positive outcome may relate to cytokines including SDF-1a, OPN, and VEGF-a secreted by UC-MSC, which has been indicated to play a crucial part in homing and engraftment.

Disclosures

No relevant conflicts of interest to declare.
Pegfilgrastim Versus Filgrastim in the Management of Therapy-Related Neutropenia in Relapsed and Refractory Multiple Myeloma in Treatment with Pomalidomide-Dexamethasone
Authors: Cerchione Claudio , Nappi Davide , Pane Fabrizio , Catalano Lucio
Published: October 2019

Aims:

The objective of this study was to compare the efficacy and safety of pegfilgrastim in patients affected by heavily pretreated MM, treated with pomalidomide-dexamethasone, in order to determine whether a single subcutaneous injection of pegfilgrastim is as effective as daily injections of standard filgrastim, in terms of haematological toxicity, febrile neutropenic episodes, antibiotic usage and hospedalization duration.

Methods:

We enrolled 33 patients (19 male and 14 female) median age at diagnosis 69 years (r. 52-84), and median age at start of treatment 76 years (r.56-90) treated with several lines of treatments (median 7, r. 2-11), every refractory to all the drugs previously received, received Pomalidomide-Dexamethasone (Pomalidomide 4 mg for 21 days, Dexamethasone 40 mg days 1,8,15,22, pegfilgrastim day +8) every 28 days, until progression.

Results:

Since first course, received in domestic setting, with a very good compliance, patients performed blood counts once weekly and received, from day +8 to day +19 ("day + 1" when the protocol starts), prophylactic oral chinolonic antibiotics and anti-fungal drugs. During neutropenia after first cycle, Filgrastim (5 μgr/kg/day for 3 days) was given if neutrophils count was <1500 x 10^9 cells/L. Median number of filgrastim administrations was 4.8 (r. 3-6); nadir neutropenia was registered after a median of 10.7 days (r. 7-14); median of nadir neutrophil count was 1.17 x 10^9 cells/L (range 0.3 - 1.5 x 10^9 cells/L), with maximum duration of 14 days. From the second course, all patients switched to prophylaxis with pegfilgrastim (6 mg), injected subcutaneously with a single administration on day +3 independently from the neutrophil count at that time. During pegfilgrastim, neutropenia was never longer than 8 days, with a consequent reduction of neutropenia-related infections. Median nadir neutrophil count, evaluated for every patients for at least three courses of therapy (r. 3-6) registered at day +11, was 1.39 (range 0.9-2.2 x 10^9 cells/L). Only 4 patients needed a supplement of 3 administrations of filgrastim. Pegfilgrastim was well tolerated in all patients: main side effects in our patients were mild fever and bone pain (21.2%).

Conclusions:

In conclusions, in patients affected by heavily pretreated MM treated with pomalidomide-dexamethasone, pegfilgrastim seems to reduce the incidence of severe neutropenia and infections and may increase the possibility to maintain the scheduled time of treatment.

Disclosures

No relevant conflicts of interest to declare.
Secondary Skin Involvement in Non-Hodgkin's Lymphoma Does Not Impact Outcomes: SEER Database Analysis
Authors: Khurana Arushi , Al-Juhaishi Taha , Shafer Danielle
Published: October 2019

Background:

Non-Hodgkin's Lymphoma (NHL) comprises a diverse group of malignancies with varied presentations and clinical behavior. Different prognostic factors have been defined including age, LDH, gender, CNS involvement and molecular/genetic characteristics. We aim to study the incidence and impact of skin involvement as a secondary site in patients with mature B and T-cell NHLs.

Methods:

The Surveillance, Epidemiology and End Results (SEER) database was used to identify patients (≥18 years) diagnosed with mature B/T/NK-cell NHL with skin as secondary site of involvement between 1973 and 2014. B-cell and T-cell lymphomas of all stages were included. Overall survival (OS) was estimated using the Kaplan-Meier method, and compared using Log-Rank test.

Results:

Total of 6429 patients were included with secondary skin involvement. B-cell NHL was more common with 76%. Median age for B-cell and T-cell NHL was 65 years (48-82), and 64 years (46-82), respectively. Slight male preponderance was noted in both subtypes (B-cell-56.7%% and T-cell-61.7%). Diffuse large B cell lymphoma (DLBCL) was the most common histology among B-cell NHLs, (DLBCL, N=1920 39.6%), followed by follicular (FL, N=1381, 28.5%) and extra nodal marginal zone (EN MZL, N=1314, 27.1%) Among T cell NHL histologies, peripheral T-cell NOS was the most common (PTCL, N=819, 51.6%) followed by anaplastic large cell ALK+ (ALCL+, N=341, 21.5%), and Sezary syndrome (N=293, 18.5%) Median overall survival was 6.1 [5.2 - 7.2.; 95% CI] years in T-cell population compared to B-cell population which was 13.3 [12.6-14.0; 95% CI] years. Survival was different by histologic subtype and correlated with SEER database relative survival for the overall population described in the SEER Cancer Statistics Review 1973-2011.

Conclusion:-

Skin involvement is relatively common in both B and T-cell NHLs. DLBCL, FL, MZL, PTCL-NOS and ALCL+ are the more commonly involved subtypes, correlating with their relative incidences. Outcomes in patients with secondary skin involvement tends to mimic known survival patterns in different histological subtypes conferring no additional prognostic significance. Skin as an extra nodal site of involvement should not change management for these patients.

Figure. Figure.

Disclosures

No relevant conflicts of interest to declare.
A role for macrophages in hematopoiesis in the embryonic head
Authors: Li Zhuan , Mariani Samanta , Rodriguez-Seoane Carmen , He Wenyan , Ning Xiaowei , Liu Bing , Vink Chris S. , Dzierzak Elaine A
Published: October 2019

Key Points

Macrophages in the hindbrain-branchial arches region of the mouse embryo play a role HS/PC expansion and/or maturation. Macrophages from embryonic head hematopoietic niches produce pro-inflammatory factor TNF-α and enhance HS/PC expansion and/or maturation
Description of a knock-in mouse model of JAK2V617F MPN emerging from a minority of mutated hematopoietic stem cells
Authors: Mansier Olivier , Kilani Badr , Guitart Amélie V. , Guy Alexandre , Gourdou-Latyszenok Virginie , Marty Caroline , Parrens Marie , Plo Isabelle , Vainchenker William , James Chloé
Published: October 2019

Key Points

PF4iCre;JAK2 V617F/WT mice develop a full MPN that mimics polycythemia vera. The PF4iCre system induces JAK2V617F mutation in a small subset of HSC.
JAK2
ex13InDel drives oncogenic transformation and is associated with chronic eosinophilic leukemia and polycythemia vera
Authors: Patel Ami B. , Franzini Anca , Leroy Emilie , Kim Soo Jin , Pomicter Anthony D. , Genet Lidvine , Xiao Michael , Yan Dongqing , Ahmann Jonathan M. , Agarwal Archana M. , Clair Phillip , Addada Juanah , Lambert Jonathan , Salmon Matthew , Gleich Gerald J. , Cross Nicholas C. P. , Constantinescu Stefan N. , O’Hare Thomas , Prchal Josef T. , Deininger Michael W.
Published: October 2019

The authors describe recurrent novel insertion/deletion mutations in the JH2 domain of JAK2 occurring in patients with eosinophilia as a prominent feature of their myeloproliferative neoplasms. Remarkably, 2 of the patients with a specific mutation (Leu583-Ala586DelInsSer) meet the criteria for both chronic eosinophilic leukemia and polycythemia vera, suggesting that this may be a distinct overlap syndrome.
Coactivation of NF-κB and Notch signaling is sufficient to induce B-cell transformation and enables B-myeloid conversion
Authors: Xiu Yan , Dong Qianze , Fu Lin , Bossler Aaron , Tang Xiaobing , Boyce Brendan , Borcherding Nicholas , Leidinger Mariah , Sardina José Luis , Xue Hai-hui , Li Qingchang , Feldman Andrew , Aifantis Iannis , Boccalatte Francesco , Wang Lili , Jin Meiling , Khoury Joseph , Wang Wei , Hu Shimin , Yuan Youzhong , Wang Endi , Yuan Ji , Janz Siegfried , Colgan John , Habelhah Hasem , Waldschmidt Thomas , Müschen Markus , Bagg Adam , Darbro Benjamin , Zhao Chen
Published: October 2019

NF-κB and Notch signaling can be simultaneously activated in a variety of B-cell lymphomas. Patients with B-cell lymphoma occasionally develop clonally related myeloid tumors with poor prognosis. Whether concurrent activation of both pathways is sufficient to induce B-cell transformation and whether the signaling initiates B-myeloid conversion in a pathological context are largely unknown. Here, we provide genetic evidence that concurrent activation of NF-κB and Notch signaling in committed B cells is sufficient to induce B-cell lymphomatous transformation and primes common progenitor cells to convert to myeloid lineage through dedifferentiation, not transdifferentiation. Intriguingly, the converted myeloid cells can further transform, albeit at low frequency, into myeloid leukemia. Mechanistically, coactivation of NF-κB and Notch signaling endows committed B cells with the ability to self renew. Downregulation of BACH2, a lymphoma and myeloid gene suppressor, but not upregulation of CEBPα and/or downregulation of B-cell transcription factors, is an early event in both B-cell transformation and myeloid conversion. Interestingly, a DNA hypomethylating drug not only effectively eliminated the converted myeloid leukemia cells, but also restored the expression of green fluorescent protein, which had been lost in converted myeloid leukemia cells. Collectively, our results suggest that targeting NF-κB and Notch signaling will not only improve lymphoma treatment, but also prevent the lymphoma-to-myeloid tumor conversion. Importantly, DNA hypomethylating drugs might efficiently treat these converted myeloid neoplasms.
A multicenter, open-label phase 3 study of emicizumab prophylaxis in children with hemophilia A with inhibitors
Authors: Young Guy , Liesner Ri , Chang Tiffany , Sidonio Robert , Oldenburg Johannes , Jiménez-Yuste Victor , Mahlangu Johnny , Kruse-Jarres Rebecca , Wang Michael , Uguen Marianne , Doral Michelle Y. , Wright Lilyan Y. , Schmitt Christophe , Levy Gallia G. , Shima Midori , Mancuso Maria Elisa
Published: October 2019

In a Plenary Paper, Young et al describe impressive favorable outcomes of emicizumab prophylaxis in children with hemophilia A and factor VIII inhibitors, reporting a 99% reduction in annualized bleeding, with 77% of patients having no treated bleeding events.
Novel drivers and modifiers of MPL-dependent oncogenic transformation identified by deep mutational scanning
Authors: Bridgford Jessica L. , Lee Su Min , Lee Christine M. M. , Guglielmelli Paola , Rumi Elisa , Pietra Daniela , Wilcox Stephen , Chhabra Yash , Rubin Alan F. , Cazzola Mario , Vannucchi Alessandro M. , Brooks Andrew J. , Call Matthew E. , Call Melissa J.
Published: October 2019

The single transmembrane domain (TMD) of the human thrombopoietin receptor (TpoR/myeloproliferative leukemia [MPL] protein), encoded by exon 10 of the MPL gene, is a hotspot for somatic mutations associated with myeloproliferative neoplasms (MPNs). Approximately 6% and 14% of JAK2 V617F− essential thrombocythemia and primary myelofibrosis patients, respectively, have “canonical” MPL exon 10 driver mutations W515L/K/R/A or S505N, which generate constitutively active receptors and consequent loss of Tpo dependence. Other “noncanonical” MPL exon 10 mutations have also been identified in patients, both alone and in combination with canonical mutations, but, in almost all cases, their functional consequences and relevance to disease are unknown. Here, we used a deep mutational scanning approach to evaluate all possible single amino acid substitutions in the human TpoR TMD for their ability to confer cytokine-independent growth in Ba/F3 cells. We identified all currently recognized driver mutations and 7 novel mutations that cause constitutive TpoR activation, and a much larger number of second-site mutations that enhance S505N-driven activation. We found examples of both of these categories in published and previously unpublished MPL exon 10 sequencing data from MPN patients, demonstrating that some, if not all, of the new mutations reported here represent likely drivers or modifiers of myeloproliferative disease.
Long-term effects of intermittent interleukin 2 therapy in patients with HIV infection: characterization of a novel subset of CD4+/CD25+ T cells
Authors: Sereti Irini , Martinez-Wilson Hector , Metcalf Julia A. , Baseler Michael W. , Hallahan Claire W. , Hahn Barbara , Hengel Richard L. , Davey Richard T. , Kovacs Joseph A. , Lane H. Clifford
Published: October 2019

The long-term immunologic effects of intermittent interleukin 2 (IL-2) therapy were evaluated in a cross-sectional study by comparing 3 groups: HIV-seronegative volunteers, HIV-infected (HIV+) patients receiving highly active antiretroviral therapy (HAART), and HIV+ patients receiving HAART and intermittent IL-2. Whole-blood immunophenotyping was performed to study expression of the IL-2 receptor chains on T lymphocytes and natural killer cells and to further characterize CD4+/CD25+ T cells. Increased CD25 expression, especially in CD4+ T cells but also in CD8+ T cells, without increases in expression of the β and γ chains of the IL-2 receptor was detected in the IL-2 group. Up to 79% of naive CD4+ T cells (median, 61%) from patients in the IL-2 group expressed CD25, and the number of naive CD4+/CD25+ T cells correlated positively with both the total and naive CD4+ T-cell counts. A discrete population of CD45 double intermediate RA+/RO+CD4+ cells was also preferentially expanded in the IL-2 group, and the number of these cells strongly correlated with the total CD4+ count. Despite increases in CD25 expression, T lymphocytes from patients treated with IL-2 did not have increased expression of early (CD69) or late (CD95) activation markers or evidence of recent proliferation (Ki67). Both CD4+/CD25+ and CD4+/CD25− cells from IL-2–treated HIV+ patients proliferated in response to mitogens, specific antigens, and T-cell-receptor–mediated stimuli. Thus, intermittent administration of IL-2 in HIV+ patients leads to preferential expansion of a unique subset of CD4+ T cells that may represent a critical population in T-cell homeostasis.
Role for macrophage inflammatory protein (MIP)-1α and MIP-1β in the development of osteolytic lesions in multiple myeloma
Authors: Abe Masahiro , Hiura Kenji , Wilde Javier , Moriyama Keiji , Hashimoto Toshihiro , Ozaki Shuji , Wakatsuki Shingo , Kosaka Masaaki , Kido Shinsuke , Inoue Daisuke , Matsumoto Toshio
Published: October 2019

Multiple myeloma (MM) cells cause devastating bone destruction by activating osteoclasts in the bone marrow milieu. However, the mechanism of enhanced bone resorption in patients with myeloma is poorly understood. In the present study, we investigated a role of C-C chemokines, macrophage inflammatory protein (MIP)–1α and MIP-1β, in MM cell-induced osteolysis. These chemokines were produced and secreted by a majority of MM cell lines as well as primary MM cells from patients. Secretion of MIP-1α and MIP-1β correlated well with the ability of myeloma cells to enhance osteoclastic bone resorption both in vitro and in vivo as well as in MM patients. In osteoclastogenic cultures of rabbit bone cells, cocultures with myeloma cells as well as addition of myeloma cell-conditioned media enhanced both formation of osteoclastlike cells and resorption pits to an extent comparable to the effect of recombinant MIP-1α and MIP-1β. Importantly, these effects were mostly reversed by neutralizing antibodies against MIP-1α and MIP-1β, or their cognate receptor, CCR5, suggesting critical roles of these chemokines. We also demonstrated that stromal cells express CCR5 and that recombinant MIP-1α and MIP-1β induce expression of receptor activator of nuclear factor-κB (RANK) ligand by stromal cells, thereby stimulating osteoclast differentiation of preosteoclastic cells. These results suggest that MIP-1α and MIP-1β may be major osteoclast-activating factors produced by MM cells.
Regeneration of Bone Marrow from Adipose-Derived Stromal Cells - a Novel Approach for the Treatment of Fibroblastic Bone Marrow Diseases.
Authors: Ogawa Rei , Mizuno Hiroshi , Hyakusoku Hiko , Migita Makoto , Shimada Takashi
Published: October 2019

BACKGROUND: Taking advantage of homogeneously marked cells from green fluorescent protein (GFP) transgenic mice, we have previously demonstrated that adipose-derived stromal cells (ASCs) and bone marrow-derived stromal cells (BSCs) differentiate into a variety of cell lineages both in vitro and in vivo. In the present study, we extended this approach to regenerate bone marrow.

METHODS: ASCs isolated from the inguinal fat pads of GFP mice were cultured in vitro for five passages and seeded onto hydroxyapatites with small pores. The hydroxyapatites including ASCs were subsequently implanted into immunocompetent mice subcutaneously. Two months later, the hydroxyapatites were extirpated for histological and immunohistochemical analyses.

RESULTS: The specimens were covered with adipose tissues including extensively developed microvessels and GFP+ cells. Histological examination showed that the pores were filled with typical bone marrow composed of adipocytes, hematopoietic cells, vasculatures, and matrix. Immunohistochemical analysis confirmed that the GFP+ ASCs had differentiated into osteoblasts and vascular endothelial cells, but not hematopoietic cells.

CONCLUSION: We demonstrated for the first time that bone marrow can be regenerated from ASCs. Bone marrow regeneration from non-bone-marrow-derived cells will have a great impact on the treatment of various bone marrow-related disorders, in particular idiopathic myelofibrosis and osteopetrosis, in which there is no place for hematopoietic stem cells to survive. Furthermore, in combination with gene transfer, bone marrow regeneration may be applied to the development of novel therapeutic approaches for various kinds of hematopoietic diseases.
IPI-504: A Novel hsp90 Inhibitor with In Vitro and In Vivo Anti-Tumor Activity.
Authors: Mitsiades Constantine S. , Mitsiades Nicholas , Rooney Melissa , Negri Joseph , Geer Corey C. , Pink Melissa , Pien Christine , Hideshima Teru , Chauhan Dharminder , Munshi Nikhil C. , Richardson Paul G. , Barrett John , Tong Jeffrey K. , Grayzel David , Palombella Vito , Adams Julian , Anderson Kenneth C.
Published: October 2019

We have previously shown that inhibitors of the hsp90 molecular chaperone (including geldanamycin, 17-allylamino-17-demethoxy-geldanamycin (17-AAG) and other members of the ansamycin family) potently induce growth arrest and apoptosis of a large panel of drug-sensitive and -resistant MM cell lines, as well as tumor cells freshly isolated from patients with relapsed refractory MM; and sensitize these cells to other pro-apoptotic anti-tumor agents. While multiple phase I clinical trials have shown that biologically active doses of 17-AAG can be administered without significant hsp90-related toxicities, the insolubility of this compound in most conventional clinical solvents, as well as the practical limitations of DMSO-based formulations that were used in the original clinical trials have generated the need to develop more effective and practical approaches to administer 17-AAG to patients. Herein we describe the in vitro and in vivo pre-clinical profile of IPI-504, a novel analog of 17-AAG, which is soluble in aqueous formulations and can bypass key limitations of the DMSO-based formulations for administration of 17-AAG. Our in vitro studies show that IPI-504 has anti-tumor activity against a broad panel of primary MM tumor cells as well as MM cell lines (including cells resistant to cytotoxic chemotherapeutics, proteasome inhibitor bortezomib, thalidomide or its immunomodulatory thalidomide derivatives, and/or Apo2L/TRAIL). Based on hierarchical clustering analyses, logistic and linear regression models, we observed that the profiles of drug sensitivity of MM cells to IPI-504 were consistent with the profiles of sensitivity to 17-AAG. Similarly, IPI-504 triggered a constellation of molecular sequelae that were consistent with hsp90 inhibition by 17-AAG, including suppression of cell surface expression and down-stream signaling (via PI-3K/Akt and Ras/Raf/MAPK) of receptors for IGF-1 and IL-6; decreased intracellular levels of several key kinases, including Akt, Raf, IKK-α; suppressed expression of several intracellular anti-apoptotic proteins (e.g. FLIP, XIAP, cIAP2); leading to tumor cell sensitization to other pro-apoptotic agents (e.g. cytotoxic chemotherapy or PS-341). Importantly, in our mouse model of diffuse MM bone lesions in SCID/NOD mice, IPI-504 (50 mg/kg, i.v. twice weekly) was able to prolong the survival of mice vs. vehicle-treated mice (p<0.01, log-rank test), without significant treatment-related toxicities. These results indicate that hsp90 inhibitors have significant anti-MM activity in vivo, which, coupled with our ex vivo mechanistic and molecular profiling studies, have provided the framework for upcoming clinical trials of this novel class of agents in patients with MM.
T-Cell Function in Patients with B-Cell Chronic Lymphocytic Leukemia (B-CLL) Following Alemtuzumab (Campath®) or Fludarabine Treatment.
Authors: Kiaii Shahryar , Mozaffari Fariba , Lundin Jeanette , Rezvany Reza , Chudhury Aniruddha , Mellstedt Håkan , Osterborg Anders
Published: October 2019

Fludarabine and alemtuzumab (humanized anti-CD52 antibody, Campath®) are both used as routine therapy for patients with B-CLL. Fludarabine and in particular alemtuzumab, in addition to their antitumor effects, induce long-lasting suppression of T-cell numbers in blood. T-cell suppression, in combination with advanced disease, may result in an increased risk of opportunistic and other infections. In addition to a decrease in circulating T cells, functional defects in T cells have been described following alemtuzumab therapy for non-malignant disorders; however, only a limited amount of information exists about the effect of alemtuzumab on T-cell function in patients with B-CLL. The aim of the present study was to characterize in detail various aspects of T-cell function in patients with B-CLL who were in stable unmaintained PR or CR following fludarabine (n = 9) and alemtuzumab (n = 10) treatment as well as in age-matched healthy control donors (n = 10). CD4 and CD8 T-cell subsets of freshly obtained peripheral blood mononuclear cells (PBMC) were analyzed by flow cytometry to measure the expression of TCR-CD3 ζ chain, p56Lck, p59Fyn, ZAP-70, and PI3 Kinase as well as intracellular production of IFN-γ and IL-4. Additional analyses were performed to measure the proliferative response capability to a recall antigen (PPD) in alemtuzumab-treated patients and (as a control) indolent, untreated B-CLL patients (n = 11). The expression of IFN-γ and IL-4 (measured as mean fluorescence intensity [MFI]), but not the total number of positive cells, was significantly higher in CD4 and CD8 T cells for both CLL groups compared to healthy donors with the highest expression observed in alemtuzumab-treated patients. The total number of T cells that expressed the five signaling molecules was significantly lower in treated patients versus control donors; however, there were no differences between fludarabine- and alemtuzumab-treated patients. MFI analysis showed a significant increase in the TCR-CD3 ζ chain in fludarabine-treated patients compared to control donors; however, MFI analysis revealed no other significant differences between control group patients and fludarabine- and alemtuzumab-treated patients. Moreover, the proliferative response to PPD was not significantly different in alemtuzumab-treated patients and indolent CLL patients. In conclusion, these results suggest that the intrinsic signal transduction machinery in T cells is relatively well-preserved, such that T cells remain functionally intact following fludarabine or alemtuzumab therapy despite a marked reduction in their numbers. These results provide a better understanding of the immune suppression induced by alemtuzumab and fludarabine therapy in B-CLL patients.
Identification of Thymus Settling Progenitors.
Authors: Schwarz Benjamin A. , Bhandoola Avinash
Published: October 2019

T cells develop in the thymus, but are ultimately derived from hematopoietic stem cells (HSCs) that reside in the bone marrow. In order to produce T cells throughout adult life, the thymus must be periodically seeded by bone marrow progenitors via the blood. The identity of progenitors that seed the adult thymus is unknown. To determine which bone marrow progenitors that have access to they thymus, we analyzed the blood of adult mice (Schwarz & Bhandoola, Nature Immunology 2004). We found that the only progenitors in blood with T lineage potential were lineage negative cells with high expression of Sca-1 and c-Kit (LSK). Such LSK cells in blood were potent T lineage progenitors, with the capacity to expand over a million fold in the thymus. Like the corresponding population in the bone marrow, the blood LSK population was heterogeneous, containing HSCs and downstream multipotent progenitors (MPPs) including RAG-expressing early lymphoid progenitors (ELPs) and CD62L+ cells. In order to determine which of these LSK subsets can settle in the thymus, we developed a quantitative assay for thymic seeding in normal adult mice. We find that the fraction of LSK cells that settle in the thymus from the blood is extremely small. Of the estimated 3,000 to 4,000 LSK cells that pass through the thymic circulation each day, less than 10 cells are able to settle in the thymus. Our data suggest that any decrease in thymic seeding, as may occur in aging, would lead to a decrease in total thymic output.
Enforced Expression of Pax-5 Results in Developmental Arrest, Immortalisation and Aberrant Expression of B Lineage Genes in Committed Myeloid Progenitors.
Authors: Anderson Kristina , Rusterholz Corinne , Mansson Robert , Jensen Christina , Bacos Karl , Sasaki Yutaka , Sigvardsson Mikael , Jacobsen Sten Eirik W.
Published: October 2019

The paired domain transcription factor Pax-5, has been demonstrated to play a crucial role in development and final commitment of the B cell lineage. In fact, otherwise committed B cell progenitors acquire multipotentiality (myelo-lymphoid) potential upon targeted deletion of Pax-5 expression (Nutt et al. Nature.1999; 401:556–562). Thus, in addition, to promoting B cell development through acting as an activator of transcription of B cell specific genes such as CD19, Pax-5 is also thought to act as a suppressor of transcription of genes involved in determination of other blood cell lineages. However, it remains unclear how Pax-5 might repress myeloid development. Thus, we investigated the effect of Pax-5 expression on lympho-myeloid differentiation by overexpressing human (h)Pax-5 (through retroviral transduction) in adult murine bone marrow Linlo/−Sca-1+c-kit+ (LSK) cells. When compared to cells transduced with a control vector, LSK cells ectopically expressing hPax-5 very efficiently developed into hPax-5+B220+CD19+ pro-B cells in response to flt3 ligand and interleukin-7 in vitro. In contrast, when hPax-5+ LSK cells were cultured under myeloid conditions, we consistently observed development of a highly proliferative and immortalised bi-phenotypic (B-myeloid) hPax-5+B220+Gr-1+Mac-1+ population that predominantly consisted of immature myeloblasts but also maturated granulocytes and monocytes. Global gene expression analysis by micro-array, and confirmation by RT-PCR, demonstrated that hPax-5+B220+Gr-1+Mac-1+ cells also possessed a bi-phenotypic gene expression pattern, characteristic for B-cell as well as myeloid lineages including the Pax-5 target B cell genes mb-1 and BLNK as well as GM-CSFRα and low levels of C/EBPα. Similar findings were observed when targeting committed myeloid progenitors. These findings suggest that in addition to promoting B cell development, Pax-5 is capable of inhibiting/blocking myeloid differentiation and inducing immortalization as well as expression of B cell specific genes in otherwise myeloid committed progenitors. These findings motivate a careful investigation of the potential involvement of Pax-5 also in myeloid leukemias.
High Level CD24 Expression Is Correlated to Higher Metastatic Potential of Breast Cancer Cells.
Authors: Park Gyeongsin , Kim Hae-Jung , Park Bo-Bae , Park Woo-Chan , Lee Kyo-Young , Kang Chang-Suk , Oh Il-Hoan
Published: October 2019

Cancer stem cells are subpopulations of cancer cells with potential to seed and develop new cancer mass causing recurrence and/or metastasis, but phenotypic characteristics of such cell populations remains uncertain. As an initial approach to identify heterogeneity in cancer mass, we performed comparative study using matched pairs of primary breast cancer masses and their immediate metastatic counterparts in regional LN.

First, in the gene expression study of primary tumor mass and their LN counterpart using cDNA microarray, about 100 genes showed differential expression between the two, but with variations depending on cases (3 Exp). In addition, when fresh individually matched surgical block from primary mass and LN mass were each inoculated into NOD/SCID mice, higher growth of LN-derived mass was observed supporting distinction between the two (2 Exp). In subsequent search for phenotypic difference between the two, 99 primary-LN tissue pairs with individual matching were immunostained for CD24, a newly proposed marker for cancer stem cell, and analysed by semiquantitative scoring (0, 1+, 2+, 3+, 4+). Among 73 cases with positive staining for CD24, higher level of CD24 expression in LN mass compared to its matched primary tumor tissue was observed in 38 cases (52.1%), while the opposite was for only 7 cases (9.6%). Furthermore, high level expression (4+) of CD24 was observed more frequently in LN tumor tissues (63.0%, 46/73) than in their primary tumor counterparts (26%, 19/73)( P = 0.000). These results support the notion that cancer cells in tumor mass are clonally heterogeneous and suggest that CD24 expression could be related to characteristic of metastatic cancer stem cells.
Significance of Marrow Fibrosis in Chronic Phase Chronic Myelogenous Leukemia (CML) Post Interferon-A Failure Treated with Imatinib Mesylate Therapy.
Authors: Alvarez Ricardo H. , Kantarjian Hagop M. , Bueso-Ramos Carlos , Talpaz Moshe , O’Brien Susan , Giles Francis , Rios Mary Beth , Shan Jianqin , Cortes Jorge
Published: October 2019

Background. Myelofibrosis in the setting of CML has been historically associated with poor prognosis and with transformation. Its significance in the context of imatinib therapy is unknown.

Study Aims. To evaluate the prognostic relevance of myelofibrosis in patients with chronic phase CML post interferon-a failure treated with imatinib.

Patients and Methods. 110 patients with Philadelphia chromosome (Ph)-positive CML were analyzed. Imatinib was given at 400 mg orally daily. Marrow biopsies obtained prior to imatinib were subjected to Snook’s reticulin staining and graded on a scale of 1 to 4 for the degree of reticulin fibrosis as previously reported (Dekmezian.Cancer 59:1739, 1987). Severe fibrosis included grades 3–4.

Results. Patients’ median age was 57 years (24 to 81). Median duration of chronic phase was 31 months (range 6–148). Reticulin fibrosis was grade 1 in 4%, grade 2 in 35%, grade 3 in 28% and grade 4 in 33%. Splenomegaly was more frequent with grade 3–4 fibrosis (p=0.04). However, patients with severe fibrosis had a similar incidence of complete cytogenetic response and survival as those with lesser degrees of fibrosis.

Parameter Fibrosis Grade 1–2 3–4 pvalue No. analyzed 43 67 No. cytogenetic CR (%) 25(58) 45(67) .45 Estimated cytogenetic CR (%) 88% 80% .27

Conclusions. Imatinib, being a highly effective therapy in CML, may have reduced or eliminated the previously known prognostic impact of myelofibrosis in CML.
Gene Expression Profiling of Philadelphia Chromosome(Ph) Negative CD34+ Hematopoietic Stem and Progenitor Cells of Patients with Ph Positive CML in Complete Molecular Remission during Therapy with Imatinib.
Authors: Neumann Frank , Teutsch Nathalie , Kliszewski Slawomir , Bork Simone , Brors Benedikt , Schimkus Nadine , Roes Nicole , Steidl Ulrich , Germing Ulrich , Hildebrandt Barbara , Royer-Pokora Brigitte , Eils Roland , Gattermann Norbert , Haas Rainer , Kronenwett Ralf
Published: October 2019

Treatment of chronic myelogenous leukemia (CML) with Imatinib has shown its efficacy and superiority to conventional therapies. Beside inhibition of BCR-ABL Imatinib also has inhibitory effects on other tyrosine kinases such as wild-type ABL, c-KIT and platelet-derived growth factor receptors (PDGF-R) alpha and beta which play a role in differentiation and proliferation of hematopoietic stem cells and are involved in DNA repair mechanisms. Inhibition of these genes might therefore result in functional disturbance of hematopoiesis or even in secondary karyotypic abnormalities. Therefore, we compared gene expression profiles of immunomagnetically isolated CD34+ bone marrow (BM) cells from six healthy volunteers with Ph negative CD34+ cells from BM of eight patients (6 males, 2 females; median age: 52 years, range: 25–68 years) with Ph positive CML in chronic phase who reached major molecular remission during imatinib therapy (400 mg/day; 28 months (range: 11–39 months)). Cytogenetics, FISH analysis and quantitative real-time RT-PCR for BCR-ABL transcripts showed that all patients were in complete cytogenetic and in major molecular remission ( ≥ 3 log reduction of the BCR-ABL/G6PDH ratio in comparison to pretreatment value). CD34+ cells (median: 5 x 105; range 2 x 105–1.8 x 106) were isolated immunomagnetically from bone marrow with a purity >98%. Total RNA (median: 550 ng; range: 130 – 990 ng) from isolated CD34+ cells was used to generate biotin-labelled cRNA (median: 7.5 μg; range: 2.6–12.3 μg). Labeled cRNA was hybridized to Affymetrix HG-Focus GeneChips covering 8793 genes representing a broad spectrum of different functional groups. For normalization and data analysis we used the statistical scripting language “R”. Raw data were normalized using a method of variance stabilizing transformations (VSN). To compare the normalized data from CD34+ cells of imatinib-treated patients and healthy volunteers we used the Significance Analysis of Microarrays (SAM) algorithm v1.21. We found that CD34+ cells of patients after reaching major molecular remission during first-line treatment with 400 mg imatinib per day showed no significantly differentially expressed genes in comparison to CD34+ cells of healthy volunteers in vivo. There was no evidence for transcriptional changes of genes involved in DNA repair or oncogenesis supporting the view that secondary chromosomal aberrations in Ph negative hematopoiesis observed during imatinib therapy might not be induced by the tyrosine kinase inhibitor itself. Our data suggest that imatinib as first-line therapy might not result in a major functional disturbance of Ph negative hematopoiesis.
Recurrent Fusion of PCM1 to JAK2 in Atypical Chronic Myeloid Leukemia and Acute Leukemia Associated with the t(8;9)(p21-22;p23-24).
Authors: Reiter Andreas , Walz Christoph , Watmore Ann , Schoch Claudia , Blau Ilona , Telford Nick , Aruliah Shilani , Yin John , Vanstraelen Danny , Barker Helen , Taylor Peter , O’Driscoli Aisling , Schlegelberger Brigitte , Berger Ute , Hochhaus Andreas , Hehlmann Rüdiger , Chase Andrew , Cross Nicholas C.P.
Published: October 2019

We have identified a novel, recurrent t(8;9)(p21–22;p23–24) in six patients with diverse hematological malignancies: atypical CML (n=4), secondary AML following idiopathic myelofibrosis (n=1) and pre B-ALL (n=1). Because of the involvement of several different tyrosine kinases in atypical CML, we focused our analysis on this class of gene. Initial FISH studies of one patient indicated that the janus kinase 2 gene (JAK2), located at 9p24, was disrupted. RACE-PCR was then used to identify the 8p21 partner gene as PCM1, a large centrosomal protein that contains multiple coiled-coil domains. RT-PCR and FISH analysis confirmed the fusion in this case, and also identified PCM1-JAK2 in the five other t(8;9) patients (RT-PCR and FISH, n=4; RT-PCR only, n=1; FISH only, n=1). Four different types of in-frame mRNA junction were identified, but in all cases the chimeric mRNA is predicted to encode a protein that retains several of the predicted coiled-coil domains from PCM1 and the entire tyrosine kinase domain of JAK2. Reciprocal JAK2-PCM1 mRNA could not be amplified in any patient. Clinically, 4 patients displayed CML-like hyperplasia with variable degrees of myelofibrosis and eosinophilia. Similar to typical CML, the clinical course of these patients was variable: one is alive 11 months after allogeneic stem cell transplantation, one transformed to acute leukemia 5 years after diagnosis, one died 4 days after presentation and one achieved a major cytogenetic response with interferon but died due to neurodegenerative disease and pneumonia at 7.5 years. Of the two remaining patients, one presented with secondary AML following idiopathic myelofibrosis and remains in remission 15 years after diagnosis following intensive chemotherapy and maintenance with interferon. The final patient died shortly after induction therapy for pre B-ALL. In conclusion, PCM1-JAK2 is a novel recurrent fusion gene in hematological malignancies that is likely to deregulate hemopoiesis in a manner similar to BCR-ABL. Patients with PCM1-JAK2 disease are attractive targets for signal transduction therapy.
Flow Cytometric FLT3 Expression in Acute Leukaemias Is of Diagnostic Value but Does Not Correlate with ITD / D835Y Mutation Status.
Authors: Ridler Colin , Smith Matthew , Milne Tim , Ebrahim Fatema , Macey Marion , Lister T. A. , Fitzgibbon Jude , Cavenagh Jamie D. , Agrawal Samir G.
Published: October 2019

High level expression of FLT3 in acute myeloid (AML) and lymphoid (ALL) leukaemias has been demonstrated by PCR techniques. This suggests that FLT3 and its ligand may play a significant role in the proliferation of leukaemic blast cells. Mutations of FLT3 are the most common genetic lesion in AML, occurring in 20–30% of cases, and confer an adverse prognosis. Flow cytometry was used as an alternative method to investigate FLT3 expression in acute leukaemia. Surface expression of FLT3 was measured in 111 bone marrow or peripheral blood samples of patients with AML or ALL using a phycoerythrin-labelled monoclonal IgG1 antibody (Coulter-Immunotech). Exons 14 – 15 and 20 were amplified from DNA extracted from mononuclear cells of 38 of the 111 samples. Internal tandem duplication (ITD) and mutation of the D835 position of the activation loop of FLT3 were identified by SSCP and direct sequencing. 82% (47/57) of AML and 81% (34/42) of B-lineage ALL samples showed ≥ 20% (range, 20 – 97%) positivity for FLT3 using flow cytometry. The Mean Fluorescence Intensity (MFI) was similar between the two groups and no significant difference was found between adult (82%) and paediatric (80%) samples. However, FLT3 expression in all 9 T-ALL samples (pro, intermediate and late subtypes) was negative with markedly reduced MFIs. Of the 25 AML cases tested for ITD/D835Y, 5 (20%) were positive (4 ITDs, 1 D835Y). Surface FLT3 expression in these 5 cases (% and MFI) was the same as in those with wild-type FLT3. No mutations of FLT3 were found in the ALL cases tested (9 B-ALL, 4 T-ALL). Of three biphenotypic leukaemias, one case with myeloid/B lineage positivity was FLT3+ by flow cytometry, while another with myeloid/T lineage markers, was FLT3−. A diagnostic conundrum was a patient with AML morphologically (cytogenetics failed): CD33+, MPO−, CD13− CD117−; T-lineage markers CD2+, CD5+, CD7+, as well as TdT+. The T-lineage specific marker cytoplasmic CD3 was negative. Only FLT3 positivity allowed a definitive diagnosis of AML to be made. In conclusion, surface FLT3 expression was present in > 80% of AML and B-lineage ALL, but in 0% (0/9) of T-ALL cases. Detection of this antigen should be included in the diagnostic immunophenotyping of acute leukaemias and is important where T-ALL needs to be excluded. Flow cytometric FLT3 expression cannot differentiate cases with or without ITD or point mutations of FLT3.
In Chronic Inflammation, There Exists an IL-6 Independent Pathway for the Induction of Hepcidin.
Authors: Rivera Seth , Gabayan Victoria , Ganz Tomas
Published: October 2019

The iron regulatory hormone hepcidin is presumed to be the mediator of anemia of inflammation (AI). Patients with hepatic adenomas producing large amounts of hepcidin had severe AI that resolved when the tumors were resected (Weinstein et al, 2002). Patients with disorders associated with AI and mice with acute inflammation also have increased hepcidin. However, hepcidin deficient mice do not develop hypoferremia in response to acute inflammation (Nicolas et al, 2002). We previously showed that IL-6 was necessary for the inflammatory induction of hepcidin and the development of hypoferremia in an acute turpentine model of inflammation (Nemeth et al, 2004). In order to determine whether IL-6 was necessary for the development of AI, we induced chronic peritoneal S. epidermidis abscesses in IL-6 deficient mice (19 with abscesses, 12 controls) and wild type mice with the same genetic background (16 with abscesses, 10 controls). In contrast to our previous results, both IL-6 deficient and wild type mice had a significant rise in hepcidin-1 expression (27.6 vs. 10.9-fold, respectively) but there was no difference between the groups (p=0.939 by 2-way ANOVA). Both IL-6 deficient and control mice demonstrated a similar small but significant fall in hematocrit (49% to 44% vs. 46% to 42%, p=0.538 by 2-way ANOVA) in response to the abscesses. IL-6 is not necessary for the induction of hepcidin or the development of AI in a physiological model of infection. Experiments are underway to define which other inflammatory mediators induce hepcidin.
Functional Studies of Human Bone Marrow Derived Side Population Cells.
Authors: Banu Naheed , Fabian Attila , Mulligan Richard C.
Published: October 2019

Rationale:

We have previously reported that staining of murine or human bone marrow with the vital dye, Hoechst 33342, identifies a rare population of dye-effluxing cells termed the “side population” (SP). In mice, competitive blood repopulation assays demonstrate that murine SP cells are highly enriched in hematopoietic stem cells (HSC). However, whether bone marrow SP cells in humans similarly possess functional stem cell characteristics is not currently known. We tested the stem cell potential of human SP cells in comparison to non-SP and AC133+ cells by transplantation of bulk or purified marrow sub-populations into irradiated NOD/SCID mice.

Methods:

Ficolled human bone marrow preparations were stained with Hoechst dye or a fluorescence-conjugated antibody against AC133. Flow cytometry was used to purify SP cells, non-SP cells designated as ‘MP”, AC133+ cells, or unfractionated mononuclear (MNC) cells. Several cell doses of each sub-population were intravenously transplanted into sub-lethally irradiated SCID/NOD recipient mice. Long-term bone marrow chimerism was analyzed 6–8 weeks post transplant to assess the SCID/NOD repopulating potential of each sub-population.

Results:

Transplantation of 1x10^6, 500, 000 and 100, 000 mononuclear cells resulted in detectable but low-level marrow chimerism 5.14%, 1.13%, 0.17%. Transplantation of same number of MP cells also resulted in low level of chemerism (3.88%, 1.20%, 0.67%) compared to SP cells. High level of chemerism was obtained with 40,000, 20,000, and 10,000 SP cells 7.09%, 1.4%, and 0.50% respectively. In comparison 40,000, 20,000, and 10,000 AC133+ cells resulted in 2.57%, 0.97, and 0.07% chimerism. To examine whether the ability of AC133+ cells to efflux Hoechst dye could better identify cells with HSC potential we transplanted 5,000 AC133+ SP cells vs. 5,000 AC133+ non-SP cells into SCID/NOD recipients and found that AC133+ cells that do not efflux Hoechst (non-SP) do not contribute to long-term marrow chimerism whereas AC133+ SP cells resulted in 0.63% chimerism.

Conclusion:

We show for the first time that human bone marrow SP cells possess functional hematopoietic stem cell characteristics. Similarly, all of the NOD/SCID repopulating activity of adult human bone-marrow AC133+ cells are found to be more enrich in the “SP”AC133+ fraction (0.1% SP) compare to total mononuclear, or “MP”AC133+ or total AC133+ cells.