bluuebunny/crossref_metadata_2025 huggingface dataset:
Outcome Of Aplastic Anemia In Adolescence. A Survey Of The Severe Aplastic Anemia Working Party (SAAWP) Of The European Blood and Bone Marrow Transplant Group (EBMT)
Authors: Dufour Carlo , Pillon Marta , Passweg Jakob , Socié Gérard , Bacigalupo Andrea , Franceschetto Genny , Oneto Rosi , Risitano Antonio M. , Peffault de la Tour Regis , Tichelli Andre , Rovo Alicia , Peters Christina , Hochsmann Britta , Schrezenmeier Hubert , Aljurf Mahmoud D. , Marsh Judith
Published: October 2019

We analyzed the outcome according to different treatments of 537 adolescents (age 12-18 years) with acquired aplastic anemia from the data-base of the SAAWP of the EBMT.

OS after front-line matched family donor (MFD) hematopoietic stem cell transplantation (HSCT) was 86% whereas after front-line immunosuppressive therapy (IST) was 82% (p= 0.53). EFS (events being death, relapse/no response, PNH/MDS/AML evolution, transplant) was 83% after MFD HSCT and 37% after IST (p=0.0001).

To evaluate the outcome of adolescents who were transplanted after failure of IST upfront (HSCT after failed IST) we extrapolated these patients from the original population receiving front-line immunosuppression and compared with those undergoing MFD HSCT upfront and IST upfront but who were not subsequently transplanted (IST up-front alone in whom transplant was not an event). OS in MFD HSCT was 86%, in IST up-front alone 90% and in HSCT post failed IST 78% (p=0.14). EFS was respectively 83%, 64% and 71% (p= 0.04) with no difference in post hoc analysis between IST alone and HSCT post IST (p=0.17). In the transplanted population, OS and EFS were significantly higher with bone marrow vs peripheral blood as source of cells. No differences in OS and EFS were seen in patients transplanted in adult, pediatric or mixed centres.

Cumulative incidence (CI) of rejection was 8 % in MFD HSCT and 9% in HSCT post failed IST (p=0.62). CI of Ac GVHD was 12% in MDF HSCT and 18% in HSCT post failed IST (p=0.18). CI of Chronic GVHD was 8% in MFD HSCT and 20% in HSCT post failed IST (p=0.0009). CI of secondary malignancies was 0.7% in MDF HSCT, 7% in HSCT post-failed IST and 21% after IST up-front alone (p= 0.0017).

In the whole cohort in multivariate analysis, the diagnosis to treatment interval of ≤2 months positively affected OS whereas IST alone negatively affected EFS. In transplanted patients interval diagnosis-treatment ≤2 months, first line MDF HSCT and BM as source of cells provided a significant advantage in OS and EFS. Aplastic anemia in adolescents has a very good outcome. If a MFD is available, HSCT using BM cells is the first choice treatment. If a MFD is not available, IST looks a reasonable second choice. If IST fails HSCT represents a very good rescue option.

Disclosures:

Dufour: Pfizer: Consultancy, Research Funding. Marsh:Sanofi: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria.
Disturbed CXCR4/CXCL12 Axis In Pediatric Precursor B-Cell Acute Lymphoblastic Leukemia
Authors: van den Berk Lieke C.J. , Veer Arian van der , Willemse Marieke E. , Theeuwes Myrte J.G.A. , Luijendijk Mirjam W. , Tong Wing H. , van der Sluis Inge M. , Pieters Rob , den Boer Monique L.
Published: October 2019

Malignant cells that infiltrate the bone marrow (BM) interfere with the normal cellular behavior of supporting cells, thereby creating an alternative malignant niche. This intercellular communication is mostly mediated by cytokines and their receptors. In this study, we find that expression of the CXCR4 receptor is significantly increased in pediatric precursor B-cell acute lymphoblastic leukemia (BCP-ALL) cells compared with normal mononuclear hematopoietic cells derived of the bone marrow (p=0.016). Furthermore, we show that high CXCR4 expression is correlated with an unfavorable clinical outcome in BCP-ALL (5-yr CIR ±SE: 38.4% ±6.9% in CXCR4-high versus 12.0% ±4.6% in CXCR4-low expressing patients, p<0.001). Interestingly, BM serum levels of the CXCR4 ligand (CXCL12) are 2.7-fold lower (p=0.005) in samples taken at initial diagnosis of BCP-ALL compared with the levels in samples taken of non-leukemic controls. We show that induction chemotherapy restores CXCL12 levels in the BM to normal levels. Blocking the CXCR4 receptor with Plerixafor (FDA-approved drug) showed that the lower CXCL12 serum levels at initial diagnosis could not be explained by consumption by the leukemic cells, nor did we observe an altered CXCL12-production capacity of BM-MSC at this time-point. We rather observed that a very high density of leukemic cells negatively affected CXCL12 production by the BM-MSC while stimulating the secretion levels of G-CSF. These results suggest that highly proliferative leukemic cells are able to down-regulate the production of cytokines involved in homing (CXCL12), while simultaneously up-regulating the production of cytokines involved in hematopoietic mobilization (G-CSF). This disbalance may stimulate the spreading of BCP-ALL outside the BM.

The data presented here suggest that interference with the CXCR4/CXCL12 axis (for instance by using Plerixafor) may be an effective way to mobilize BCP-ALL cells; the more ALL cells become mobilized, the less ALL cells may escape from combination chemotherapy. In proof-of concept studies, this hypothesis needs to be validated to pave the way for implementation in future treatment protocols for children with ALL.

Disclosures:

No relevant conflicts of interest to declare.
BCR-ABL1+ Leukemic Stem Cells Are Dependent On Selectin-Ligand Interactions For Engraftment In The Bone Marrow Niche
Authors: Krause Daniela S. , Lazarides Katherine , Lewis Juliana B. , von Andrian Ulrich H. , Scadden David T. , Van Etten Richard A.
Published: October 2019

Leukemic progenitors from patients with chronic myelogenous leukemia (CML) have defects in the adhesive function of β1 integrins and in their response to the chemokine CXCL12 (Stem Cells 2002;3:259), pathways that are critical for engraftment and maintenance of normal hematopoietic stem cells (HSC) in the bone marrow (BM) niche. Previous studies in a mouse BCR-ABL1 retroviral transduction/transplantation model of CML demonstrated that BCR-ABL1+ leukemic stem cells, but not normal murine HSC, are dependent on expression of the CD44 adhesion molecule for BM homing and engraftment (Nat Med 2006;12:1175). To investigate further other adhesion molecules required for stable engraftment of CML stem cells, we employed donor and recipient mice with targeted mutations in genes encoding selectins and those required for expression of selectin ligands in the mouse retroviral CML model. Neither the β1 integrin ligand VCAM-1 nor P-selectin was required in the BM endothelium of recipient mice for efficient engraftment and induction of CML-like myeloproliferative neoplasia (MPN) by BCR-ABL1-expressing stem cells. By contrast, loss of recipient E-selectin significantly impaired engraftment of BCR-ABL1+ stem/progenitor cells, as demonstrated by decreased frequency of BM proviral leukemia-initiating clones, a defect overcome by direct intrafemoral injection of the leukemic cells. We also demonstrated a requirement for expression of selectin ligands on the leukemic stem cells, as BCR-ABL1-expressing stem cells lacking enzymes contributing to biosynthesis of selectin ligands (Core2 or Fucosyltransferases IV and VII) exhibited decreased efficiency of engraftment, resulting in attenuated disease. Further, donor cells deficient in P-selectin glycoprotein ligand-1 (PSGL-1) or in both PSGL-1 and CD44, a glycoform of which can function as a selectin ligand on hematopoietic stem cells (J. Biol Chem 2001;278:47623), displayed intermediate or profound impairment in engraftment of CML-like leukemia, respectively. Removal of selectin ligands by neuraminidase treatment of BCR-ABL1-transduced HSC blocked engraftment of leukemic stem cells and resulted in long-term survival of transplant recipients. Together, these results demonstrate that BCR-ABL1-expressing stem cells are dependent on selectin ligands, expressed on PSGL-1 and possibly on CD44, and their interaction with BM E-selectin for engraftment and retention in the BM microenvironment. Disrupting these niche interactions could represent new avenues for targeting CML stem cells that are resistant to ABL kinase inhibitors like imatinib.

Disclosures:

Van Etten: Bristol Myers Squibb: Consultancy; Deciphera Pharmaceuticals: Consultancy; TEVA Pharmaceuticals: Consultancy, Research Funding.
A Phase I Study Of Nilotinib As a c-Kit Inhibitor Combined With Re-Induction Chemotherapy For Relapsed and Refractory c-Kit Positive Acute Myeloid Leukemia
Authors: Brandwein Joseph M. , Hedley David , Chow Sue , Yee Karen , Schuh Andre C , Gupta Vikas , Schimmer Aaron , Minden Mark D.
Published: October 2019

Background

A previous study in relapsed/refractory AML, combining re-induction chemotherapy with imatinib as a c-kit inhibitor, demonstrated that effective inhibition of downstream pAkt was associated with a higher CR rate (Brandwein et al, 2011). Nilotinib is also a c-kit inhibitor which diffuses into cells and is therefore not affected by potential defects in active cellular influx mechanisms.

Methods

Patients age 18-65 with acute myeloid leukemia (AML) who had persistent leukemia after 3+7 induction with cytarabine and daunorubicin, or relapsed within 24 months of achieving CR, and whose blasts were c-kit (CD117) positive, were enrolled in a phase I clinical trial with nilotinib on Days 1-12 combined with mitoxantrone 10 mg/m2/day IV Days 6-10, etoposide 100 mg/m2/day IV Days 6-10 and cytarabine 1.5 g/m2 IV q12h x 4 doses on Days 11-12. None of the cases harbored c-kit mutations. Nilotinib was escalated through 2 dose levels (400 mg daily and 400 mg BID) in successive 6 patient cohorts; up to 2/6 dose-limiting toxicities (DLTs) were permitted.

Results

At the 400 mg once daily nilotinib dose there were 2 DLTs, both hematologic and consisting of prolonged platelet and neutrophil recovery. At the 400 mg BID dose there was one DLT, consisting of grade 3 transient liver enzyme elevation; 5/6 patients experienced transient grade I-II bilirubin elevations at this dose. The median time to ANC > 0.5 x109/L was 38 days (range 28-54) and to platelets > 20 x109/L was 30 days (range 28-54). Complete responses were seen in 10 patients, 5/6 at each dose level.

AML blasts from peripheral blood were assayed for pAKT, pERK and pS6 by flow cytometry, after stimulation with stem cell factor (SCF), on Days 1 and 6, pre- and 1.5 and 3 hours post-nilotinib dosing on each day. A decrease in the level of SCF-stimulated pAkt of at least 50% on Day 6 post-nilotinib, as compared to baseline, was seen in 1/6 patients at the 400 mg BID dose; the remaining 11 cases did not demonstrate an appreciable decrease in pAkt and other intermediates as compared to pre-nilotinib levels. Ex vivo dose titration curves showed that nilotinib inhibited c-kit mediated ERK and Akt activation in the blast cells of all cases, but concentrations >10 micromolar were needed to produce 50% inhibition in most cases.

Conclusions

The results indicate that nilotinib can be safely combined with this re-induction chemotherapy regimen, at doses up to 400 mg BID. Although the CR rate was high, in most cases nilotinib did not demonstrate significant inhibition of the c-kit pathway in leukemia cells in vivo. Dose titration curves showed that concentrations needed to produce 50% inhibition were generally above those achievable in vivo.

Disclosures:

Brandwein: Novartis: Honoraria, Research Funding. Off Label Use: nilotinib for acute myeloid leukemia.
HLA Ligandome Analysis Of Chronic Myeloid Leukemia (CML), Revealed Novel Tumor Associated Antigens For Peptide Based Immunotherapy
Authors: Stickel Juliane S. , Kowalewski Daniel J. , Schemionek Mirle , Kanz Lothar , Brümmendorf Tim H. , Salih Helmut R. , Rammensee Hans-Georg , Stevanovic Stefan
Published: October 2019

Despite the success of targeted therapy using tyrosine kinase inhibitors (TKIs), chronic myeloid leukemia (CML) remains largely incurable, most likely due to the treatment resistance of leukemic stem cells. Specific T-cell based immunotherapies for patients with CML might be able to eliminate these residual CML cells. For this goal the identification of tumor associated HLA-presented peptides, which are able to induce a tumor-specific T cell response, is indispensable. However, only few tumor associated antigens for CML are described till date. Thus the aim of this study was to identify for the first time naturally processed and presented HLA ligands from the cell surface of primary CML cells. HLA class I ligands from primary CML cells as well as from bone marrow mononuclear cell (BMNCs) and peripheral blood PBMCs of healthy donors were analyzed using the approach of direct isolation and identification of naturally presented HLA peptides by affinity chromatography and mass spectrometry (LC-MS/MS). LC-MS/MS peptide analysis provided qualitative and semi-quantitative information regarding the composition of the respective ligandomes. Comparative analysis of malignant and benign samples served to identify ligandome-derived tumor associated antigens (LiTAAs) and to select peptide vaccine candidates. So far 10 CML patients (8 chronic phase, 2 accelerated phase) and 30 healthy donors were analyzed within this study. We were able to identify a total of more than 8200 CML derived HLA ligands representing >4500 different source proteins, of which 734 were exclusively represented in CML, but not in healthy PBMCs/BMNCs. 55 of these CML exclusive antigens are presented on 3 or more of all examined CML patients, representing, as broadly represented LiTAAs, promising targets for peptide vaccination. For the first time, previously predicted CML tumor associated antigens for example Myeloperoxidase (10 peptide sequences on 7 CML patients) and Proteinase 3 (5 peptide sequences on 4 CML patients) were here confirmed by direct elution from primary CML cells, which also served to validate our methodological approach. Notably, beyond that we also identified a vast array of novel proteins (e.g. Carcinoembryonic antigen-related cell adhesion molecule 8, CEACAM8; Matrix metallopeptidase 8, MMP8; intracellular adhesion molecule 3, ICAM3) that are broadly and exclusively represented in the ligandome of CML cells.

By providing for the first time HLA class I tumor associated antigens, directly obtained from the HLA ligandomes of CML patients, this study may pave the way for the future development of a multipeptide-based immunotherapy approach to eradicate residual CML cells after conventional TKI therapy.

Disclosures:

No relevant conflicts of interest to declare.
Activating Mutations In Fyn Kinase In Peripheral T-Cell Lymphomas
Authors: Couronne Lucile , Palomero Teresa , Khiabanian Hossein , Kim Mi-Yeon , Ambesi Alberto , Carpenter Zachary , Abate Francesco , Allegretta Maddalena , Lossos Izidore S. , Nicolas Concha , Balbin Milagros , Bastard Christian , Bhagat Govind , Piris Miguel A. , Campo Elias , Bernard Olivier A. , Rabadan Raul , Ferrando Adolfo A.
Published: October 2019

Peripheral T-cell lymphomas (PTCLs) are a heterogeneous group of aggressive non Hodgkin lymphomas with poor prognosis. Molecular and cytogenetic studies have shown a prominent role for kinase fusion oncogenes, mostly NPM-ALK, in ALK+ anaplastic large cell lymphomas (ALCLs) and ITK-SYK kinase in unspecified PTCLs. To gain further insight on the genetics and pathogenic mechanisms of aggressive PTCLs we performed an integrated mutation analysis using whole exome sequencing (n=12) and RNAseq (n=35) data. This analysis identified 288 candidate coding somatic mutations in 268 genes including known recurrent mutations in the TET2, DNMT3A and IDH2 epigenetic factor genes and pointed to the FYN kinase gene as a new recurrently mutated oncogene in T-cell transformation. The FYN tyrosine kinase is, with LCK, the predominant SRC family kinase found in T lymphocytes and plays an important role in T-cell activation upon T-cell receptor (TCR) stimulation. FYN mutations in PTCL included a FYN L174R mutation detected in one AITL patient sample, a FYN R176C allele recurrently found in two PTCL NOS cases and a FYN Y531H mutation present in a PTCL NOS sample. Notably, each of these alleles are predicted to specifically disrupt the inhibition of FYN kinase activity by the C terminal SRC kinase (CSK). Thus, structure analysis of FYN and FYN mutant proteins predicted that FYN L174R and, most prominently, FYN R176C and FYN Y531H can disrupt the inhibitory interaction of the FYN SH2 domain with the CSK-phosphorylated Y531. Consistently, pull down assays using GST-FYN-SH2 recombinant proteins and biotinylated C-terminal FYN peptides encompassing Y531 showed abrogation of the interaction between FYN-SH2 and P-Y531 in each of these mutants. In agreement with these results, expression of FYN L174R, FYN R176C and FYN Y531H resulted in increased levels of FYN activation. Moreover, CSK expression effectively inhibited wild type FYN, but failed to abrogate FYN L174R, FYN R176C or FYN Y531H activation. In contrast, pharmacologic kinase inhibition with dasatinib, a multikinase inhibitor which blocks ABL1 and SRC kinases, effectively abrogated the activity of FYN L174R, FYN R176C and FYN Y531H mutant proteins and suppressed the growth of cells transformed via expression of activated FYN mutant alleles. Overall these results support an oncogenic role for FYN activating mutations in the pathogenesis of PTCL and support a role for SRC kinase inhibitors for the treatment of this disease.

Disclosures:

No relevant conflicts of interest to declare.
Therapeutic Efficacy and Cure Of Sensitive and T315I Pan-Resistant Human Ph+ Leukemia In Mice Using a TCR-Like Antibody To WT1/HLA-A0201 Alone, Or In Combination With Tyrosine Kinase Inhibitors
Authors: Dubrovsky Leonid , Brea Elliott , Pankov Dmitry , Veomett Nicholas , Dao Tao , Yan Su , Liu Cheng , Scheinberg David A.
Published: October 2019

Acute and chronic leukemias, including CD34+ CML stem cells, overexpress the Wilms tumor gene 1 (WT1) protein, making WT1 an attractive therapeutic target. ESKM is a fully human IgG1 antibody that targets a 9 amino acid sequence (RMF) of the protein WT1 in the context of HLA-A0201, allowing it to target an undruggable, widely expressed, intracellular oncogene product. BV173 is an HLA-A0201+, human Ph+ ALL cell line that expresses WT1, and tagged by our lab with luciferase. We engineered a tyrosine kinase inhibitor (TKI) resistant BV173-R cell line by transducing BV173 with the resistant T315I Bcr-Abl plasmid. Antibody-dependent cellular cytotoxicity (ADCC) was evaluated in vitro by chromium release assay, utilizing human PBMC effectors. Tumor growth in vivo was assessed in NOD/SCID gamma (NSG) mice with bioluminescence imaging (BLI). RT-PCR was used to evaluate minimal residual disease in mice with negative BLI signal at the end of therapy. Imatinib, dasatinib, and ponatinib were used at up to maximally tolerated doses, given IP once daily. ESKM was administered at 100 µg twice weekly IP. ESKM mediated ADCC against both BV173 and BV173-R cell lines in vitro. In a BV173 engrafted human leukemia xenograft model, ESKM was more potent than imatinib, with median tumor growth reduction of 78% vs 52%. Combination of imatinib and ESKM therapy resulted in a 94% reduction in leukemic growth. High dose dasatinib (40 mg/kg daily) was more potent than ESKM, but discontinuation of therapy due to dasatinib toxicity resulted in relapse. Combination with ESKM therapy with dasatinib resulted in cure in 75% of mice, confirmed by bone marrow RT-PCR three weeks after termination of therapy. For mice cytoreduced with dasatinib followed by consolidation therapy with ESKM, delayed relapse was observed, but no cures. ESKM was highly superior to imatinib and dasatinib against the T315I BV173-R leukemia in vivo. Cures were not achieved with combination therapy of ESKM and either first or second generation TKIs against resistant T315I leukemia. Ponatinib at 10 mg/kg had higher efficacy than ESKM alone against BV173-R, but mice treated with combination of ESKM and ponatinib had superior tumor reduction. CONCLUSION: ESKM is an effective therapeutic antibody for sensitive and T315I Ph+ ALL. Resistant T315I Ph+ leukemic growth is inhibited more effectively by ESKM therapy compared to imatinib and dasatinib, and combination therapy with ESKM is superior to ponatinib.

Supported by the Leukemia and Lymphoma Society, NIH R01CA55349, P01 23766 and T32CA62948-18.

Disclosures:

Yan: Eureka Therapeutics: Employment. Liu:Eureka Therapeutics: Employment, Equity Ownership.
Human Reticulocytes Extrude Inside-Out, Phosphatidylserine-Exposed, Autophagic Vesicles During The Final Step In The Formation Of Erythrocytes
Authors: Mankelow Tosti J. , Griffiths Rebecca E. , Flatt Joanna F. , Anstee David J.
Published: October 2019

During maturation to an erythrocyte, a reticulocyte must degrade or eliminate its residual cytosolic organelles and reduce its surface area. Using confocal microscopy, we show that in late reticulocytes produced from an in vitro culture system, this is achieved through a novel form of exocytosis whereby large (∼1.4um) intact, inside-out phosphatidylserine-exposed vesicles are expelled from the maturing reticulocyte. The vesicles contain organelle marker proteins and numerous erythroid membrane proteins, notably CD71, CD147 and stomatin. The presence of ubiquitin within these vesicles suggests a recognised mechanism for the targeting of proteins for extracellular export or degradation. The exocytosed vesicles are identical to intracellular GPA-decorated autophagic vesicles previously identified in cultured reticulocytes (Griffiths et al 2012). GPA-decorated vesicles are also seen in some cells in peripheral blood. Proteins involved in vesicle trafficking, SNARE (VAMP7), ESCRT (CHMP4B) and myosin, locate to defined positions at the point of vesicle extrusion. We hypothesize that the exposed “eat me” phosphatidylserine signal ensures that released autophagic vesicles are rapidly removed from circulation by professional phagocytic cells within the spleen thus ensuring maturation to erythrocytes without the release of potentially toxic material into circulation. Our results describe a previously unrecognised mode of exocytosis which may have significance beyond erythropoiesis particularly with respect to apoptosis and autophagy and reveal the final step in the formation of human erythrocytes.

Reference

Griffiths RE, Kupzig S, Cogan N, Mankelow TJ, Betin VM, Trakarnsanga K, Massey EJ, Lane JD, Parsons SF, Anstee DJ.Maturing reticulocytes internalize plasma membrane in glycophorin A-containing vesicles that fuse with autophagosomes before exocytosis. Blood. 2012 Jun 28;119(26):6296-306.

Disclosures:

No relevant conflicts of interest to declare.
How Does CD47-SIRPα ‘Don’t Eat Me Signal’ Physically Signal Self
Authors: Nisha Sosale , Discher Dennis E.
Published: October 2019

Resident macrophages in spleen and liver are particularly adept at recognizing foreign pathogens through recognition of ‘non-self’ proteins on the pathogen surface but also through the absence of ‘self’ proteins that are highly displayed on circulating blood cells. Red blood cells display a ‘marker of self’ protein CD47 which increases the in vivo half-life and decreases red-pulp splenic macrophage uptake of mouse RBC (Oldenborg et al, Science 2000) and also of particles displaying human-CD47 in recent studies by our group (Rodriguez et al, Science 2013). CD47 signals self through its counter receptor SIRPa, which is highly expressed on the surfaces of myeloid cells but also highly polymorphic. The CD47 protein functions in vitro as a marker of self toward human SIRPa on human macrophages and monocytes, inhibiting accumulation of myosin II motor protein to the phagocytic synapse (Tsai 2008). The work here aims to clarify when and how CD47-SIRPa inhibition physically signals ‘self’ during macrophage phagocytosis uptake. While it is clear that CD47 reduces the number of uptake events, here we use time-lapse and confocal microscopy to examine the forces of distortion imparted by phagocytes on opsonized red blood cell targets during uptake. Glutaraldehyde-fixed RBC are also used as a model to assess the affects of cell rigidity in this self-recognition process, since rigidity is relevant to processes as diverse as RBC aging and sickle RBC to malaria but also because adhesion (by macrophages) is expected to activate the myosin-II contractility system and oppose CD47 signaling. Through blocking and pharmacological approaches, we parse the pathways between foreign, self, and rigidity sensing.

Disclosures:

No relevant conflicts of interest to declare.
Consolidation Therapy Is Associated with Significantly Lower Bleeding Risk Compared to Induction Therapy in Patients with Acute Myeloid Leukemia
Authors: Schaefer-Eckart Kerstin , Wendelin Knut , Pilz Bettina , Kramer Michael , Ehninger Gerhard , Wilhelm Martin , Wandt Hannes
Published: October 2019

Background: Between 2005 und 2010 we conducted a multicenter randomized study comparing a therapeutic and a prophylactic (morning platelet (ptl) trigger < 10/nl) platelet transfusion strategy in patients with hematological disorders. (The Lancet Vol 380, No 9850, pp 1309-16). Briefly, we could show that a therapeutic plt transfusion strategy, where platelets are transfused in clinical stable patients (pts) only if bleeding ≥ WHO grade II occurs is safe in patients after autologous transplantation. In patients with acute myeloid leukemia (AML) we observed significantly more severe bleedings (WHO IV°) with the therapeutic regimen. To proof, if there is a difference in bleeding risk related to the remission status of the patients, we conducted a post-hoc analysis to compare the risk of severe bleeding (WHO III° and IV°) in induction with consolidation therapy.

Patients and Methods: We analyzed 175 pts with 175 cycles of induction therapy, 90 with a prophylactic transfusion regimen and 85 cycles with a therapeutic regimen. 131 pts received 268 cycles of consolidation therapy, 155 with a prophylactic and 113 with a therapeutic transfusion strategy.

Results: Bleedings WHO III° were neither different between the two strategies nor between induction and consolidation therapy. In contrast, there were significantly more bleedings WHO IV° in induction (7,4%) compared to consolidation therapy (1,5%; p=0,01). In addition, there were significantly more bleedings WHO IV° with the therapeutic regimen (11,8%) compared to the prophylactic strategy (3,3%) in induction therapy (p=0,012). This difference was less pronounced in consolidation therapy. But even in the prophylactic arm most WHO IV° bleedings occurred in pts with more than 10/nl ptl, which shows, that the morning platelet count should not be the only trigger for a platelet transfusion.

Conclusions: In consolidation therapy the risk of bleeding is significantly less compared to induction therapy, even with a therapeutic platelet transfusion strategy. During induction therapy the prophylactic strategy should remain the standard of care. The therapeutic transfusion strategy in consolidation therapy will be proven prospectively in an ongoing multicenter study.

Disclosures

No relevant conflicts of interest to declare.
Day 21 Chimerism Is a Critical Prognostic Factor for Graft Failure and Survival in Recipients of Myeloablative Conditioning and Umbilical Cord Blood (UCB) Transplantation
Authors: Bejanyan Nelli , Brunstein Claudio G , Cao Qing , Thyagarajan Bharat , Sandhu Karamjeet S. , Arora Mukta , Weisdorf Daniel J. , Wagner John E
Published: October 2019

Compared to other hematopoietic stem cell sources, UCB carries an increased risk of graft failure (GF), a life-threatening complication of transplant. Timely recognition of GF could help plan potentially curative salvage therapy, such as second grafting [Ruggeri et al, Haematologica 2014]. This study was aimed at identifying patients at high risk of GF. We hypothesized that early chimerism results might be a useful prognosticator. Therefore, we examined the results of day 21 bone marrow (BM) and peripheral blood (PB) chimerism testing in 327 (123 single UCB and 204 double UCB) patients receiving myeloablative conditioning (2000-2013) for various hematological malignancies. Recursive partitioning analysis was used to determine the optimal cut off of day 21 chimerism for neutrophil engraftment. Chimierism ≥85% was observed in 283 and <85% in 44 patients. For all patients surviving >21 days without evidence of disease relapse the incidence of neutrophil recovery was 92% (95% CI 89-95%) by day 42. However, for those with <85% chimerism, the rate of GF was 42% (n=20) as compared to only 3% (n=9) among patients with ≥85% chimerism at day 21 (p<0.01). As expected, 1-year overall survival (OS) was very poor in the <85% chimerism (21% with complications of GF as the most common cause of death). In multiple regression analysis, <85% chimerism was associated with 3.7-fold less likelihood of achieving neutrophil recovery by day 42 (HR 0.27, 95% CI 0.18-0.41; p<0.01) after adjustment for number of UCB units (p=0.04), HLA disparity (p=0.15) and year of transplant (p=0.10). Chimerism was also an independent risk factor for 1-year treatment related mortality (TRM, 19% vs. 39%; p<0.01), disease-free survival (63% vs. 42%; p=0.03), and OS (69% vs. 42%; p<0.01) among engrafted subset of patients with chimerism ≥85% versus <85%, respectively. In multivariable analysis, <85% chimerism remained prognostic for increased 1-year TRM (HR 2.14, 95% CI 1.06-4.31; p=0.03), treatment failure (HR 1.85, 95% CI 1.05-3.25; p=0.03) and mortality (HR 2.19, 95% CI 1.23-3.87; p<0.01) for all engrafted patients with myeloablative UCB transplant. Chimerism, however, was not an independent predictor of grade II-IV acute GVHD at 100 days (p=0.41), 1-year relapse (p=0.78) or chronic GVHD (p=0.41). Our data indicate that chimerism <85% at day 21 is critically important prognostic factor for graft failure and survival after UCB transplant. These results argue for early evaluation in order to intervene at an earlier time point to enhance survival.

Disclosures

No relevant conflicts of interest to declare.
Efficacy of Mesenchymal Stem Cells for the Treatment of Steroid-Refractory aGVHD
Authors: Watakabe Kyoko , Miyamura Koichi , Ozawa Yukiyasu , Okada Masaya , Yamashita Takuya , Murata Makoto , Ishikawa Takayuki , Uike Naokuni , Hidaka Michihiro , Kobayashi Ryoji , Imamura Masahiro , Tanaka Junji , Ohashi Kazuteru , Taniguchi Shuichi , Ikeda Takashi , Eto Tetsuya , Mori Masaki , Muroi Kazuo , Ozawa Keiya
Published: October 2019

Background: Steroid-refractory acute graft vs. host disease (SR-aGVHD) remains a significant complication of allogenic hematopoietic stem cell transplantation (allo-HSCT). In this phase II-III multicenter clinical study we evaluated the efficacy and safety of MSCs(JR-031; JCR Pharmaceuticals Co., Ltd., Japan) for SR-aGVHD.

Method: 25 patients (age: 5-66 years, median 33years; male 15, female 10) who developed SR-aGVHD (grade III 22, grade IV 3) after allo-HSCT (Nov. 2011-Sept. 2012) were enrolled and given 8 biweekly infusions of 2x106 cells/kg of JR-031 for 4 weeks, with an additional 4 infusions weekly after 28 days in patients with partial response (PR). They were followed up to 24 weeks.

Results: At 4 weeks after the first dose, 6 (24%) patients showed complete response (CR), 9 (36%) patients showed PR, 4 (16%) patients showed mixed response (MR) and 1(4%)patient showed no change (NC). During 24 weeks of observation period, 12 (48%) patients achieved durable CR (CR >=28days). As for response by organ, 80% (16/20) of GI, 66.7% (8/12) of skin and 66.7%(4/6) of liver GVHD completely responded (stage 0). 15 (60%) patients survived up to 24 weeks after the first dose.All patients experienced at least one adverse event(AE). Common AEs were leukopenia (12 patients), thrombopenia (9 patients), sepsis, anemia, TMA and hepatic dysfunction (6 patients). Among 10 death, causal relationship with JR-031 were not completely ruled out in 4 cases (TTP, pneumonia, sepsis and relapse of underlying malignancy), however there was no case in which the causality was strongly suggested.

Conclusion: It was suggested that JR-031 is a safe and effective therapy in the treatment of patients with SR-aGVHD.

Disclosures

Miyamura: Novartis: Honoraria, Speakers Bureau.
Androgen Function in Long-Term Survivors of Haematopoietic Stem Cell Transplantation
Authors: Topping Jonathon E , Todd Jeannie , Apperley Jane F. , Salooja Nina
Published: October 2019

Endocrinopathies are well known complications of haematopoietic stem cell transplants (HSCT) and their onset may occur many years after the transplant process. Infertility in men is common and occurs soon after exposure to high doses of radiation/chemotherapy as a result of Sertoli cell damage. Despite this testosterone levels are usually normal. We have investigated testosterone levels in male patients surviving a minimum of 10 years following of HSCT to see whether there are long term effects of chemoradiotherapy on testosterone production.

We have investigated testosterone levels in 46 long-term survivors of allogeneic HSCT attending a dedicated late effects clinic over a 12 month period. Data were compared to age-matched non-transplant controls in a paired case-control analysis. The controls were being investigated for fatigue but had no specific underlying illness. Where testosterone levels were low, repeat measurements were taken with LH and SHBG.

The median age at HSCT was 30 years (range 12-53) and the median duration of follow up was 19 years (range 10-34). 44/46 received cyclophosphamide/TBI conditioning. 32 patients had CML, 5 had ALL, 7 had AML, 1 had aplastic anaemia and 1 had lymphoma.

26/46 (57%) patients had a low testosterone reading at some point compared to15% in the control group (p < 0.0001). Two of these patients had a low SHBG and a normal free androgen index. 16/46 patients had persistently low levels which for 8 was primary testosterone deficiency with elevated LH and for 8 was secondary with inappropriate low gonadotrophins. In four patients the testosterone fluctuated around the normal level and for four it was an isolated low reading. The transplanted patient group had a significantly lower mean testosterone level at 10.8 nmol/l (range 3.2-20.3) compared to 15.2 nmol/l (range 7.5-28.1) in the control group (p = 0.00009). The probability of developing true hypogonadism (defined as consistently low testosterone levels after repeat measurements) increased from 0.087 at 10 years to 0.554 after 25 years.

We have shown that true hypogonadism is a common finding between 10 and 25 years post transplant. Implications include reduced energy levels, decreased libido, decreased bone density and sexual dysfunction. Our data indicate that for male patients surviving 10 years or more after HSCT annual biochemical surveillance is required.

Disclosures

Apperley: Ariad: Research Funding; Bristol Myers Squibb: Research Funding; Novartis: Research Funding; Pfizer: Research Funding.
Safety and Efficacy of Total Dose Infusion of Low Molecular Weight (LMW) Iron Dextran in a Large Population of Anemic Patients Across a Broad Spectrum of Diagnoses Associated with Iron Lack
Authors: Mehmood Tahir , Swe Khine , Das Gopimohan , Gozu Aysegul , Auerbach Michael , Aung Sein
Published: October 2019

Background: Physicians are reluctant to recommend high dose intravenous (IV) iron infusion for iron deficient (ID) patients due to concerns of adverse events (AEs) including anaphylaxis. This retrospective analysis evaluates efficacy and AEs with LMW iron dextran (ID) total dose infusion (TDI) in patients with iron deficiency.

Methods: We retrospectively evaluated the Electronic Medical Records of 468 consecutive patients with iron deficient anemia (IDA) who received IV LMW ID between 01/2008 and 07/2011 in an ambulatory infusion center. Diagnostic parameters for IDA included hemoglobin [Hgb] <11 g/dL, transferrin saturation [TSAT] 19%, ferritin <100 ng/mL. The majority had documented intolerance of or inadequate response to oral iron. All received TDI of LMW ID (1130 ± 217 mg) over 1 hour after a test dose of 25 mg. Pre-medication was not used. Patients were closely monitored for AEs during and immediately after infusions. Laboratory data for baseline and repeat hematological parameters within 8 weeks following infusions were analyzed. End points were safety and efficacy. Safety data was available for all 468 patients. Follow-up data at 8 weeks was available on 315.

Figure 1 Figure 1.

Results: There were 103 males and 365 female patients. Most common diagnoses were GI bleed, chemotherapy, menorrhagia and chronic kidney disease. At baseline, mean Hgb was 9.8 g/dL, ferritin 92.08ng/mL, and TSAT 12.37%. At 8 weeks the mean hemoglobin increased to 10.97 g/dL (p =0.0001), Ferritin 279.63 ng/mL (p =0.0001), and TSAT 21.85% (p=0.0001). All tolerated the infusion well. There were no incidents of fever, chills, wheezing, hypotension, stridor, periorbital edema, anaphylaxis or death. One complained of chest tightness, but completed the infusion after several minutes and the administration of diphenhydramine and steroids. One developed a rash. Fatigue and myalgia were reported in 17.31% and 8.33% of patients respectively.

Conclusions: This study demonstrates TDI of LMW ID can be given safely and conveniently over 1 hour in an outpatient setting without premedication to ID patients. Easily manageable minor reactions did not affect the therapeutic plan. No allergic or anaphylactic reactions were observed. This study demonstrated TDI of LMW ID improved hemoglobin levels more than 1 g/dL within 8 weeks. Consistent with the preponderance of published evidence TDI of LMW ID should be considered an important therapeutic option for iron replacement in patients with IDA.

Figure 1 Figure 1.

Disclosures

No relevant conflicts of interest to declare.
Myeloproliferative Syndrome with Leukemia Preceding Polycythemia Vera: A Case Report
Authors: CALABRESI PAUL
Published: October 2019

A case of chronic erythroleukemia is reported in which a picture of chronic granulocytic leukemia preceded the erythremic proliferation by one to three years. The patient was followed throughout his entire 10-year course and the clinical impressions were confirmed initially by biopsy and eventually at necropsy.

The rare examples of this occurrence usually cited in the literature are reviewed and briefly discussed. In every case the development of erythremia was preceded by radiation therapy for the leukemic dyscrasia.

A set of criteria for evaluating such cases in order to provide definite evidence for this sequence of events is suggested.

The transformation of chronic granulocytic leukemia into polycythemia vera or chronic erythroleukemia lends further support to the concept of fundamental similarity and close inter-relation of these myeloproliferative disorders.
Hematologic Findings in Human Beings Given Therapeutic Doses of Gallium-72
Authors: WOLINS W. , BOND V. P.
Published: October 2019

The hematologic effects of Ga72 administered intravenously to patients with diffuse bony metastases or primary bone malignancy were studied.

1. The effects of this internally administered isotope were, in general, similar to those resulting from total-body external radiation. The degree of effect was greater than that anticipated from estimates of the total-body dose received from the isotope. Localization in the bone may have been responsible for this finding.

2. The depth and duration of platelet count depression probably represented the best indices of the degree of marrow damage after radiation, and may be indicative also of the total damage sustained by the marrow from previous exposure.

3. The effects of large doses of radiation on the marrow appeared to be cumulative to a point of no return, beyond which regeneration may not be possible.

4. It was found that a total white count below 1,000 and a platelet count below 25,000 could be tolerated for weeks without infection or gross bleeding and with ultimate recovery. Therapy because of these findings alone did not appear to be indicated.
Folate Deficiency in Chronic Liver Disease
Authors: KLIPSTEIN FREDERICK A. , LINDENBAUM JOHN
Published: October 2019

Fifty-five patients with liver disease of varied etiology and severity have been studied. Serum folate concentrations were subnormal and folic acid clearances rapid, when studied, in 19 actively imbibing alcoholic cirrhotics who had a megaloblastic anemia. Eleven patients, from both the nonalcoholic and alcoholic groups, had rapid folic acid clearances, with subnormal serum folate levels in seven, in the absence of morphologic evidence of folate deficiency. Serum B12 concentrations were uniformly normal or elevated.

Dietary deficiency appeared to be the major cause of folate deficiency; all 19 patients who had megaloblastic changes were considered to have an inadequate dietary intake. Increased requirement for folate due to hyperactivity of the bone marrow secondary to gastrointestinal bleeding. hypersplenism, or hemolysis appeared to contribute to the development of abnormal folic acid determinations in many patients in both the alcoholic and nonalcoholic groups. Two of 10 patients studied had malabsorption of folic acid. Such factors as the presence of ascites, an expanded plasma volume, a Patent portacaval shunting procedure, the type of alcoholic beverage imbibed, and the severity of impairment of liver function did not appear to be of significance in the development of folate deficiency.
Expansion of the growth fraction in multiple myeloma with alkylating agents
Authors: Salmon SE
Published: October 2019

Patients with IgG multiple myeloma underwent serial studies of tumor cell kinetics including (1) estimation of the total body myeloma cell number (TBMC), (2) measurement of the myeloma cell tritiated thymidine labeling index (LI), and (3) calculation of the total number of myeloma cells undergoing DNA synthesis. Intermittent courses of chemotherapy with cycle-non-specific agents such as melphalan resulted in a marked increase in the LI of myeloma cells in patients who had a 75% reduction in TBMC. The long “plateau” phase of partial remission of myeloma in these patients was associated with a continued high LI: this suggests that the plateau resulted from a balance between the cytoreductive effects of chemotherapy and expansion of the growth fraction (GF) of the tumor. Preliminary attempts to capitalize therapeutically on this expansion of the GF in several patients included administration of the cycle-active agents vincristine and cytosine arabinoside. Vincristine appeared to induce a further reduction in tumor in several patients, although cytosine arabinoside appeared to be ineffective despite clear evidence of its inhibition of DNA synthesis in myeloma cells in vivo. Further clinical studies of the effects of cycle-active drugs on myeloma appear to be warranted; however, successful exploitation of the dynamic change in myeloma cell kinetics with chemotherapy will require the use of cycle-active agents with marked selective toxicity for myeloma cells.
Transient neutropenia induced by transfusion of blood exposed to nylon fiber filters
Authors: Schiffer CA , Aisner J , Wiernik PH
Published: October 2019

During the course of granulocyte collection by continuous-flow filtration leukopheresis, an abrupt fall in neutrophil count was noted (mean decrease 77%, range 64%-95%). Neutropenia occurred within 5 min of return of blood exposed to the nylon fiber filters and lasted less than 30 min. Saline exposed to the fibers, withdrawal and reinfusion of whole blood, and heparin did not cause neutropenia. Heparinized blood passed by gravity through isolated filters and reinfused immediately also induced neutropenia (mean decrease 64% +/- 8%, range 11%-19%). Blood anticoagulated with ACD (decrease 19.5% +/- 6%, range 6%-56%), heparinized plasma (N = 10, decrease 15% +/- 3%, range 3%-29%) and platelet-rich plasma exposed to the filters failed to produce neutropenia. 91% +/- 2% of the neutrophils adhered to the fibers using heparinized blood as compared to 21% +/- 5% using ACD (p less than 0.001). All donors were asymptomatic during the infusions. These results suggest that during neutrophil adherence a substance is released which produces profound, transient neutropenia perhaps by inducing margination of cells.
Inhibition of sickling in erythrocytes by amino acids
Authors: Rumen NM
Published: October 2019

Sickling of erythrocytes was rapidly and quantitatively inhibited at room temperature by 3.8 mM homoserine, asparagine, and glutamine but by no other amino acid.
The anemia of chronic disorders: studies of marrow regulation and iron metabolism
Authors: Douglas SW , Adamson JW
Published: October 2019

Marrow regulation and iron metabolism were evaluated in 17 patients with mild or moderate anemia associated with chronic disorders. In addition, whole blood P50 and red cell 2,3-diphosphoglycerate (DPG) levels were measured. The study group consisted of seven patients with non-hematologic malignancies, nine with infection or inflammation, and one with idiopathic hypoproliferative anemia. The mean whole blood P50 and DPG levels were elevated to 28.5 +/- 1.9 mm Hg and 7.03 +/- 0.83 mumole/ml packed RBC, respectively, as compared to normal values of 26.6 +/- 0.6 mm Hg and 4.83 +/- 0.33 mumole/ml packed RBC. Erythropoietin (ESF) excretion was variable (1.1–28.7 IRP U, day), clearly elevated above normal in only three patients and, within the study group, bore no relation to hematocrit. While nine of the 17 subjects had ESF excretion rates within the 95% limits predicted by hematocrit, the remaining eight had lower than expected values. No significant differences in ferrokinetics, ESF excretion, or hematologic profile were found between patients with malignancy and those with inflammation. Marrow transit times correlated inversely with both serum and urine ESF activity (r = -0.57, p less than 0.02; and r = -0.63, p less than 0.01, respectively), indicating that the marrow reticulocyte release response to ESF stimulation was unimpaired. Erythroid iron turnovers were unrelated to serum or urinary ESF activity but were significantly correlated with serum iron levels expressed as microgram/100 ml whole blood (r = 0.56, p less than 0.02). These studies suggest that there is an intraerythrocytic response to the anemia in this group of patients, document that reduced ESF production is not a uniform finding with the anemia of chronic disorders, and provide evidence that the marrow proliferative response to anemia is limited in many patients primarily by the availability of iron.
Peroxidase-H2O2-halide system: Cytotoxic effect on mammalian tumor cells
Authors: Clark RA , Klebanoff SJ , Einstein AB , Fefer A
Published: October 2019

Myeloperoxidase, H2O2, and a halide constitute a potent antimicrobial system. A cytotoxic effect of this system on a line of mouse ascitic lymphoma cells (LSTRA) is demonstrated here using four different assay systems: 51Cr release, trypan blue exclusion, inhibition of glucose C-1 oxidation, and loss of oncogenicity for mice. Deletion of each component of the system, preheating the peroxidase, or addition of azide, cyanide, or catalase abolished the cytotoxicity. Myeloperoxidase was effective with either chloride or iodide as the halide, while lastoperoxidase was effective with iodide but not chloride. The iodinated thyroid hormones, triiodothyronine and thyroxine, could substitute for the halide, and H2O2 could be replaced by a peroxide- generating enzyme system such as glucose and glucose oxidase or by H2O2 producing bacteria such as pneumococci or streptococci. The possibility is raised that the peroxidases of inflammatory cells and certain biologic fluids may affect tumor initiation or growth in vivo.